Dosing regimens using heterodimeric relaxin fusions

Abstract

The present disclosure relates to dosing regimens, methods, and pharmaceutical compositions for treating heart failure with pulmonary hypertension, comprising administering a heterodimeric relaxin fusion.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 63 / 386,762 (filed December 9, 2022), 63 / 387,359 (filed December 14, 2022), and 63 / 497,169 (filed April 19, 2023), the contents of each of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present disclosure relates to dosing regimens and methods for treating subjects with heart failure accompanied by pulmonary hypertension, comprising administering a heterodimeric relaxin fusion. [Background technology]

[0003] Heart failure is associated with significant morbidity and mortality. It is characterized by increased myocardial cell death and complex tissue remodeling, including interstitial fibrosis. A significant number of heart failure patients suffer from pulmonary hypertension (PH). It has been estimated that approximately 50% of heart failure patients with preserved ejection fraction (HFpEF) also suffer from pulmonary hypertension (PH), and approximately 60% of heart failure patients with reduced ejection fraction (HFrEF) also suffer from PH (Guazzi, (2014) Circ Heart Fail., 7:367-377; Miller et al., (2013) JACC Heart Fail., 1(4):290-299). Patients with heart failure and pulmonary hypertension (HF+PH) have been shown to have reduced survival rates compared with patients with heart failure without PH (Barnett and De Marco, (2012) Heart Fail. Clin. 8:447-459). In patients with heart failure, a 3 mmHg increase or decrease in estimated pulmonary artery diastolic pressure (ePAD), corresponding to an increase or decrease of approximately 4 mmHg in mean pulmonary arterial pressure (mPAP), was associated with a 24% increase or a 19% decrease in cardiovascular mortality, respectively (Zile MR, et al. (2017) Circ Heart Fail. 10:e003594). A 4 mmHg decrease in mPAP was also associated with an improvement in dyspnea in patients with HF+PH (Solomonica A, et al. (2013) Circ Heart Fail., 6:53-60).

[0004] Pulmonary hypertension (PH) group 2 results from left-sided cardiac dysfunction and is the most common cause of PH. Left-sided dysfunction increases pulmonary pressure, leading to pulmonary edema, pulmonary vascular damage, and ultimately right ventricular failure. PH group 2 consists mostly of HF New York Heart Association (NYHA) class III and IV patients (HF patients with limited physical activity who experience fatigue, palpitations, or dyspnea). 40% to 75% of HF patients have HFrEF, and 36% to 83% of HF patients with heart failure HFpEF also have PH. PH group 2 is associated with impaired exercise capacity and reduced survival. PH group 2 patients live with significantly limited physical activity and quality of life despite optimal guideline-directed management. Currently, there is no dedicated treatment for this group of patients.

[0005] Relaxin is a peptide hormone belonging to the insulin superfamily. In humans, the relaxin peptide family includes seven peptides with high structural similarity but low sequence similarity: relaxins 1, 2, and 3, and insulin-like peptides INSL3, INSL4, INSL5, and INSL6. Naturally occurring relaxins consist of A and B polypeptide chains covalently linked by two interchain disulfide bonds. The A chain has an additional intrachain disulfide bond. The relaxin gene encodes a prohormone with the structure BCA (B and A polypeptide chains connected by a C peptide). The prohormone undergoes endoproteolytic cleavage by PC1 and PC2 enzymes to remove the C peptide before secretion of mature relaxin.

[0006] Without being bound by theory, relaxin is understood to be a pleiotropic hormone that mediates systemic hemodynamic and renal adaptive changes during pregnancy. During pregnancy, relaxin mediates systemic hemodynamic and renal adaptive changes to meet the increased metabolic demands of pregnancy without causing cardiac injury, possessing the unique ability to simultaneously reduce both systemic vascular resistance and renal resistance (Conrad 2011). Without being bound by theory, if relaxin mimics these hemodynamic adaptations in the setting of heart failure, it could reduce myocardial demand and improve end-organ perfusion. Relaxin also possesses antifibrotic properties and has been shown to have beneficial effects in heart failure, including acute decompensated heart failure (ADHF). Relaxin activates several signaling cascades that have been shown to be beneficial in the setting of ischemia-reperfusion and heart failure. These signaling pathways include activation of the phosphoinositide 3-kinase pathway and activation of the nitric oxide signaling pathway (Bathgate RA et al. (2013) Physiol. Rev. 93(1):405-480, Mentz RJ et al. (2013) Am. Heart J. 165(2):193-199, Tietjens J et al. (2016) Heart 102:95-99, Wilson SS et al. (2015) Pharmacology 35:315-327).

[0007] Clinical trials have been conducted using unmodified recombinant human relaxin-2, serelaxin. Continuous intravenous administration of serelaxin to hospitalized patients improved markers of cardiac, renal, and liver injury and congestion (Felker GM et al. (2014) J. Am. Coll. Cardiol. 64(15):1591-1598; Metra M et al. (2013) J. Am. Coll. Cardiol. 61(2):196-206; Teerlink JR et al. (2013) Lancet 381(9860):29-39). Serelaxin also improved pulmonary artery pressure, cardiac output, and systemic and pulmonary vascular resistance, although these results required a continuous infusion of 30 μg / kg / day for approximately 20 hours (Ponikowski et al., (2014) European Heart Journal 35:431-441). The rapid clearance of serelaxin from patients' circulation limited its therapeutic effect, which rapidly disappeared when intravenous infusion was stopped. In addition, approximately one-third of patients experienced a significant drop in blood pressure (>40 mmHg) after receiving intravenous serelaxin, and as a result, the infusion rate had to be reduced by more than half.

[0008] WO 2013 / 004607 and WO 2018 / 138170 describe recombinant relaxin polypeptides in which relaxin A and relaxin B are fused in a single chain with a linker peptide. WO 2013 / 004607 describes recombinant relaxin with a linker peptide of at least 5 amino acids and less than 15 amino acids. WO 2018 / 138170 describes recombinant relaxin with a linker peptide of at least 15 amino acids.

[0009] In contrast, WO 2021 / 255127, incorporated herein by reference in its entirety, describes heterodimeric fusions comprising a relaxin chain polypeptide and a heterodimerization domain that exhibit relaxin activity without requiring the fusion of a relaxin A chain polypeptide and a relaxin B chain polypeptide in a single chain. WO 2021 / 255127 describes that heterodimerization of the heterodimerization domain induces correct folding and heterodimerization of the relaxin A and relaxin B chain polypeptides. Additionally, unlike wild-type relaxin proteins, the fusions of WO 2021255127 do not require endoproteolytic processing for biological activity and exhibit extended half-lives.

[0010] Patients with HF+PH may require long-term treatment, which can affect patient compliance, quality of life, and can result in adverse side effects when doses are high and / or need to be administered frequently (e.g., continuously, with respect to serelaxin). Thus, there remains a need to develop improved treatment regimens for patients with HF+PH that are administered less frequently and / or at lower doses to not only minimize side effects but also promote patient compliance, quality of life, and / or other therapeutic benefits. Summary of the Invention

[0011] Disclosed in part herein are low-dose and / or less frequent dosing regimens for heart failure subjects, optionally HF+PH subjects, using heterodimeric relaxin fusion proteins. The dosing regimens provided herein can improve patient quality of life while maintaining efficacy and minimizing side effects.

[0012] In one aspect, the disclosure provides a method of treating a subject having heart failure, optionally with heart failure accompanied by pulmonary hypertension, comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable excipient and about 1 mg to about 30 mg of a heterodimeric fusion, wherein the heterodimeric fusion is (i) a first heterodimerization domain connected to at least one relaxin A chain polypeptide or variant thereof; (ii) a second heterodimerization domain connected to at least one relaxin B chain polypeptide or variant thereof; The first heterodimerization domain heterodimerizes with the second heterodimerization domain, and the heterodimeric fusion has relaxin activity.

[0013] In some embodiments, the amount of heterodimeric fusion in the pharmaceutical composition is about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

[0014] In some embodiments, the method comprises administering the heterodimeric fusion to a subject once every two weeks (also referred to herein as "biweekly" administration). In some embodiments, the method comprises biweekly administration, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg. In some embodiments, the method comprises biweekly administration, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1 mg. In some embodiments, the method comprises biweekly administration, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1.1 mg. In some embodiments, the method comprises biweekly administration, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 5.4 mg. In some embodiments, the method comprises biweekly administration, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 30 mg.

[0015] In some embodiments, the method comprises subcutaneous administration to a subject. In some embodiments, the method comprises subcutaneous administration to a subject, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg. In some embodiments, the method comprises subcutaneous administration, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1 mg. In some embodiments, the method comprises subcutaneous administration, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1.1 mg. In some embodiments, the method comprises subcutaneous administration, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 5.4 mg. In some embodiments, the method comprises subcutaneous administration, and the amount of the heterodimeric fusion in the pharmaceutical composition is about 30 mg.

[0016] In some embodiments, the method comprises biweekly subcutaneous administration to a subject, wherein the amount of the heterodimeric fusion in the pharmaceutical composition is about 1 mg. In some embodiments, the method comprises biweekly subcutaneous administration to a subject, wherein the amount of the heterodimeric fusion in the pharmaceutical composition is about 1.1 mg. In some embodiments, the method comprises biweekly subcutaneous administration to a subject, wherein the amount of the heterodimeric fusion in the pharmaceutical composition is about 5.4 mg. In some embodiments, the method comprises biweekly subcutaneous administration to a subject, wherein the amount of the heterodimeric fusion in the pharmaceutical composition is about 30 mg.

[0017] In some embodiments, the subject may have heart failure with reduced ejection fraction, heart failure with intermediate ejection fraction, or heart failure with preserved ejection fraction. In some embodiments, the subject meets one or more of the following criteria: New York Heart Association (NYHA) functional class II-IV, mean pulmonary artery pressure (mPAP) greater than 20 mmHg, and pulmonary artery wedge pressure (PAWP) greater than 15 mmHg. In some embodiments, the subject meets one or more of the following criteria: chest congestion, dyspnea at rest or on minimal exertion, N-terminal prohormone of brain natriuretic peptide (NT-proBNP) greater than 125 pg / mL or brain natriuretic peptide (BNP) greater than 35 pg / mL, systolic blood pressure >125 mmHg, mild to moderate renal insufficiency, and a body mass index of at least 18 kg / m. 2 and a reduced ejection fraction (HFrEF) of 40 percent or less.

[0018] In some embodiments, the endogenous relaxin plasma level in the subject is about 10,000-fold lower than the endogenous relaxin plasma level in a first-trimester pregnant subject. In some embodiments, the amount of heterodimeric fusion in the pharmaceutical composition is sufficient to provide the subject with a relaxin exposure corresponding to at most about 0.5-fold, at most about 2.5-fold, or at most about 15-fold the endogenous relaxin level in the plasma of a first-trimester pregnant subject. In some embodiments, the amount of heterodimeric fusion in the pharmaceutical composition is sufficient to provide the subject with a relaxin exposure corresponding to at most about 0.5-fold, at most about 3.5-fold, or at most about 7-fold the endogenous relaxin level in the plasma of a first-trimester pregnant subject. Relaxin levels, e.g., endogenous relaxin levels in the plasma of a first-trimester pregnant subject, can be measured using a relaxin detection assay, e.g., by the use of an anti-relaxin antibody. An example of a suitable assay is described in Example 10. In some embodiments, the average endogenous relaxin level in the plasma of first trimester pregnant subjects may be at or about 0.2 ng / mL (e.g., as measured using a relaxin detection assay, e.g., the assay described in Example 10).

[0019] In some embodiments, administration of the pharmaceutical composition is sufficient to result in a minimum steady-state plasma concentration of the heterodimeric fusion in the subject of about or at least 0.026 μg / mL. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a maximum steady-state plasma concentration of the heterodimeric fusion in the subject of about or at least 1-2 μg / mL. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a maximum steady-state plasma concentration of the heterodimeric fusion in the subject of about or at least 1.6 μg / mL. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a plasma concentration of the heterodimeric fusion in the subject of 0.026 μg / mL to 1.6 μg / mL (i.e., inclusive of about 0.026 μg / mL to up to about 1.6 μg / mL in the subject).

[0020] In some embodiments, administration of the pharmaceutical composition is sufficient to result in a dose-dependent increase in renin of at least 2-fold compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a dose-dependent increase in renin of at least 2.5-fold compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a dose-dependent increase in renin of at least 3-fold compared to baseline (pre-administration) levels.

[0021] In some embodiments, the hematocrit level in the subject is reduced by 1 percent or less after administration of the pharmaceutical composition compared to the baseline (pre-administration) level, e.g., after administration of a 1-30 mg dose of the heterodimeric fusion. In some embodiments, the hematocrit level in the subject is reduced by 2 percent or less after administration of the pharmaceutical composition compared to the baseline (pre-administration) level. In some embodiments, the hematocrit level in the subject is reduced by 3 percent or less after administration of the pharmaceutical composition compared to the baseline (pre-administration) level. In some embodiments, the hematocrit level in the subject is reduced by 4 percent or less after administration of the pharmaceutical composition compared to the baseline (pre-administration) level. In some embodiments, the hematocrit level in the subject is reduced by 5 percent or less after administration of the pharmaceutical composition compared to the baseline (pre-administration) level. In some embodiments, the hematocrit level in the subject is reduced by 6 percent or less after administration of the pharmaceutical composition compared to the baseline (pre-administration) level. In some embodiments, the hematocrit level in the subject is reduced by 7 percent or less after administration of the pharmaceutical composition compared to the baseline (pre-administration) level. In some embodiments, the hematocrit level in the subject is reduced by 8 percent or less after administration of the pharmaceutical composition compared to the baseline (pre-administration) level.

[0022] In some embodiments, the hemoglobin level in the subject is reduced by 2 percent or less after administration of the pharmaceutical composition compared to baseline (pre-administration) levels, e.g., after administration of a 1-30 mg dose of the heterodimeric fusion. In some embodiments, the hemoglobin level in the subject is reduced by 3 percent or less after administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, the hemoglobin level in the subject is reduced by 4 percent or less after administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, the hemoglobin level in the subject is reduced by 5 percent or less after administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, the hemoglobin level in the subject is reduced by 6 percent or less after administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, albumin levels are not significantly reduced in the subject after administration of the pharmaceutical composition compared to baseline levels.

[0023] Administration of the pharmaceutical compositions described herein may result in: (a) Increase in stroke volume (SV), (b) a decrease in systemic vascular resistance (SVR) and / or an increase in estimated glomerular filtration rate (eGFR); (c) increased ejection fraction; (d) Increased cardiac output The changes in SV, SVR, eGFR, ejection fraction, and / or cardiac output may each occur after 1 to 24 weeks of treatment. In some embodiments, the changes in SV, SVR, eGFR, ejection fraction, and / or cardiac output occur after 24 weeks of treatment.

[0024] Administration of the pharmaceutical compositions described herein may also result in an increase in: (a) Reduction of pulmonary vascular resistance (PVR), (b) reduction of mean pulmonary artery pressure (mPAP); (c) Reduction of estimated pulmonary artery diastolic pressure (ePAD) The changes in PVR, mPAP, and / or ePAD may each occur after 1 to 24 weeks of treatment. In some embodiments, the changes in PVR, mPAP, and / or ePAD occur after 24 weeks of treatment.

[0025] Thus, administration of the pharmaceutical compositions described herein may result in: a) Reduction of PVR, (b) reduction of mPAP; (c) reduction of ePAD; (d) Increased cardiac stroke volume (SV), (e) reduction in systemic vascular resistance (SVR) and / or increase in estimated glomerular filtration rate (eGFR); (f) increased ejection fraction; (g) Increased cardiac output The changes in PVR, mPAP, ePAD, SV, SVR, eGFR, ejection fraction, and / or cardiac output may each occur after 1 to 24 weeks of treatment. In some embodiments, the changes in PVR, mPAP, ePAD, SV, SVR, eGFR, ejection fraction, and / or cardiac output occur after 24 weeks of treatment.

[0026] In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in stroke volume (SV) in a subject, e.g., after 1 to 24 weeks of treatment with, e.g., a 1 to 30 mg dose of the heterodimeric fusion. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to 55 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to 50 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to 25 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to 20 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to 10 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to 5 percent compared to baseline (pre-administration) levels. In some embodiments, administration is for at least 10 weeks. In some embodiments, the administration is for at least 24 weeks.

[0027] In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to 1 percent compared to baseline (pre-administration) levels, optionally after 24 weeks of treatment, for example, at a biweekly dose of about 1 mg or about 1.1 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to about 5 percent compared to baseline (pre-administration) levels, optionally after 24 weeks of treatment, for example, at a biweekly dose of about 5.4 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to about 10 percent compared to baseline (pre-administration) levels, optionally after 24 weeks of treatment, for example, at a biweekly dose of about 30 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to about 25 percent compared to baseline (pre-administration) levels, optionally after 24 weeks of treatment, for example, at a biweekly dose of about 5.4 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of up to about 60 percent compared to baseline (pre-administration) levels, optionally after 24 weeks of treatment, e.g., at a biweekly dose of about 30 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to result in an increase in SV of at least 10 percent compared to baseline (pre-administration) levels, optionally after 24 weeks of treatment.

[0028] In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in systemic vascular resistance (SVR) in a subject, e.g., after 1 to 24 weeks of treatment. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in SVR of up to about 35 percent compared to baseline (pre-administration) levels, e.g., after administration of a 1 to 30 mg dose of the heterodimeric fusion. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in SVR of up to about 30 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in SVR of up to about 25 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in SVR of up to about 20 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in SVR of up to about 15 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in SVR of up to about 10 percent compared to baseline (pre-administration) levels. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a reduction in SVR of up to about 8 percent compared to baseline (pre-administration) levels. In some embodiments, administration is for at least 10 weeks. In some embodiments, administration is for at least 24 weeks.

[0029] In some embodiments, administration of the pharmaceutical composition is sufficient to result in a reduction in SVR of up to about 20 percent compared to baseline (pre-administration) levels after 24 weeks of treatment, optionally at a biweekly dose of, for example, about 1 mg or about 1.1 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a reduction in SVR of up to about 30 percent compared to baseline (pre-administration) levels after 24 weeks of treatment, optionally at a biweekly dose of, for example, about 5 mg or about 5.4 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a reduction in SVR of up to about 30 percent compared to baseline (pre-administration) levels after 24 weeks of treatment, optionally at a biweekly dose of, for example, about 30 mg.

[0030] In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in systemic vascular resistance (SVR) in the subject, e.g., after 1 to 10 weeks of treatment. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in SVR of up to about 10 percent compared to baseline (pre-administration) levels, optionally after 10 weeks of treatment, e.g., at a biweekly dose of about 5 mg or about 5.4 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a decrease in SVR of up to about 15 percent compared to baseline (pre-administration) levels, optionally after 10 weeks of treatment, e.g., at a biweekly dose of about 15 mg up to 30 mg.

[0031] In some embodiments, the heterodimeric fusion agonizes relaxin family peptide receptor 1 (RXFP1). In some embodiments, at least one relaxin A chain polypeptide or variant thereof of the heterodimeric fusion and at least one relaxin B chain polypeptide or variant thereof of the heterodimeric fusion are covalently linked by at least one interchain disulfide bond. In some embodiments, at least one relaxin A chain polypeptide or variant thereof of the heterodimeric fusion and at least one relaxin B chain polypeptide or variant thereof of the heterodimeric fusion are not covalently linked to each other by an amino acid linker. In some embodiments, at least one relaxin A chain polypeptide is a relaxin-2 A chain polypeptide and / or at least one relaxin B chain polypeptide is a relaxin-2 B chain polypeptide. In some embodiments, at least one relaxin A chain polypeptide is a relaxin-2 A chain polypeptide and at least one relaxin B chain polypeptide is a relaxin-2 B chain polypeptide. In some embodiments, at least one relaxin-2 A chain polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and / or at least one relaxin-2 B chain polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, at least one relaxin-2 A chain polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and at least one relaxin-2 B chain polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0032] In some embodiments, at least one relaxin A chain polypeptide or variant thereof is connected to a first heterodimerization domain of the heterodimeric fusion via a connector, and at least one relaxin B chain polypeptide or variant thereof is connected to a second heterodimerization domain via a connector. In some embodiments, at least one connector is a polypeptide. In some embodiments, both connectors are polypeptides. In some embodiments, at least one connector is a polypeptide having a length of 6 to 40 amino acids. In some embodiments, both connectors are polypeptides having a length of 6 to 40 amino acids. In some embodiments, at least one connector is a polypeptide having a length of 21 amino acids. In some embodiments, both connectors are polypeptides having a length of 21 amino acids.

[0033] In some embodiments, at least one connector is a G4S / G5S amino acid linker. In some embodiments, both connectors are G4S / G5S amino acid linkers. In some embodiments, at least one connector has the amino acid sequence of SEQ ID NO: 5. In some embodiments, both connectors have the amino acid sequence of SEQ ID NO: 5.

[0034] In some embodiments, the first heterodimerization domain is derived from a first immunoglobulin Fc region and the second heterodimerization domain is derived from a second immunoglobulin Fc region, and the first and second Fc regions comprise constant domains CH2 and CH3. In some embodiments, the CH2 and CH3 domains are derived from an IgG1 immunoglobulin. In some embodiments, the CH2 and / or CH3 domains are mutated.

[0035] In some embodiments, the C-terminus of the first Fc region is connected to the N-terminus of at least one relaxin A chain polypeptide, and the C-terminus of the second Fc region is connected to the N-terminus of at least one relaxin B chain polypeptide.

[0036] In some embodiments, the first and second Fc regions comprise heterodimerization-promoting amino acid mutations. In some embodiments, the heterodimerization-promoting amino acid mutations are present in the CH3 domain of the first and second Fc regions.

[0037] In some embodiments, the heterodimerization-promoting amino acid mutations in the first Fc region comprise S354C and T366W in the CH3 domain, and the heterodimerization-promoting amino acid mutations in the second Fc region comprise Y349C, T366S, L368A, and Y407V in the CH3 domain, where amino acid numbering is according to the EU index as in Kabat. In some embodiments, the first and / or second Fc region further comprise amino acid mutations L234F, L235E, and P331S, where amino acid numbering is according to the EU index as in Kabat. In some embodiments, the first Fc region comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the second Fc region comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the first Fc region comprises the amino acid sequence of SEQ ID NO:4, and the second Fc region comprises the amino acid sequence of SEQ ID NO:3.

[0038] In some embodiments, the heterodimeric fusion comprises the amino acid sequences of SEQ ID NO: 11 (a relaxin-2 A chain polypeptide connected to a first Fc region via a connector) and SEQ ID NO: 20 (a relaxin-2 B chain polypeptide connected to a second Fc region via a connector). In some embodiments, the heterodimeric fusion consists of the amino acid sequences of SEQ ID NO: 11 (a relaxin-2 A chain polypeptide connected to a first Fc region via a connector) and SEQ ID NO: 20 (a relaxin-2 B chain polypeptide connected to a second Fc region via a connector), and may be referred to as "AZD3427."

[0039] In some embodiments, the heterodimeric fusion further comprises a second relaxin A chain polypeptide or variant thereof connected to the N-terminus of the first Fc region and a second relaxin B chain polypeptide or variant thereof connected to the N-terminus of the second Fc region. In some embodiments, the second relaxin A chain is connected to the first Fc region via a connector polypeptide. In some embodiments, the connector polypeptide has the amino acid sequence of SEQ ID NO: 5. In some embodiments, the second relaxin B chain is connected to the second Fc region via a connector polypeptide. In some embodiments, the connector polypeptide has the amino acid sequence of SEQ ID NO: 5.

[0040] In some embodiments, the present disclosure provides a method of treating a subject having heart failure with pulmonary hypertension, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable excipient and about 1 mg to about 30 mg of a heterodimeric fusion, wherein the heterodimeric fusion (i) an FcX-con-A fusion polypeptide; (ii) an FcY-con-B fusion polypeptide; and Including, A is a relaxin A chain polypeptide or variant thereof, e.g., a relaxin-2 A chain or variant thereof; B is a relaxin B chain polypeptide or variant thereof, e.g., a relaxin-2 B chain or variant thereof; FcX is an Fc region comprising CH2 and CH3 of a human IgG1 immunoglobulin, optionally comprising the amino acid mutations S354C and T366W; FcY is the Fc region comprising CH2 and CH3 of a human IgG1 immunoglobulin, and optionally comprises the amino acid mutations Y349C, T366S, L368A, and Y407V; con is optionally a connector polypeptide having the amino acid sequence of SEQ ID NO:5; Amino acid numbering is according to the EU index as in Kabat, FcX heterodimerizes with FcY, and the heterodimeric fusion has relaxin activity. In some embodiments, the heterodimeric fusion comprises AZD3427. In some embodiments, the heterodimeric fusion is AZD3427. In some embodiments, the heterodimeric fusion is present in the pharmaceutical composition in an amount of about 1 mg to about 30 mg. In some embodiments, the heterodimeric fusion is present in the pharmaceutical composition in an amount of about 1 mg. In some embodiments, the heterodimeric fusion is present in the pharmaceutical composition in an amount of about 1.1 mg. In some embodiments, the heterodimeric fusion is present in the pharmaceutical composition in an amount of about 5.4 mg. In some embodiments, the heterodimeric fusion is present in the pharmaceutical composition in an amount of about 30 mg. In some embodiments, the pharmaceutical composition is administered to the subject via biweekly administration, e.g., at a dose of about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg of the heterodimeric fusion (e.g., AZD3427). In some embodiments, the pharmaceutical composition is administered to the subject via biweekly subcutaneous administration, e.g., at a dose of about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg of the heterodimeric fusion (e.g., AZD3427).

[0041] In some embodiments, AZD3427 is administered subcutaneously to a subject every other week at a dose of about 1 mg. In some embodiments, AZD3427 is administered subcutaneously to a subject every other week at a dose of about 1.1 mg. In some embodiments, AZD3427 is administered subcutaneously to a subject every other week at a dose of about 5.4 mg. In some embodiments, AZD3427 is administered subcutaneously to a subject every other week at a dose of about 30 mg.

[0042] In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered chronically to a subject. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered to a subject at least four times. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered to a subject at least five times. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered to a subject at least 12 times. [Brief explanation of the drawings]

[0043] [Figure 1] Exemplary formats of heterodimeric fusions provided herein are shown. The format of each fusion polypeptide of the heterodimeric fusion is given in terms of FcX, FcY, A, B, con, and L, where FcX and FcY are two Fc regions containing heterodimerization-promoting amino acid mutations and / or modifications, A ("RlxA") and B ("RlxB") are relaxin A chain and relaxin B chain polypeptides, "con" is a connector polypeptide, L is a linker polypeptide, HC X and HC Y represent antibody heavy chains, and LC represents antibody light chains. [Figure 2] 1 shows the pharmacokinetic (PK) profile of an exemplary heterodimeric fusion, AZD3427, in cynomolgus monkeys after intravenous (IV) and subcutaneous (SC) administration. LLOQ (lowest level of quantitation) = lowest level of quantitation. [Figure 3A] Figure 3 shows cardiac function and systemic vascular resistance in non-human primates (NHPs) after administration of AZD3427: Figure 3A shows ejection fraction (EF), Figure 3B shows cardiac output, Figure 3C shows systemic vascular resistance (SVR), and Figure 3D shows plasma renin concentration as a percentage of pre-dose. The dotted line in Figure 3D represents the final dose. [Figure 3B]Figure 3 shows cardiac function and systemic vascular resistance in non-human primates (NHPs) after administration of AZD3427: Figure 3A shows ejection fraction (EF), Figure 3B shows cardiac output, Figure 3C shows systemic vascular resistance (SVR), and Figure 3D shows plasma renin concentration as a percentage of pre-dose. The dotted line in Figure 3D represents the final dose. [Figure 3C] Figure 3 shows cardiac function and systemic vascular resistance in non-human primates (NHPs) after administration of AZD3427: Figure 3A shows ejection fraction (EF), Figure 3B shows cardiac output, Figure 3C shows systemic vascular resistance (SVR), and Figure 3D shows plasma renin concentration as a percentage of pre-dose. The dotted line in Figure 3D represents the final dose. [Figure 3D] Figure 3 shows cardiac function and systemic vascular resistance in non-human primates (NHPs) after administration of AZD3427: Figure 3A shows ejection fraction (EF), Figure 3B shows cardiac output, Figure 3C shows systemic vascular resistance (SVR), and Figure 3D shows plasma renin concentration as a percentage of pre-dose. The dotted line in Figure 3D represents the final dose. [Figure 4] Phase 1 study design is shown. SAD = single ascending dose (SAD). MAD = multiple ascending dose (MAD). HV = healthy volunteers. HF = heart failure patients. HF with EF >= 41% = heart failure with ejection fraction >= 41%. HFrEF = heart failure with reduced ejection fraction. JD = Japanese descent. F / U = follow-up. IV = intravenous. SC = subcutaneous. [Figure 5] Geometric mean serum AZD3427 concentrations (μg / mL) over time in hours (h) after a single dose in the Phase 1 SAD cohort. SC = subcutaneous. [Figure 6A]Figure 6 shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in pooled subjects, Figure 6D shows systemic vascular resistance (SVR) in pooled subjects, Figure 6E shows stroke volume (SV) in pooled subjects, Figure 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 6G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 6H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 61 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 6B] Figure 6 shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in pooled subjects, Figure 6D shows systemic vascular resistance (SVR) in pooled subjects, Figure 6E shows stroke volume (SV) in pooled subjects, Figure 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 6G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 6H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 61 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 6C]Figure 6 shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in pooled subjects, Figure 6D shows systemic vascular resistance (SVR) in pooled subjects, Figure 6E shows stroke volume (SV) in pooled subjects, Figure 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 6G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 6H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 61 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 6D] Figure 6 shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in pooled subjects, Figure 6D shows systemic vascular resistance (SVR) in pooled subjects, Figure 6E shows stroke volume (SV) in pooled subjects, Figure 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 6G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 6H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 61 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 6E]Figure 6 shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in pooled subjects, Figure 6D shows systemic vascular resistance (SVR) in pooled subjects, Figure 6E shows stroke volume (SV) in pooled subjects, Figure 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 6G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 6H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 61 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 6F] Figure 6 shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in pooled subjects, Figure 6D shows systemic vascular resistance (SVR) in pooled subjects, Figure 6E shows stroke volume (SV) in pooled subjects, Figure 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 6G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 6H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 61 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 6G]Figure 6 shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in pooled subjects, Figure 6D shows systemic vascular resistance (SVR) in pooled subjects, Figure 6E shows stroke volume (SV) in pooled subjects, Figure 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 6G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 6H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 61 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 6H] Figure 6 shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in pooled subjects, Figure 6D shows systemic vascular resistance (SVR) in pooled subjects, Figure 6E shows stroke volume (SV) in pooled subjects, Figure 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 6G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 6H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 61 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 6I]Figure 6 shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in pooled subjects, Figure 6D shows systemic vascular resistance (SVR) in pooled subjects, Figure 6E shows stroke volume (SV) in pooled subjects, Figure 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 6G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 6H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 61 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 7A] Figure 7 shows interim clinical results for plasma renin, hematocrit, and hemoglobin levels in the MAD cohort following administration of AZD3427 in MAD subjects: Figure 7A shows the percentage of renin, Figure 7B shows the percentage of hemoglobin, and Figure 7C shows the percentage of hematocrit. Observed data are presented as geometric means with 90% confidence intervals, and the solid lines represent the fitted Emax dose-response model (Figures 7B and 7C). [Figure 7B] Figure 7 shows interim clinical results for plasma renin, hematocrit, and hemoglobin levels in the MAD cohort following administration of AZD3427 in MAD subjects: Figure 7A shows the percentage of renin, Figure 7B shows the percentage of hemoglobin, and Figure 7C shows the percentage of hematocrit. Observed data are presented as geometric means with 90% confidence intervals, and the solid lines represent the fitted Emax dose-response model (Figures 7B and 7C). [Figure 7C]Figure 7 shows interim clinical results for plasma renin, hematocrit, and hemoglobin levels in the MAD cohort following administration of AZD3427 in MAD subjects: Figure 7A shows the percentage of renin, Figure 7B shows the percentage of hemoglobin, and Figure 7C shows the percentage of hematocrit. Observed data are presented as geometric means with 90% confidence intervals, and the solid lines represent the fitted Emax dose-response model (Figures 7B and 7C). [Figure 8] The phase 2b study design is shown. [Figure 9] Dosing comparisons are shown for AZD3427. QW = weekly (QW). Q2W = every other week. Q4W = every 4 weeks. Shaded areas indicate 90 percent prediction intervals. The dashed line (0.08 μg / mL) represents the estimated concentration of AZD3427 equivalent to the reference pregnancy level of relaxin in the first trimester (0.2 ng / mL, in-house assay). [Figure 10] Figure 1 shows the dose response on cardiac function and hemodynamic levels after biweekly AZD3427 administration using concentration-response relationships from the NHP HFrEF model and human PK data based on interim clinical data. [Figure 11A]

[0033] Figure 11A shows interim clinical data for hematocrit, hemoglobin, and albumin levels following AZD3427 administration: Figure 11A shows the percentage change from baseline in hematocrit, Figure 11B shows the percentage change from baseline in hemoglobin, and Figure 11C shows the percentage change from baseline in albumin. Observed data for MAD subjects are shown as geometric means with 90% confidence intervals, and the solid line represents the fitted Emax dose-response model. [Figure 11B]

[0033] Figure 11A shows interim clinical data for hematocrit, hemoglobin, and albumin levels following AZD3427 administration: Figure 11A shows the percentage change from baseline in hematocrit, Figure 11B shows the percentage change from baseline in hemoglobin, and Figure 11C shows the percentage change from baseline in albumin. Observed data for MAD subjects are shown as geometric means with 90% confidence intervals, and the solid line represents the fitted Emax dose-response model. [Figure 11C]

[0033] Figure 11A shows interim clinical data for hematocrit, hemoglobin, and albumin levels following AZD3427 administration: Figure 11A shows the percentage change from baseline in hematocrit, Figure 11B shows the percentage change from baseline in hemoglobin, and Figure 11C shows the percentage change from baseline in albumin. Observed data for MAD subjects are shown as geometric means with 90% confidence intervals, and the solid line represents the fitted Emax dose-response model. [Figure 12A] Figure 12 shows final clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A shows ejection fraction (EF) in HFpEF subjects, Figure 12B shows EF in HFrEF subjects, Figure 12C shows cardiac output in pooled subjects, Figure 12D shows systemic vascular resistance (SVR) in pooled subjects, and Figure 12E shows stroke volume (SV) in pooled subjects. , Figure 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 12G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 12H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 121 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 12B]Figure 12 shows final clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A shows ejection fraction (EF) in HFpEF subjects, Figure 12B shows EF in HFrEF subjects, Figure 12C shows cardiac output in pooled subjects, Figure 12D shows systemic vascular resistance (SVR) in pooled subjects, and Figure 12E shows stroke volume (SV) in pooled subjects. , Figure 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 12G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 12H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 121 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 12C] Figure 12 shows final clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A shows ejection fraction (EF) in HFpEF subjects, Figure 12B shows EF in HFrEF subjects, Figure 12C shows cardiac output in pooled subjects, Figure 12D shows systemic vascular resistance (SVR) in pooled subjects, and Figure 12E shows stroke volume (SV) in pooled subjects. , Figure 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 12G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 12H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 121 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 12D]Figure 12 shows final clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A shows ejection fraction (EF) in HFpEF subjects, Figure 12B shows EF in HFrEF subjects, Figure 12C shows cardiac output in pooled subjects, Figure 12D shows systemic vascular resistance (SVR) in pooled subjects, and Figure 12E shows stroke volume (SV) in pooled subjects. , Figure 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 12G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 12H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 121 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 12E] Figure 12 shows final clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A shows ejection fraction (EF) in HFpEF subjects, Figure 12B shows EF in HFrEF subjects, Figure 12C shows cardiac output in pooled subjects, Figure 12D shows systemic vascular resistance (SVR) in pooled subjects, and Figure 12E shows stroke volume (SV) in pooled subjects. , Figure 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 12G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 12H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 121 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 12F]Figure 12 shows final clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A shows ejection fraction (EF) in HFpEF subjects, Figure 12B shows EF in HFrEF subjects, Figure 12C shows cardiac output in pooled subjects, Figure 12D shows systemic vascular resistance (SVR) in pooled subjects, and Figure 12E shows stroke volume (SV) in pooled subjects. , Figure 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 12G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 12H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 121 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 12G] Figure 12 shows final clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A shows ejection fraction (EF) in HFpEF subjects, Figure 12B shows EF in HFrEF subjects, Figure 12C shows cardiac output in pooled subjects, Figure 12D shows systemic vascular resistance (SVR) in pooled subjects, and Figure 12E shows stroke volume (SV) in pooled subjects. , Figure 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 12G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 12H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 121 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 12H]Figure 12 shows final clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A shows ejection fraction (EF) in HFpEF subjects, Figure 12B shows EF in HFrEF subjects, Figure 12C shows cardiac output in pooled subjects, Figure 12D shows systemic vascular resistance (SVR) in pooled subjects, and Figure 12E shows stroke volume (SV) in pooled subjects. , Figure 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 12G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 12H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 121 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 12I] Figure 12 shows final clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A shows ejection fraction (EF) in HFpEF subjects, Figure 12B shows EF in HFrEF subjects, Figure 12C shows cardiac output in pooled subjects, Figure 12D shows systemic vascular resistance (SVR) in pooled subjects, and Figure 12E shows stroke volume (SV) in pooled subjects. , Figure 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, Figure 12G shows systolic blood pressure (SBP) in (i) patients with EF≦40% and (ii) patients with EF>40%, Figure 12H shows stroke volume in (i) patients with EF≦40% and (ii) patients with EF>40%, and Figure 121 shows eGFR in (i) patients with EF≦40% and (ii) patients with EF>40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1). SE = standard error. [Figure 13A]Figure 13 shows final clinical data for plasma renin, hematocrit, and hemoglobin levels in the MAD cohort following administration of AZD3427 in MAD subjects: Figure 13A shows the percentage of renin, Figure 13B shows the percentage of hemoglobin, and Figure 13C shows the percentage of hematocrit. Observed data are presented as geometric means with 90% confidence intervals, and the solid lines represent the fitted Emax dose-response model (Figures 13B and 13C). [Figure 13B] Figure 13 shows final clinical data for plasma renin, hematocrit, and hemoglobin levels in the MAD cohort following administration of AZD3427 in MAD subjects: Figure 13A shows the percentage of renin, Figure 13B shows the percentage of hemoglobin, and Figure 13C shows the percentage of hematocrit. Observed data are presented as geometric means with 90% confidence intervals, and the solid lines represent the fitted Emax dose-response model (Figures 13B and 13C). [Figure 13C] Figure 13 shows final clinical data for plasma renin, hematocrit, and hemoglobin levels in the MAD cohort following administration of AZD3427 in MAD subjects: Figure 13A shows the percentage of renin, Figure 13B shows the percentage of hemoglobin, and Figure 13C shows the percentage of hematocrit. Observed data are presented as geometric means with 90% confidence intervals, and the solid lines represent the fitted Emax dose-response model (Figures 13B and 13C). [Figure 14] Figure 1 shows the dose response on cardiac function and hemodynamic levels after biweekly AZD3427 administration using concentration-response relationships from the NHP HFrEF model and human PK data based on final clinical data. [Figure 15A]Final clinical data for hematocrit, hemoglobin, and albumin levels following AZD3427 administration are shown: Figure 15A shows the percentage change from baseline in hematocrit, Figure 15B shows the percentage change from baseline in hemoglobin, and Figure 15C shows the percentage change from baseline in albumin. Observed data for MAD subjects are shown as geometric means with 90% confidence intervals, and the solid line represents the fitted Emax dose-response model. [Figure 15B] Final clinical data for hematocrit, hemoglobin, and albumin levels following AZD3427 administration are shown: Figure 15A shows the percentage change from baseline in hematocrit, Figure 15B shows the percentage change from baseline in hemoglobin, and Figure 15C shows the percentage change from baseline in albumin. Observed data for MAD subjects are shown as geometric means with 90% confidence intervals, and the solid line represents the fitted Emax dose-response model. [Figure 15C] Final clinical data for hematocrit, hemoglobin, and albumin levels following AZD3427 administration are shown: Figure 15A shows the percentage change from baseline in hematocrit, Figure 15B shows the percentage change from baseline in hemoglobin, and Figure 15C shows the percentage change from baseline in albumin. Observed data for MAD subjects are shown as geometric means with 90% confidence intervals, and the solid line represents the fitted Emax dose-response model. DETAILED DESCRIPTION OF THE INVENTION

[0044] Disclosed herein are methods for treating heart failure subjects, optionally HF+PH subjects, comprising low-dose and / or infrequent administration of heterodimeric relaxin fusions.

[0045] Heterodimeric relaxin fusions As used herein, a "heterodimeric fusion" (also referred to as a "heterodimeric fusion protein") refers to a heterodimer of at least a first and a second fusion polypeptide, wherein the first fusion polypeptide comprises a first heterodimerization domain connected to a first subunit of a heterodimeric protein (e.g., a relaxin A chain polypeptide or a variant thereof), and the second fusion polypeptide comprises a second heterodimerization domain connected to a second subunit of a heterodimeric protein (e.g., a relaxin B chain polypeptide or a variant thereof).

[0046] In some embodiments, a heterodimeric fusion of the present disclosure comprises a first heterodimerization domain connected to at least one relaxin A chain polypeptide or variant thereof and a second heterodimerization domain connected to at least one relaxin B chain polypeptide or variant thereof, wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and the heterodimeric fusion has relaxin activity. In some embodiments, at least one relaxin A chain polypeptide or variant thereof is connected to the first heterodimerization domain via a connector, e.g., a connector polypeptide. In some embodiments, at least one relaxin B chain polypeptide or variant thereof is connected to the first heterodimerization domain via a connector, e.g., a connector polypeptide.

[0047] Relaxin chain polypeptide In some embodiments, the heterodimeric fusion of the present disclosure comprises relaxin A and B chain polypeptides selected from relaxin-1, relaxin-2, and relaxin-3, or variants thereof. In some embodiments, the relaxin A chain polypeptide is a relaxin-2 A chain polypeptide. In some embodiments, the relaxin B chain polypeptide is a relaxin-2 B chain polypeptide. In some embodiments, the relaxin A chain polypeptide comprises a human relaxin-2 A chain polypeptide. In some embodiments, the relaxin B chain polypeptide is a human relaxin-2 B chain polypeptide.

[0048] As used herein, a "variant" of a relaxin chain polypeptide differs from the wild-type relaxin chain polypeptide while retaining relaxin activity.

[0049] In some embodiments, relaxin A or relaxin B chain polypeptide variants that retain relaxin activity contain at least one conserved motif associated with relaxin activity. In some embodiments, relaxin-2 B chain polypeptide variants include the conserved motif Arg-XXX-Arg-XX-Ile (Claasz AA et al. (2002) Eur. J. Biochem. 269(24):6287-6293) or Arg-XXX-Arg-XX-Val (Bathgate RA et al. (2013) Physiol Rev. 93(1):405-480).

[0050] Variants may contain one or more amino acid substitutions and / or insertions. In some embodiments, relaxin-2 A chain polypeptide variants contain one or more amino acid substitutions selected from K9E, K9H, K9L, K9M, R18E, R18H, R22A, R22I, R22M, R22Q, R22S, R22Y, F23E, F23A, and F23I. In some embodiments, relaxin-2 A chain polypeptide variants contain the amino acid substitution K9H. In some embodiments, relaxin-2 B chain polypeptide variants contain one or more additional amino acids, e.g., K30 and R31, and the N-terminus V-2, A-1, and M-1, compared to SEQ ID NO: 62.

[0051] In some embodiments, the variant comprises one or more amino acid derivatives. In some embodiments, the first amino acid of the relaxin-2 B chain polypeptide variant is pyroglutamate.

[0052] In some embodiments, the relaxin-2 A chain polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the relaxin-2 B chain polypeptide comprises the amino acid sequence of SEQ ID NO:2.

[0053] In some embodiments, the relaxin chain polypeptide of the heterodimeric fusion agonizes relaxin family peptide receptor 1 (RXFP1). In some embodiments, the relaxin chain polypeptide is the relaxin chain polypeptide of AZD3427.

[0054] Relaxin activity The heterodimeric fusions provided herein exhibit relaxin activity. As used herein, "relaxin activity" (used interchangeably with "biological activity") refers to the ability of relaxin to bind to a relaxin receptor and / or activate a relaxin receptor and / or initiate a signaling cascade inside a cell. In some embodiments, relaxin activity is relaxin-2 activity, and relaxin activity refers to the ability to bind to and / or activate the receptor RXFP1 and / or the receptor RXFP2. Relaxin activity can be determined in vitro and / or in vivo. In some embodiments, relaxin activity is determined in vitro.

[0055] Heterodimeric fusions of the present disclosure can be determined to have relaxin activity if they exhibit at least a percentage of the activity of the reference relaxin protein. For example, heterodimeric fusions of the present disclosure can be determined to have relaxin activity if the ratio of the activity of the heterodimeric fusion to the activity of the reference relaxin protein is about 10 -5 ~ about 1, about 10 -4 ~ about 1, about 10 -3 ~ about 1, about 10 -2 to about 1, about 1 / 50 to about 1, about 1 / 20 to about 1, about 1 / 15 to about 1, about 1 / 10 to about 1, about 1 / 5 to about 1, or about 1 / 2 to about 1. In some embodiments, a heterodimeric fusion of the present disclosure may have relaxin activity if the ratio of the activity of the heterodimeric fusion to the activity of a reference relaxin protein is between about 1 and about 10 5 , about 1 to about 10 4 , about 1 to about 10 3 , about 1 to about 100, about 1 to about 50, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 2. In some embodiments, the heterodimeric fusion has at least half the activity of the reference relaxin protein. In some embodiments, the heterodimeric fusion has at least three-quarters the activity of the reference relaxin protein. In some embodiments, the heterodimeric fusion has at least the same level of activity as the reference protein.

[0056] In some embodiments, the reference relaxin protein is a wild-type protein. In some embodiments, the reference relaxin protein is a recombinant protein. In some embodiments, the reference relaxin protein is a relaxin protein having the relaxin A chain and relaxin B chain array of a mature relaxin protein. Recombinant relaxins having the relaxin A chain and relaxin B chain array of a mature relaxin protein are commercially available. For example, recombinant human relaxin-2, mouse relaxin-1, and INSL3 are available from R&D systems (catalog numbers 6586-RN, 6637-RN, and 4544-NS, respectively). In some embodiments, the reference relaxin protein has the same relaxin A chain and relaxin B chain as the heterodimeric fusion of the present disclosure, or differs from the relaxin A chain and relaxin B chain of the heterodimeric fusion of the present disclosure by up to 10 amino acids, e.g., 1 or 2 amino acids. In some embodiments, the first amino acid of the B chain of the reference relaxin-2 is D, and this amino acid is absent at the first position of the relaxin B chain of the heterodimeric fusion. In some embodiments, the reference relaxin protein is a relaxin-2 protein having the relaxin-2 A chain and relaxin-2 B chain array of the mature relaxin-2 protein disclosed in UniProtKB / Swiss-Prot Accession No. P04090.1. Relaxin activity can be determined by measuring the binding of relaxin to a relaxin receptor and / or by measuring downstream events from binding to a relaxin receptor. In some embodiments, relaxin activity is determined by measuring the amount and / or presence of molecules downstream from relaxin activation of the receptor. In some embodiments, relaxin activity is determined by measuring cAMP production after relaxin activation of the receptor. Methods for detecting relaxin-induced cAMP production are known in the art. Such methods include the cAMP ELISA, the HTRF cAMP assay, and the HitHunter® cAMP assay.In some embodiments, relaxin activity is determined by measuring relaxin-induced cAMP production in an HTRF cAMP assay. In some embodiments, relaxin activity is determined by measuring nitric oxide (NO) production following relaxin activation of the receptor.

[0057] In some embodiments, relaxin activity is determined by measuring activation of molecules downstream from relaxin activation of the receptor, hi some embodiments, relaxin activity is determined by measuring activation of p42 / 44 MAPK.

[0058] In some embodiments, relaxin activity is determined by measuring the activation of relaxin target genes. In some embodiments, relaxin activity is determined, for example, by measuring the transcriptional activation of vascular endothelial growth factor (VEGF) in THP-1 cells. Methods for determining gene transcriptional activation are known in the art and include quantitative PCR analysis of mRNA. In some embodiments, the relative expression of VEGF mRNA is measured by quantitative real-time PCR induction of VEGF transcripts after incubation of THP-1 cells with relaxin, for example, as described in Xiao et al. (2013) Nat Commun. 4:1953.

[0059] In some embodiments, relaxin activity is determined by measuring one or more downstream effects of relaxin. For example, reduction in cardiac hypertrophy can be measured by echocardiography, left ventricular weight relative to body weight, and / or tibia length according to standard methods. In some embodiments, relaxin activity is determined by measuring fibrosis reduction by Masson's trichrome staining. In some embodiments, relaxin activity is determined by measuring modulation of connective tissue metabolism, for example, inhibition of profibrotic factors (e.g., transforming growth factor-beta (TGF-β)), inhibition of fibroblast proliferation and differentiation, and / or activation of matrix metalloproteinase (MMP)-mediated extracellular matrix degradation (Bathgate RA et al. (2013) Physiol Rev. 93(1):405-480).

[0060] Heterodimerization domain The heterodimeric fusions of the present disclosure comprise a first heterodimerization domain and a second heterodimerization domain. In some embodiments, the first and second heterodimerization domains are derived from an immunoglobulin Fc region. The first and second Fc regions comprise immunoglobulin domains CH2 and CH3. In some embodiments, either or both of the first and second Fc regions further comprise a CH4 domain. The Fc regions provided herein can be derived from an immunoglobulin from any species (e.g., IgG), optionally human (e.g., human IgG). In embodiments where the Fc region is derived from an IgG, the Fc region can be derived from any subclass of IgG (e.g., IgG1, IgG2, IgG3, IgG4). In some embodiments, the first and second Fc regions are derived from an IgG1. In some embodiments, the first and second Fc regions are derived from a human IgG1 immunoglobulin. In some embodiments, the first and second Fc regions are derived from an IgG4. In some embodiments, the first and second Fc regions are derived from a human IgG4 immunoglobulin.

[0061] In some embodiments, the first and second Fc regions comprise heterodimerization-promoting amino acid mutations. In some embodiments, the mutations comprise asymmetric, complementary modifications of the first and second Fc regions (e.g., creating an Fc knob and an Fc hole structure) such that both chains can fit together and thus form heterodimers, but each chain cannot dimerize with itself. Such modifications can include insertions, deletions, conservative substitutions, non-conservative substitutions, and rearrangements.

[0062] In some embodiments, the heterodimerization-promoting amino acid mutations are present in the CH3 domains of the first and second Fc regions.

[0063] In some embodiments, the first Fc region (FcX) and the second Fc region (FcY) are derived from a human IgG1 immunoglobulin and comprise mutations in the CH3 domain, wherein the mutations are selected from the combinations set forth in Table 1, or conservative substitutions thereof, and the positions are according to the EU index as in Kabat.

[0064] [Table 1]

[0065] In some embodiments, the FcY mutations include Y349C, T366S, L368A, and Y407V, or conservative substitutions thereof, and the FcX mutations include S354C and T366W, or conservative substitutions thereof, where amino acid numbering is according to the EU index as in Kabat. In some embodiments, the FcY mutations include Y349C, T366S, L368A, and Y407V, and the FcX mutations include S354C and T366W, where amino acid numbering is according to the EU index as in Kabat.

[0066] It will be understood that the Fc region may further comprise other amino acid modifications relative to the wild-type Fc region. The Fc region may be modified, for example, to increase the affinity of the IgG molecule for FcRn. WO 02 / 060919 discloses modified immunoglobulins comprising an Fc region with one or more amino acid modifications, and is incorporated herein by reference in its entirety. Methods for producing Fc regions with one or more amino acid modifications are known in the art. In some embodiments, the first and / or second Fc regions provided herein comprise one or more amino acid modifications that reduce or abolish an effector function of the Fc region. In some embodiments, the first and / or second Fc regions provided herein comprise one or more amino acid modifications that reduce or avoid cytotoxicity, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In some embodiments, the first Fc region further comprises the mutations L234F, L235E, and P331S, or conservative substitutions thereof, where amino acid numbering is according to the EU index as in Kabat. In some embodiments, the second Fc region further comprises the mutations L234F, L235E, and P331S, or conservative substitutions thereof, where amino acid numbering is according to the EU index as in Kabat. In some embodiments, the first and second Fc regions further comprise the mutations L234F, L235E, and P331S, or conservative substitutions thereof, where amino acid numbering is according to the EU index as in Kabat.

[0067] In some embodiments, the first Fc region comprises the amino acid sequence of SEQ ID NO: 4 and the second Fc region comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the first Fc region consists of the amino acid sequence of SEQ ID NO: 4 and the second Fc region consists of the amino acid sequence of SEQ ID NO: 3.

[0068] In some embodiments, the cysteine ​​at position 354 of the first Fc region (e.g., of SEQ ID NO: 4) and the cysteine ​​at position 349 of the second Fc region (e.g., of SEQ ID NO: 3) form a stabilizing disulfide bond.

[0069] connector One or both of the relaxin A and B chain polypeptides can be connected to their respective heterodimerization domains by a connector polypeptide. In some embodiments, the relaxin A chain polypeptide is connected to a first heterodimerization domain (e.g., a first Fc region) via a connector polypeptide, and the relaxin B chain polypeptide is connected to a second heterodimerization domain (e.g., a second Fc region) via a connector polypeptide.

[0070] In some embodiments, the heterodimeric fusion further comprises a second relaxin A chain polypeptide or variant thereof connected to the N-terminus of the first Fc region and a second relaxin B chain polypeptide or variant thereof connected to the N-terminus of the second Fc region. In some embodiments, the second relaxin A chain is connected to the first Fc region via a connector polypeptide.

[0071] The connector polypeptide can be of any suitable length, for example, about 6 to 40 amino acids in length, optionally about 6 to 21 amino acids in length. In some embodiments, the connector polypeptide is at least 6 amino acid residues in length, optionally at least 11 amino acids in length, and even optionally at least 16 amino acids in length. In some embodiments, the connector polypeptide is less than 40 amino acids in length. Connector polypeptides of different or the same length can be used in each arm of the heterodimeric fusions of the present disclosure. In some embodiments, at least one connector polypeptide has a length of 21 amino acids. In some embodiments, both connector polypeptides (i.e., the connector polypeptide connecting the relaxin A chain polypeptide with the first Fc region and the relaxin B chain polypeptide with the second Fc region) have a length of 21 amino acids.

[0072] Connector polypeptides of different or the same amino acid composition can be used in each arm of the heterodimeric fusions of the present disclosure.

[0073] In some embodiments, one or optionally both connector polypeptides comprise glycerine and serine repeats, such as those described in Chen X et al. (2013) Adv. Drug. Deliv. Rev. 65(10):1357-1369. In some embodiments, one or both connector polypeptides comprise the motif (GGGGS)n (SEQ ID NO: 69), where n can be 1-8, e.g., n is 4. In some embodiments, one or more connector polypeptides consist of the 10 amino acid sequence GGGSGGGSGG (SEQ ID NO: 60). In some embodiments, one or more connector polypeptides consist of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5). In some embodiments, both connector polypeptides (i.e., the connector polypeptide connecting the relaxin A chain polypeptide with the first Fc region and the relaxin B chain polypeptide with the second Fc region) consist of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5).

[0074] In some embodiments, one or both of the relaxin A and B chains may be connected to their respective heterodimerization domains by a synthetic connector polypeptide, such as a polyethylene glycol (PEG) polymer chain. PEGylation can be performed according to methods known in the art.

[0075] Exemplary Heterodimeric Fusions In some embodiments, the heterodimeric fusion comprises (i) a relaxin A chain polypeptide comprising the amino acid sequence of SEQ ID NO: 1 connected to a first Fc region comprising the amino acid sequence of SEQ ID NO: 4 by a connector polypeptide comprising the amino acid sequence of SEQ ID NO: 5, and (ii) a relaxin B chain polypeptide comprising the amino acid sequence of SEQ ID NO: 2 connected to a second Fc region comprising the amino acid sequence of SEQ ID NO: 3 by a connector polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0076] In some embodiments, the heterodimeric fusion comprises the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:20, where SEQ ID NO:11 is the amino acid sequence of a relaxin A chain polypeptide connected to a first Fc region by a connector polypeptide and SEQ ID NO:20 is the amino acid sequence of a relaxin B chain polypeptide connected to a second Fc region by a connector polypeptide, wherein the first and second Fc regions heterodimerize and the relaxin A and relaxin B chain polypeptides heterodimerize. In some embodiments, the heterodimeric fusion comprises a stabilizing disulfide bond between cysteine ​​at position 349 of the second Fc region and cysteine ​​at position 354 of the first Fc region, where position numbering is according to the EU index as in Kabat.

[0077] An exemplary heterodimeric fusion, AZD3427, consists of a fusion relaxin A-connector-Fc region of amino acid sequence SEQ ID NO: 11 heterodimerized with a fusion relaxin B-connector-Fc region of amino acid sequence SEQ ID NO: 20. In some embodiments, the heterodimeric fusion comprises AZD3427. In some embodiments, the heterodimeric fusion is AZD3427. The sequence used in AZD3427 is shown in Table 2.

[0078] [Table 2]

[0079] Pharmaceutical Composition The heterodimeric fusions of the present disclosure may be provided in pharmaceutical compositions.

[0080] The pharmaceutical compositions of the present disclosure may contain one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are known in the art, see, for example, Remington's Pharmaceutical Sciences (by Joseph P. Remington, 18th ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference in its entirety.

[0081] kit A kit containing the pharmaceutical composition of the present disclosure may be provided. The kit may include a package containing the pharmaceutical composition of the present disclosure and instructions. In some embodiments, the pharmaceutical composition of the present disclosure is formulated in a single-dose vial or a container closure system (e.g., a pre-filled syringe). Such a container may optionally be accompanied by a notice in the form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, and this notice reflects the agency's approval of the manufacture, use, or sale for human administration.

[0082] Medication and treatment methods The present disclosure provides methods of treating a subject with heart failure, optionally with pulmonary hypertension, by administering to the subject a heterodimeric relaxin fusion or a pharmaceutical composition comprising a heterodimeric relaxin fusion provided herein. It should be understood that any method of treatment disclosed herein also provides a disclosure of the corresponding use of a heterodimeric relaxin fusion or a pharmaceutical composition comprising a heterodimeric relaxin fusion to treat a subject with heart failure, optionally with pulmonary hypertension. It should be understood that features of any method of treatment described herein are disclosed mutatis mutandis in the context of the use of a heterodimeric relaxin fusion or a pharmaceutical composition comprising a heterodimeric relaxin fusion to treat a subject with heart failure, optionally with pulmonary hypertension. It should also be understood that any method of treatment disclosed herein also provides a disclosure of the corresponding use of a heterodimeric relaxin fusion or a pharmaceutical composition comprising a heterodimeric relaxin fusion in the manufacture of a medicament for treating a subject with heart failure, optionally with pulmonary hypertension. It should be understood that any method of treatment features described herein are disclosed mutatis mutandis in the context of the use of a heterodimeric relaxin fusion or a pharmaceutical composition comprising a heterodimeric relaxin fusion in the manufacture of a medicament for treating a subject with heart failure, optionally with heart failure accompanied by pulmonary hypertension. It should also be understood that any method of treatment disclosed herein provides a corresponding disclosure of a heterodimeric relaxin fusion or a pharmaceutical composition comprising a heterodimeric relaxin fusion for use in treating a subject with heart failure, optionally with heart failure accompanied by pulmonary hypertension. It should also be understood that any method of treatment features described herein are disclosed mutatis mutandis in the context of a heterodimeric relaxin fusion or a pharmaceutical composition comprising a heterodimeric relaxin fusion for use in treating a subject with heart failure, optionally with heart failure accompanied by pulmonary hypertension. The subject treated with the heterodimeric relaxin fusion or pharmaceutical composition can be an animal, optionally a mammal, and optionally a human.

[0083] In some embodiments, the subject meets one or more of the following criteria: New York Heart Association (NYHA) functional class II-IV, mean pulmonary artery pressure (mPAP) greater than 20 mmHg, and pulmonary artery occlusion pressure (PAWP) greater than 15 mmHg. In some embodiments, the subject meets one or more of the following criteria: chest congestion, dyspnea at rest or on minimal exertion, N-terminal prohormone of brain natriuretic peptide (NT-proBNP) greater than 125 pg / mL or brain natriuretic peptide (BNP) greater than 35 pg / mL, systolic blood pressure >125 mmHg, mild to moderate renal insufficiency, and a body mass index (BMI) of at least 18 kg / m. 2 and a reduced ejection fraction (HFrEF) of 40 percent or less.

[0084] As used herein, a subject with "pulmonary hypertension" is a subject with a mean pulmonary artery pressure of at least 20 mmHg. In some embodiments, the pulmonary hypertension is classified as Group 2 pulmonary hypertension as defined by the World Health Organization. This may also be referred to as "heart failure with pulmonary hypertension due to left heart disease." In some embodiments, the pulmonary hypertension is classified as Group 1 pulmonary arterial hypertension as defined by the World Health Organization (see Ryan et al., 2012, Pulm. Circ. 2(1):107-121).

[0085] Pulmonary hypertension and heart failure parameters can be measured or estimated using techniques known in the art. For example, these include echocardiography, pulmonary artery catheters, and implantable monitoring devices. In certain embodiments, the subject may be wearing a blood pressure monitoring device, optionally a pulmonary artery pressure monitoring device, as known in the art. In some embodiments, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. Typically, the device is worn before treatment with the heterodimeric fusions of the present disclosure provided herein. The subject wears the device during or after treatment.

[0086] A heterodimeric relaxin fusion or pharmaceutical composition of the present disclosure can be administered to a subject by injection, such as intravenous, subcutaneous, or intramuscular injection. In some embodiments, the heterodimeric fusion or pharmaceutical composition is administered subcutaneously. In some embodiments, the heterodimeric fusion is administered every other week.

[0087] In some embodiments, the heterodimeric relaxin fusion administered to a subject is administered chronically to the subject. In some embodiments, the heterodimeric fusion is administered to the subject at least four times. In some embodiments, the heterodimeric fusion is administered to the subject at least five times. In some embodiments, the heterodimeric fusion is administered to the subject at least 12 times. In some embodiments, the heterodimeric fusion is administered subcutaneously to the subject every other week. In some embodiments, the heterodimeric fusion is administered subcutaneously to the subject every other week, e.g., at about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

[0088] In some embodiments, the heterodimeric fusion consists of a fusion relaxin A-connector-Fc region of amino acid sequence SEQ ID NO: 11 heterodimerized with a fusion relaxin B-connector-Fc region of amino acid sequence SEQ ID NO: 20. In some embodiments, the heterodimeric fusion comprises AZD3427. In some embodiments, the heterodimeric fusion is AZD3427.

[0089] In some embodiments, AZD3427 is administered chronically to a subject. In some embodiments, AZD3427 is administered subcutaneously to a subject every other week. In some embodiments, AZD3427 is administered subcutaneously to a subject every other week, e.g., at about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

[0090] In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is about 1 mg to about 30 mg. In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg. In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is about 1 mg. In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is 0.9 mg to 1.1 mg. In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is about 1.1 mg. In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is 1.0 mg to 1.2 mg. In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is about 5.4 mg. In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is 5.3 mg to 5.5 mg. In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is about 5 mg. In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is about 30 mg. In some embodiments, the amount of heterodimeric fusion, eg, AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is between 29.9 mg and 30.1 mg.

[0091] In some embodiments, the amount of heterodimeric fusion, e.g., AZD3427, administered to a subject or in a pharmaceutical composition administered to a subject is about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, or about 30 mg.

[0092] Without being bound by theory, subcutaneous administration, e.g., at a biweekly dose of about 1-30 mg, may provide convenience and compliance benefits for the subject. For example, biweekly administration of a heterodimeric fusion, e.g., AZD3427, by subcutaneous injection at a lower, less frequent dose may provide advantages such as greater comfort for the subject or patient, improved patient compliance, and the opportunity to administer to the subject or patient outside of a hospital setting, providing a better quality of life.

[0093] In some embodiments, subcutaneous administration of the heterodimeric fusion is at a dose sufficient to increase relaxin levels, e.g., an increase of at least 0.5-fold that measured in the first trimester of pregnancy, while minimizing adverse effects such as decreases in hemoglobin, hematocrit, and / or albumin levels. Unless otherwise specified, relaxin levels during pregnancy refer to relaxin levels in the first trimester of pregnancy. In some embodiments, subcutaneous administration is at a dose sufficient to improve cardiac output. In some embodiments, subcutaneous administration is at a dose sufficient to improve organ perfusion. In some embodiments, subcutaneous administration is at a dose sufficient to increase blood flow through the kidneys. In some embodiments, subcutaneous administration is at a dose sufficient to increase stroke volume, e.g., by at least a 1 percent increase. In some embodiments, subcutaneous administration is at a dose sufficient to decrease systemic vascular resistance, e.g., by at least an 8 percent decrease. In some embodiments, subcutaneous administration is at a dose sufficient to decrease systemic vascular resistance, e.g., by at least a 10 percent decrease. In some embodiments, subcutaneous administration is at a dose sufficient to decrease systemic vascular resistance, e.g., by at least a 15 percent decrease.

[0094] In some embodiments, the dose of the heterodimeric fusion, e.g., AZD3427, is selected to provide a subject with a relaxin exposure that corresponds to at most about 0.5-fold, at most about 2.5-fold, or at most about 15-fold the endogenous relaxin level in the plasma of the first-trimester pregnant subject. In some embodiments, the dose of the heterodimeric fusion, e.g., AZD3427, is selected to provide a subject with a relaxin exposure that corresponds to at most about 0.5-fold, at most about 3.5-fold, or at most about 7-fold the endogenous relaxin level in the plasma of the first-trimester pregnant subject. Relaxin levels, e.g., endogenous relaxin levels in the plasma of a first-trimester pregnant subject, can be measured using a relaxin detection assay, e.g., by the use of an anti-relaxin antibody. An example of a suitable assay is described in Example 10. In some embodiments, the average endogenous relaxin level in the plasma of a first-trimester pregnant subject can be at most about 0.2 ng / mL (e.g., as measured using a relaxin detection assay, e.g., the assay described in Example 10).

[0095] In some embodiments, subcutaneous administration of a biweekly dose of about 1-30 mg of a heterodimeric fusion, e.g., AZD3427, results in a dose-dependent increase in renin of at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 1.7-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, or at least 4-fold. In some embodiments, subcutaneous administration of a biweekly dose of about 1-30 mg of a heterodimeric fusion, e.g., AZD3427, results in a dose-dependent increase in renin of at least 2-fold. In some embodiments, the dose of the heterodimeric fusion, e.g., AZD3427, is selected to result in a dose-dependent increase in renin of at least 2-fold.

[0096] In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 to 30 mg increases cardiac output in a subject by about 1 percent, about 2 percent, about 3 percent, about 4 percent, about 5 percent, about 6 percent, about 7 percent, about 8 percent, about 9 percent, about 10 percent, about 11 percent, about 12 percent, about 13 percent, about 14 percent, about 15 percent, about 16 percent, about 17 percent, about 18 percent, about 19 percent, about 20 percent, about 21 percent, about 22 percent, about 23 percent, about 24 percent, about 25 percent, about 26 percent, about 27 percent, about 28 percent, about 29 percent, or about 30 percent, e.g., after 10 to 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 mg increases cardiac output in a subject by about 10 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1.1 mg increases cardiac output in a subject by about 10 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 5.4 mg increases cardiac output in a subject by about 10 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 30 mg increases cardiac output in a subject by at least about 10 percent, e.g., after 24 weeks of treatment.

[0097] In some embodiments, subcutaneous administration of about 1 to 30 mg of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of results in an increase in stroke volume (SV) in about 0.5 percent, 1 percent, about 2 percent, about 3 percent, about 4 percent, about 5 percent, about 6 percent, about 7 percent, about 8 percent, about 9 percent, about 10 percent, about 11 percent, about 12 percent, about 13 percent, about 14 percent, about 15 percent, or about 20 percent of subjects, e.g., after 10 or 24 weeks of treatment. In some embodiments, subcutaneous administration of about 1 to 30 mg of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of results in an increase in stroke volume (SV) in about 5 to 60 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 mg results in an increase in stroke volume (SV) in about 1 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1.1 mg results in an increase in SV in about 1 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 5.4 mg results in an increase in SV in about 5 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 30 mg results in an increase in SV in about 10 percent of subjects, e.g., after 24 weeks of treatment.

[0098] In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg of a heterodimeric fusion, e.g., AZD3427, results in a reduction in systemic vascular resistance (SVR) in about 5 percent, about 10 percent, or about 15 percent of subjects, e.g., after 10 weeks of treatment.

[0099] In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 to 30 mg results in a reduction in systemic vascular resistance (SVR) in about 1 percent, about 2 percent, about 3 percent, about 4 percent, about 5 percent, about 6 percent, about 7 percent, about 8 percent, about 9 percent, about 10 percent, about 11 percent, about 12 percent, about 13 percent, about 14 percent, about 15 percent, about 16 percent, about 17 percent, about 18 percent, about 19 percent, about 20 percent, about 21 percent, about 22 percent, about 23 percent, about 24 percent, about 25 percent, about 26 percent, about 27 percent, about 28 percent, about 29 percent, about 30 percent, about 31 percent, about 32 percent, about 33 percent, about 34 percent, about 35 percent, about 36 percent, about 37 percent, about 38 percent, about 39 percent, or about 40 percent of subjects, e.g., after 24 weeks of treatment.

[0100] In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 mg results in a reduction in SVR in about 8 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1.1 mg results in a reduction in SVR in about 8 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 or 1.1 mg results in a reduction in SVR in up to about 20 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 5.4 mg results in a reduction in SVR in about 15 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 5.4 mg results in a reduction in SVR in up to about 30 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 30 mg results in a reduction in SVR in up to about 20 percent of subjects, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 30 mg results in a reduction in SVR in up to about 40 percent of subjects, e.g., after 24 weeks of treatment.

[0101] In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in an increase in estimated glomerular filtration rate (eGFR) in about 1 percent, about 2 percent, about 3 percent, about 4 percent, about 5 percent, about 6 percent, about 7 percent, about 8 percent, about 9 percent, about 10 percent, about 11 percent, about 12 percent, about 13 percent, about 14 percent, about 15 percent, about 16 percent, about 17 percent, about 18 percent, about 19 percent, about 20 percent, about 21 percent, about 22 percent, about 23 percent, about 24 percent, or about 25 percent of subjects, e.g., after 24 weeks of treatment.

[0102] In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in a reduction in pulmonary vascular resistance (PVR) in a subject of at least 1 to 10 percent, 1 to 20 percent, 1 to 30 percent, 1 to 40 percent, or 1 to 50 percent or more. In some embodiments, the reduction in PVR in a subject can be about 1 percent, about 2 percent, about 3 percent, about 4 percent, about 5 percent, about 6 percent, about 7 percent, about 8 percent, about 9 percent, about 10 percent, about 11 percent, about 12 percent, about 13 percent, about 14 percent, about 15 percent, about 16 percent, about 17 percent, about 18 percent, about 19 percent, about 20 percent, about 21 percent, about 22 percent, about 23 percent, about 24 percent, or about 25 percent, for example, after 24 weeks of treatment.

[0103] In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, reduces mean pulmonary artery pressure (mPAP) in a subject by at least 1 mmHg, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, reduces mean pulmonary artery pressure in a subject by at least 1 mmHg, at least 2 mmHg, at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 11 mmHg, at least 12 mmHg, at least 13 mmHg, at least 14 mmHg, or at least 15 mmHg, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, reduces estimated pulmonary artery diastolic pressure (ePAD) in a subject by at least 1 mmHg to 15 mmHg, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, reduces estimated pulmonary artery diastolic pressure in a subject by at least 1 mmHg, at least 2 mmHg, at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 11 mmHg, at least 12 mmHg, at least 13 mmHg, at least 14 mmHg, or at least 15 mmHg, e.g., after 24 weeks of treatment.

[0104] In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 to 30 mg increases ejection fraction (EF) percentage in a subject by about 1 percent to about 10 percent compared to placebo, e.g., after 10 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 to 30 mg increases ejection fraction (EF) percentage in a subject by about 1 percent to about 5 percent compared to placebo, e.g., after 10 weeks of treatment.

[0105] In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg increases ejection fraction (EF) percentage in a subject by about 1 percent to about 40 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg increases EF percentage in a subject by at least 5 percent, at least 10 percent, at least 20 percent, at least 30 percent, or at least 40 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 mg increases EF in a subject by at least 25 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1.1 mg increases EF in a subject by at least 25 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 5.4 mg increases EF in a subject by at least 30 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of a heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 30 mg increases EF in a subject by at least 35 percent, e.g., after 24 weeks of treatment.

[0106] In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, does not result in a change in hematocrit, hemoglobin, and / or albumin levels in a subject. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg of a heterodimeric fusion, e.g., AZD3427, does not result in a significant decrease in hematocrit, hemoglobin, and / or albumin levels in a subject. In some embodiments, subcutaneous administration of a biweekly dose of 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in a 1 percent or less, 2 percent or less, 3 percent or less, 4 percent or less, 5 percent or less, 6 percent or less, 7 percent or less, or 8 percent or less reduction in hematocrit levels in a subject. In some embodiments, subcutaneous administration of a biweekly dose of 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in a 5 percent or less reduction in hematocrit levels in a subject. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in a 7 percent or less reduction in hematocrit levels in a subject. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in an 8 percent or less reduction in hematocrit levels in a subject. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in a 1 percent or less, 2 percent or less, 3 percent or less, 4 percent or less, or 5 percent or less reduction in hemoglobin levels in a subject.In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in a 3 percent or less reduction in hemoglobin levels in a subject. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in a 1 percent or less, 2 percent or less, 3 percent or less, 4 percent or less, or 5 percent or less change in albumin levels. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in a 2 percent or less change in albumin levels compared to baseline levels. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in a decrease in albumin levels of no more than 1 percent, no more than 2 percent, no more than 3 percent, no more than 4 percent, or no more than 5 percent. In some embodiments, subcutaneous administration of a biweekly dose of about 1 to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) of a heterodimeric fusion, e.g., AZD3427, results in an increase in albumin levels of no more than 1 percent, no more than 2 percent, no more than 3 percent, no more than 4 percent, or no more than 5 percent.

[0107] In some embodiments, the change (eg, increase or decrease) in biomarker levels or patient outcome following administration of the heterodimeric fusion is relative to a baseline (pre-administration) measurement.

[0108] In some embodiments, administration of the heterodimeric fusion, e.g., AZD3427, to a patient does not cause a significant change in the patient's blood pressure (e.g., systolic blood pressure). This can be no significant change (e.g., percentage change) relative to the patient's baseline blood pressure (i.e., before administration) and / or compared to placebo. In some embodiments, no significant change in the patient's blood pressure (e.g., systolic blood pressure) means a change of 20% or less relative to the patient's baseline blood pressure (e.g., systolic blood pressure). This can be a decrease of 20% or less relative to the patient's baseline blood pressure (e.g., systolic blood pressure).

[0109] Exemplary Dosing Regimen In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered subcutaneously to a subject every other week. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered subcutaneously to a subject every other week at a dose of about 1 mg to about 30 mg. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered subcutaneously to a subject every other week at a dose of about 1 mg. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered subcutaneously to a subject every other week at a dose of about 1.1 mg. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered subcutaneously to a subject every other week at a dose of about 5.4 mg. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered subcutaneously to a subject every other week at a dose of about 30 mg.

[0110] Manufacturing method The heterodimeric fusions of the present disclosure can be produced by any method known in the art. In some embodiments, the heterodimeric fusions of the present disclosure are produced by recombinant expression of a nucleic acid molecule encoding the heterodimeric fusion (or one or more fragments thereof) in a host cell. For example, the fragment of the heterodimeric fusion can be a relaxin A-connector-first Fc region fusion or a relaxin B-connector-second Fc region fusion.

[0111] Methods known to those skilled in the art can be used to construct expression vectors containing nucleic acid molecules encoding the heterodimeric fusions of the present disclosure. Suitable vectors include, for example, plasmid, phagemid, phage, or viral vectors.

[0112] The vector containing the nucleic acid molecule encoding the heterodimeric fusion of the present disclosure can be transferred into a host cell by conventional techniques. Suitable host cells are known in the art. The host cell can be a mammalian cell, such as a HEK293 cell or a CHO cell.

[0113] The transfected cells can be cultured by conventional techniques to produce the fusion polypeptide of this disclosure.

[0114] Once a heterodimeric fusion of the present disclosure, or a fragment thereof, is produced, for example, by recombinant expression, it can be purified by any method known in the art. Exemplary protein purification techniques include chromatography (e.g., ion exchange, affinity, and / or sizing column chromatography), centrifugation, and differential solubility. The present disclosure provides isolated heterodimeric fusions separated from cell culture, optionally by at least one purification step.

[0115] definition As used herein, the articles "a" and "an" may refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0116] "About" and "approximately" are used interchangeably herein to refer to an acceptable degree of variation in the amount measured given the nature or precision of measurements based on pharmaceutical preparations. For example, with respect to the doses of the heterodimeric fusions provided herein, "about" allows for a variation of ±0.1 from the stated value.

[0117] As used herein, the term "biweekly" administration refers to administration once every two weeks.

[0118] Embodiments provided herein as "comprising" one or more features may also be considered to disclose corresponding embodiments "consisting of" such features.

[0119] The term "EU index as in Kabat" refers to the numbering system for the human IgG1 EU antibody as described in Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service. National Institutes of Health. Bethesda, MD. All amino acid positions referenced in this application refer to EU index positions.

[0120] As used herein, the term "heart failure" includes acute heart failure, chronic heart failure (CHF), and acutely decompensated heart failure (ADHF). The term "heart failure" also includes more specific diagnoses, such as heart failure with preserved ejection fraction (HFpEF), heart failure with an ejection fraction ≧41% (HF with EF ≧41%), heart failure with intermediate ejection fraction, or heart failure with reduced ejection fraction (HFrEF). This also includes heart failure due to hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0121] As used herein, the term "heart failure with pulmonary hypertension" refers to the subset of heart failure subjects who also suffer from pulmonary hypertension (HF+PH subjects).

[0122] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

[0123] The term "plasma concentration" refers to the average plasma concentration at steady state.

[0124] The term "treatment" refers to the amelioration and / or elimination of one or more symptoms or causes of a target disease or condition. In some embodiments, this includes modulating the level of one or more biological markers or functions, e.g., compared to a diseased state, e.g., to within a non-diseased range (compared to a healthy cohort).

[0125] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format. It should be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​expressly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were expressly recited. Ranges should be understood to include the endpoints of the range, unless otherwise specified.

[0126] The above embodiments and the following examples should be understood as illustrative and non-limiting. Further embodiments are contemplated. It should be understood that any feature provided in connection with any one embodiment may be used alone or in combination with other features provided, in combination with one or more features of any other embodiment, or in any combination of any other embodiment. Furthermore, equivalents and modifications not provided above may be employed without departing from the scope of the present disclosure, as defined in the appended claims.

[0127] In the context of this disclosure, other examples and variations of the fusion polypeptides and methods provided herein will be apparent to those of skill in the art.

[0128] Other examples and variations are within the scope of this disclosure, as set forth in the following claims. All documents cited herein are each incorporated by reference in their entirety, including all data, tables, figures, and text presented in the cited documents. [Example]

[0129] Example 1: Production of recombinant heterodimeric Fc-relaxin-2 fusion protein Relaxin-2 chains A and B were genetically fused to two complementary Fc moieties (at the N- and / or C-termini of the Fc) via connectors, as shown in Figure 1. CHO cells were then co-transfected with two expression vectors containing each of the single Fc-relaxin chains (A and / or B). The two complementary Fc moieties assemble within the CHO cells, thereby promoting the assembly and correct folding of relaxin-2.

[0130] The heterodimeric Fc-relaxin-2 fusion protein was secreted into the supernatant and then purified using an automated system by affinity chromatography, in which the Fc region of the protein binds to a column matrix.

[0131] Example 2: PK profile of RELAX0023 (AZD3427) in cynomolgus monkeys The pharmacokinetic (PK) profile of RELAX0023 (AZD3427) in cynomolgus monkeys was determined using a sandwich ELISA-based immunoassay. RELAX0023 was administered to a total of 12 female cynomolgus monkeys, randomly assigned to four groups of three animals per group. Animals in groups 1, 2, and 3 received 0.1, 1, and 10 mg / kg of RELAX0023 SC, respectively. Animals in group 4 received a 10 mg / kg IV bolus of RELAX0023. Serum samples were collected at 0.25 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 96 hours, 7 days, 14 days, and 21 days after drug administration.

[0132] Assay plates were coated with goat anti-human IgG antibody and incubated with cynomolgus monkey serum from animals in groups 1 to 4. Plate-bound RELAX0023 was detected with an anti-relaxin antibody conjugated to HRP. Cynomolgus monkey serum was diluted 1:10 and then added to the plate. The lower limit of quantitation in 100% serum was 0.010 μg / mL, and the upper limit of quantitation was 0.300 μg / mL.

[0133] Figure 2 shows the mean serum concentration-time profiles of AZD3427 in cynomolgus monkeys after a single dose. After a single dose was administered SC, AZD3427 exhibited linear PK over the dose range of 0.01 to 10 mg / kg. A dose-proportional increase in Cmax was observed. The mean Cmax values ​​were 0.400, 4.69, and 34.8 μg / mL for the 0.1, 1, and 10 mg / kg SC dose groups, respectively. A dose-proportional increase in AUC0-last values ​​was also observed in the SC groups from 0.1 mg / kg to 10 mg / kg. The mean AUC0-last values ​​were 2.01, 25.5, and 193 μg·day / mL for the 0.1, 1, and 10 mg / kg SC dose groups, respectively. Overall, AZD3427 PK was linear over the range of 0.1 mg / kg to 10 mg / kg, with a mean CL / F of 51.0 mL / day.

[0134] Example 3: Evaluation of the long-term efficacy of AZD3427 in cynomolgus monkeys (Macaca fascicularis) with heart failure and reduced left ventricular ejection fraction (LVEF) The long-term efficacy of AZD3427 on cardiac function was evaluated in obese and elderly cynomolgus monkeys (Macaca fascicularis). Cynomolgus monkeys were chosen as the test species over other lower mammalian species because of their close phylogenetic and physiological relationship to humans. Elderly cynomolgus monkeys fed a high-fat diet for at least two years share risk factors with human patients predisposed to cardiovascular disease and develop metabolic syndrome, which can characteristically progress to heart failure and reduced left ventricular ejection fraction (LVEF). The effect of AZD3427 on LVEF was evaluated when administered by subcutaneous (SC) injection at different dose levels over a 20-week period. The first dose was administered during week 1 of the study, followed by an 18-week observation period. From a pool of approximately 100 obese and elderly cynomolgus monkeys, aged 12–20 years and weighing 6–15 kg, who had been fed a high-fat diet for at least two years, 38 monkeys were identified by 2D echocardiographic screening as having an LVEF of 30–60%. Healthy monkeys of this age weigh 5–8 kg and have an LVEF of 70–75%; therefore, an LVEF of 60% or less represents a HFrEF model. Identified animals were selected and randomly assigned to three treatment groups, each with eight monkeys, and a vehicle group with 14 monkeys. The dosing period consisted of once-weekly (QW) SC administration of AZD3427 at three escalating dose levels (0.015 mg / kg, 0.15 mg / kg, and 3 mg / kg).

[0135] Cardiac function measurements by 2D echocardiography were determined nine times: baseline (week 2) and weeks 5, 9, 13, 17, 21, 25, 29, and 33 during the dosing and post-dose observation periods. An additional 2D echocardiogram was scheduled for week 39 (end of study). Parameters, including LVEF, were based on apical two- and four-chamber views and biplane views. High-definition oscillometry (HDO) was used to measure parameters including mean arterial pressure (MAP) and heart rate (HR).

[0136] AZD3427 significantly improved LVEF at all AZD3427 dose levels compared to vehicle control at weeks 5, 9, 13, 17, and 21 (Figures 3A-3D) without affecting heart rate or blood pressure. Remarkably, improved LVEF after AZD3427 treatment compared to week 0 (baseline) was observed throughout the washout period from the end of treatment to week 33 of the study. There was also a clear increase in stroke volume (SV) and a decrease in systemic vascular resistance (SVR). A dose-dependent increase in renin was also observed, likely a compensatory response to vasodilation after AZD3427 treatment. These striking results demonstrate a significant improvement in hemodynamics in treated animals and clearly demonstrate the efficacy of AZD3427 in treating heart failure in this model. Furthermore, the magnitude of the sustained response after treatment is, to the best of our knowledge, not previously achieved by any other known compound targeting this mechanism of action pathway. The monkeys continued to be monitored through the 39th week of the study.

[0137] Example 4: Phase 1 (Ph1) study in healthy volunteers and patients with heart failure Study D8330C00001 was a Phase Ia / b, randomized, single-blind, placebo-controlled, first-time-in-human (FTIH) study (ClinicalTrials.gov identifier NCT04630067). The primary objective of the study was to evaluate the safety and tolerability of single and multiple ascending doses of AZD3427, and the secondary objectives were to evaluate (i) the pharmacokinetics (PK) and (ii) the immunogenicity of single and multiple ascending doses of AZD3427.

[0138] The study was conducted in two parts, Part A and Part B (Figure 4). Part A was a single ascending dose (SAD) study in healthy participants (men and women of non-childbearing potential), and Part B was a multiple ascending dose (MAD) study in participants (men and women of non-childbearing potential) with HF.

[0139] Part A included 56 healthy participants across seven cohorts (eight participants in each cohort) who received a single dose of AZD3427 or placebo. Within each cohort, six participants were randomized to receive AZD3427 and two participants were randomized to receive placebo. One cohort consisted exclusively of participants of Japanese descent (parents and all grandparents were Japanese). The seven cohorts were as follows: Cohorts 1a, 2a, 3a, and 4a: SC doses at dose levels of 5, 10, 30, and 90 mg, respectively Cohort 5a: 15 mg IV dose of AZD3427 at the dose level Cohort 6a: SC dose at the 270 mg dose level (participants of Japanese ancestry) Cohort 7a: A single SC AZD3427 dose of approximately 270 mg.

[0140] Part B included 48 patients across six cohorts (eight participants in each cohort). Of these, three cohorts consisted of participants with HFrEF (Cohorts 1b, 3b, and 5b), and three cohorts consisted of participants with HF with EF ≥ 41% (Cohorts 2b, 4b, and 6b). The dose levels in the HFrEF and HF with EF ≥ 41% cohorts were 5 mg (Cohorts 1b and 2b), 15 mg (Cohorts 3b and 4b), and 45 mg (Cohorts 5b and 6b), administered once weekly (QW) for 5 weeks (i.e., a total of 5 doses).

[0141] For Part B, which included 48 patients across six cohorts, inclusion criteria included: (i) all cohorts: patients with a known clinical diagnosis of stage C HF (NYHA classification I-III) and stable medical therapy for at least 12 weeks prior to screening, with no significant dose changes or addition of new medications during that period; (ii) cohorts 1b, 3b, and 5b: patients with a diagnosis of HFrEF, defined as EF ≤ 40%; (iii) cohorts 2b, 4b, and 6b: patients with a diagnosis of HF with EF ≥ 41% (including patients with a diagnosis of HFpEF, defined as EF ≥ 50%); and (iv) all cohorts: patients with a blood pressure of 18–40 kg / m. 2 (v) All cohorts: have a BMI of at least 55 kg and weigh less than or equal to 120 kg (including borderline values); and (v) All cohorts: have a prior documented NT-proBNP>125 pg / mL or BNP>35 pg / mL.

[0142] Example 5: Serum samples for determining AZD3427 concentrations Serum samples for determination of AZD3427 concentrations were analyzed by an electrochemiluminescent (ECL) method that has been validated to accurately and precisely quantify AZD3427 levels ranging from 0.10 μg / mL to 25.60 μg / mL in human serum samples.

[0143] AZD3427 was captured with a biotin-labeled antibody directed against the relaxin moiety of AZD3427 (clone AB1510209) coated at 2.0 μg / mL onto a Meso Scale Discovery (MSD) streptavidin-coated standard binding plate. Calibrators, quality controls (QCs), and samples were diluted to a method minimum required dilution (MRD) of 1:400 in assay buffer, and 50 μL / well was incubated on the plate with shaking (600 rpm) at room temperature for approximately 1 hour. After washing the assay plate, a ruthenium-labeled anti-AZD3427 antibody was added, whose knob binds to the hole Fc portion, a site on AZD3427 distinct from the site bound by the capture antibody. After incubating the assay plate for approximately 1 hour, excess reagent was washed off the plate. Addition of MSD read buffer to the plate resulted in an ECL reaction that luminesced the bound ruthenium molecules, which was measured as relative light units. The concentration of AZD3427 in the sample is determined by 1 / ECL, which relates light intensity to the concentration of AZD3427. 2 Determined by interpolation from the standard curve using a four-parameter curve fit with weighting.

[0144] Pharmacokinetic Data: AZD3427 serum concentration versus time profiles from the SAD cohort after SC dosing of 5 mg, 10 mg, 30 mg, 90 mg, and 270 mg are shown in Figure 5. After SC administration of AZD3427 solution, AZD3427 was absorbed and reached Cmax within 3-4 days. Non-compartmental analysis of the PK data from the SAD cohort was used to determine the terminal t 1 / 2 was estimated to be 7 to 9 days.

[0145] In the MAD cohort, C trough values ​​indicated that steady state was not completely reached after five weekly doses: C trough values ​​after the last dose were 329 ng / mL, 1005 ng / mL, and 2321 ng / mL for the 5 mg, 15 mg, and 45 mg dose levels, respectively.

[0146] Example 6: Ph1 AZD3427 MAD Study Results in HF Patients In the Part B MAD cohort, data for HFpEF and HFrEF patients were first pooled. Trends indicate that AZD3427 improved cardiac function, including improved cardiac output and stroke volume (SV), reduced systemic vascular resistance (SVR), and improved organ perfusion (SVR and eGFR) (Figures 6A-6F). This time, patients were divided into the following groups: (i) patients with baseline EF ≤ 40% and (ii) patients with baseline EF > 40%. Additional analyses showed that the observed trends in improved (increased) stroke volume and eGFR after treatment with AZD3427 in pooled patients were also observed in both (i) patients with baseline EF ≤ 40% and (ii) patients with baseline EF > 40% (Figures 6H-6I). These were observed without any apparent effect on blood pressure (Figure 6G). AZD3427 administration also resulted in a dose-dependent increase in plasma renin levels (Figure 7A). For test doses below 45 mg, the resulting hemoglobin and hematocrit levels were within acceptable safety profiles (Figures 7B, 7C). Advantageously, therefore, doses of AZD3427 between 1 mg and 30 mg, selected as the predicted pharmacodynamically effective dose range for the Ph 2 study based on the modeling and Ph 1 data described herein, appear to be sufficient to enable hemoglobin and hematocrit levels to remain within acceptable safety limits.

[0147] Example 7: Phase 2B (Ph2b) Study Design for AZD3427 Dosing Regimen The Phase 2b study (design shown in Figure 8, Study ID number: D8330C00003) will evaluate Q2W doses of AZD3427 at about 1.0 mg (e.g., 1.1 mg), about 5.4 mg, and about 30 mg. Participants will receive a single subcutaneous dose of AZD3427 or placebo (in a 1:1:1:1 ratio) once every two weeks for 24 weeks from Day 1 to Day 155.

[0148] Dose selection for AZD3427 was based on PK, pharmacodynamic, and safety data from the SAD / MAD study in healthy participants and HFrEF / HFpEF participants (Study D8330C00001 / NCT04630067) and from a study in NHPs with reduced LVEF. Dosing once every two weeks ("biweekly") was determined by modeling weekly, biweekly, and biweekly dosing using the data, as shown in Figure 9. The dashed line in Figure 9 (0.08 μg / mL) represents the estimated concentration of AZD3427 equivalent to the mean observed concentration of relaxin in first-trimester pregnant subjects (0.2 ng / mL, in-house assay (see also Example 10)) and serves as the reference level. This reference level of 0.08 μg / mL was calculated as follows, based on the 40-fold potency difference and 10-fold larger molecular weight of AZD3427 relative to relaxin: 0.2 * 40 * 10=80ng / mL=0.08μg / mL

[0149] Similar Cmax / Cmin ratios were predicted for weekly and biweekly dosing, both of which were shown to be adequate to maintain C trough levels above the reference level, as shown in Figure 9. Biweekly dosing is preferred because it minimizes the dose that needs to be administered to the patient, thereby promoting better patient compliance.

[0150] The mean steady-state concentrations (Css,ave) at the three dose levels are predicted to range from lower to higher levels of the corresponding relaxin levels during pregnancy: 0.5-fold, 2.5-fold, and 15-fold the relaxin levels during pregnancy, respectively. This correlation between AZD3427 and relaxin levels accounts for the difference in in vitro potency between serelaxin and AZD3427 (approximately 40-fold) based on cAMP production in CHO cells expressing the recombinant human RXFP1 receptor, as well as the difference in molecular weight between serelaxin and AZD3427 (AZD3427 is 10-fold greater), assuming a relaxin concentration of 0.2 ng / mL during pregnancy (in-house data). Additionally, using concentration-response relationships for ejection fraction, stroke volume, and systemic vascular resistance from the NHP HFrEF model and PK data from the SAD / MAD study (Study D8330C00001), it is predicted that the dose range selected in the Ph2b study will allow for characterization of the dose response for these parameters (Figure 10, Figures 11A-C).

[0151] The primary outcome measure is the change from baseline in pulmonary vascular resistance (PVR) after 24 weeks of treatment. The effect of AZD3427 on PVR parameters, as measured by right heart catheterization (RHC), will also be evaluated compared to placebo after 24 weeks of treatment in participants with HF and PH Group 2.

[0152] Secondary outcome measures include: Change from baseline in mean pulmonary artery pressure (mPAP) Change from baseline in pulmonary artery occlusion pressure (PAWP) Change from baseline in cardiac output Change from baseline in stroke volume (SV) Change from baseline in ejection fraction (EF) Change from baseline in left ventricular global longitudinal strain (LVGLS) Change from baseline in pulmonary arterial systolic pressure (PASP) Change from baseline in right ventricle / left ventricle (RV / LV) ratio Change from baseline in right ventricular outflow tract acceleration time (RVOT AT) Change from baseline in tricuspid regurgitation velocity (TRV) Change from baseline in TAPSE / PASP (Tricuspid annular plane systolic excursion / Pulmonary arterial systolic pressure) Change from baseline in right ventricular strain / pulmonary arterial systolic pressure (RVS / PASP) Change from baseline in inferior vena cava (IVC) diameter with inspiratory collapse Change from baseline in systemic vascular resistance Change from baseline in 6-minute walking distance (6MWD) Change from baseline in Kansas City Cardiomyopathy Questionnaire total symptom score (KCCQ TSS) Change from baseline in New York Heart Association Functional Class (NYHA FC) Change from baseline in serum creatinine Change from baseline in N-terminal prohormone brain natriuretic peptide (NT-proBNP) Change from baseline in cystatin C Change from baseline in eGFR (estimated glomerular filtration rate)

[0153] Inclusion Criteria: 1. Participants must be 18 years of age or older (inclusive). 2. Participants must have a pre-existing diagnosis of HF, NYHA functional class (FC) II-IV, and a pre-existing diagnosis of PH-LHD, or a high or intermediate probability of pulmonary hypertension due to left heart disease (PH-LHD) according to the 2022 European Society of Cardiology / European Respiratory Society (ESC / ESR) guidelines. Participants must be receiving stable standard HF treatment medication, including diuretics. 3. Participants must have a combination of echocardiographic parameters indicating an intermediate or high probability of PH according to the 2022 ESC / ERS guidelines. 4. Participants must have an elevated pulmonary artery pressure (PAWP) ≥ 15 mmHg or mPAP ≥ 20 mmHg during the RHC performed according to the RHC manual provided by the client at Screening Visit 2. 5. Minimum weight 50 kg (inclusive). 6. Able to give signed informed consent.

[0154] Exclusion Criteria: 1. Diagnosis of PH in World Health Organization (WHO) Group 1, WHO Group 3, WHO Group 4, or WHO Group 5. 2. History or current evidence of clinically significant disease or disorder. 3. Decompensated HF or any hospitalization. 4. Any contraindication to RHC. 5. History of hypersensitivity to SC injection or device. 6. History of hypersensitivity to drugs with a similar chemical structure or class to AZD3427 or any component of the AZD3427 drug product, or ongoing clinically significant allergy / hypersensitivity. 7. Known lung disease with forced expiratory volume in the first second / vital capacity (FEV1 / VC) <30%. 8. Congenital long QT syndrome. 9. Ventricular arrhythmia requiring treatment. Participants with atrial fibrillation or flutter and controlled ventricular rate are allowed. 10. History or anticipation of heart transplant or ventricular assist device implantation. 11. Any known planned (scheduled) highly invasive cardiovascular (CV) procedure (e.g., coronary artery revascularization, atrial fibrillation / flutter ablation, valve repair / replacement, aortic aneurysm surgery, etc.). 12. Participants who have previously received AZD3427.

[0155] Without being limited by theory, AZD3427 is expected to have vasodilatory, anti-inflammatory, and anti-fibrotic effects, improve left ventricular function, and halt and / or reverse remodeling of the diseased cardiac and pulmonary vasculature, thereby reducing PAP and PVR and improving cardiovascular outcomes.

[0156] Example 8: Final Ph 1 AZD3427 MAD Study Results in HF Patients The Phase 1 data and modeling derived therefrom described in the above examples were based on interim data sets. Examples 8 and 9 below are based on the final data set available from the Phase 1 trial, allowing for data checking and validation and for inclusion in the analysis of additional patient data as they become available. Analysis of the final data set for the Part B MAD cohort, pooled for HFpEF and HFrEF patients, fully supports the conclusions drawn from the interim data, particularly the trends observed for AZD3427 to improve cardiac function, including improved cardiac output and stroke volume (SV), reduced systemic vascular resistance (SVR), and improved organ perfusion (SVR and eGFR) compared to placebo (Figures 12A-12F). Additional analyses, this time dividing patients into the following groups: (i) patients with baseline EF ≤ 40% and (ii) patients with baseline EF > 40%, showed that the observed trends in improved (increased) stroke volume and eGFR after treatment with AZD3427 in pooled patients were also observed in both (i) patients with baseline EF ≤ 40% and (ii) patients with baseline EF > 40% (Figures 12H-12I). There was no clear effect on blood pressure (Figure 12G). AZD3427 administration also resulted in a dose-dependent increase in plasma renin levels (Figure 13A). For test doses below 45 mg, the resulting hemoglobin and hematocrit levels were within the acceptable safety profile (Figures 13B, 13C). AZD3427 at doses from 1 mg to 30 mg appeared to maintain acceptable safety limits for hemoglobin and hematocrit levels.

[0157] Example 9: Phase 2B (Ph2b) AZD3427 Dosing Regimen A Ph2b study (design shown in Figure 8, Study ID number: D8330C00003) will be conducted to evaluate AZD3427, as discussed in Example 7. The dose range selected in the Ph2b study will allow for characterization of the dose response for ejection fraction, stroke volume, renin, and systemic vascular resistance based on the NHP HFrEF model and PK data from the SAD / MAD study (Study D8330C00001) (Figure 14). The dose response will also characterize hematocrit, hemoglobin, and albumin levels (Figures 15A-15C). The primary and secondary outcomes described in Example 7 will be evaluated. The inclusion and exclusion criteria from Example 7 also apply.

[0158] Example 10: Relaxin-2 detection assay Relaxin-2 capture antibody (MAB2804, R&D Systems, USA) was used as the coating antibody. Relaxin-2 biotinylated antibody (R&D Systems, USA) was used as the detection antibody. Relaxin-2 standard (3596-RN-025 / CF, R&D Systems, USA) was used as the calibrator. The calibrator was prepared by resuspending in 200 μL of assay diluent. A dilution curve was generated by diluting the standard from 36,000 fg / mL to 10 fg / mL.

[0159] Single molecule array (Simoa™) technology was used according to the following steps: 0.1 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Thermo Fisher Scientific, USA) was added to 1.4 × 10 6Magnetic homemade carboxylated beads (Quanterix, USA) were activated by adding 0.3 mg / mL of beads to a bead solution. After 30 minutes of incubation at room temperature (RT), the beads were washed on a magnetic separator, and 0.3 mg / mL of ice-cold capture antibody was added. The beads were then incubated for 2 hours at 4°C on a mixer (HulaMixer, Invitrogen, USA). The beads were then washed, and blocking solution was added. After further washing, the conjugated beads were resuspended in the corresponding bead diluent and stored at 4°C for future use. Human plasma samples and relaxin-2 standards (370 μL) were analyzed in duplicate on a Simoa HD-X Analyzer (Quanterix, Lexington, MA). Samples and standards were plated onto a 96-well plate (NUNC, Thermo Fisher Scientific, USA). The conjugated beads were washed twice in bead dilution buffer and then resuspended in the corresponding volume of bead diluent. An IgG blocker (MSD Blocker DM, Mesoscale Discovery, USA) was added to the sample at a 1:10 ratio of total volume. The detection antibody was diluted in assay diluent to a final concentration of 0.25 μg / mL, and the enzyme SBG (streptavidin β-galactosidase, Quanterix, USA) was diluted in SBG diluent to a final concentration of 150 pM. The reagents, samples, and calibrators were run on the HD-X Analyzer using the two-step Assay Neat 2.0 protocol with 25 μL of conjugated beads, 20 μL of biotinylated antibody, 100 μL of SBG, and 50 μL of resorufin β-D-galactopyranoside (RGP, Quanterix, Lexington, MA).

[0160] [Table 3-1]

[0161] Table 3-2

[0162] Table 3-3

[0163] Table 3-4

[0164] Table 3-5

[0165] Table 3-6

[0166] Table 3-7

[0167] Table 3-8

[0168] Table 3-9

[0169] Table 3-10

[0170] Table 3-11

[0171] Table 3-12

[0172] Table 3-13

[0173] Table 3-14

Claims

1. 1. A method of treating a subject having heart failure, optionally heart failure accompanied by pulmonary hypertension, comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable excipient and about 1 mg to about 30 mg of a heterodimeric fusion, wherein the heterodimeric fusion: (i) a first heterodimerization domain connected to at least one relaxin A chain polypeptide or variant thereof; (ii) a second heterodimerization domain connected to at least one relaxin B chain polypeptide or variant thereof; The method, wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and the heterodimeric fusion has relaxin activity.

2. 10. The method of claim 1, wherein the amount of said heterodimeric fusion in said pharmaceutical composition is about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

3. 10. The method of claim 1, wherein the amount of said heterodimeric fusion in said pharmaceutical composition is about 1 mg or about 1.1 mg.

4. 10. The method of claim 1, wherein the amount of said heterodimeric fusion in said pharmaceutical composition is about 5.4 mg.

5. 10. The method of claim 1, wherein the amount of said heterodimeric fusion in said pharmaceutical composition is about 30 mg.

6. The method of any one of claims 1 to 5, wherein the pharmaceutical composition is administered to the subject every two weeks.

7. The method of any one of claims 1 to 6, wherein the pharmaceutical composition is administered subcutaneously to the subject.

8. The object is (a) New York Heart Association (NYHA) functional class II-IV; (b) mean pulmonary artery pressure (mPAP) greater than 20 mmHg, and (c) a pulmonary artery wedge pressure (PAWP) greater than 15 mmHg.

9. 9. The method of any one of claims 1 to 8, wherein the endogenous relaxin plasma level in the subject is about 10,000 times lower than the relaxin plasma level in a first trimester pregnant subject.

10. 10. The method of any one of claims 1-9, wherein the amount of the heterodimeric fusion in the pharmaceutical composition is sufficient to provide a subject with a relaxin exposure corresponding to up to about 0.5-fold, up to about 2.5-fold, or up to about 15-fold the endogenous relaxin level in the plasma of a first trimester pregnant subject.

11. 11. The method of any one of claims 1-10, wherein administration of said pharmaceutical composition is sufficient to result in a plasma concentration of said heterodimeric fusion in said subject of between 0.026 μg / mL and 1.6 μg / mL.

12. 12. The method of any one of claims 1 to 11, wherein administration of the pharmaceutical composition is sufficient to produce a dose-dependent increase in renin of at least 2-fold compared to baseline levels.

13. 13. The method of any one of claims 1 to 12, wherein after administration of the pharmaceutical composition, the hematocrit level in the subject is reduced by 5 to 7 percent or less, for example, 5 percent or less or 7 percent or less, compared to the baseline level.

14. 14. The method of any one of claims 1 to 13, wherein after administration of the pharmaceutical composition, the hemoglobin level in the subject is reduced by no more than 3 percent or no more than 5 percent compared to the baseline level.

15. The method of any one of claims 1 to 14, wherein albumin levels in the subject are not significantly reduced after administration of the pharmaceutical composition compared to baseline levels.

16. 16. The method of any one of claims 1 to 15, wherein the subject's blood pressure (e.g., systolic blood pressure) does not change significantly after administration of the pharmaceutical composition compared to baseline blood pressure (e.g., systolic blood pressure).

17. administration of the pharmaceutical composition results in: (a) reduction of pulmonary vascular resistance (PVR); (b) a reduction in mean pulmonary artery pressure (mPAP); (c) reduction in estimated pulmonary artery diastolic pressure (ePAD); (d) increased stroke volume (SV); (e) a decrease in systemic vascular resistance (SVR) and / or an increase in estimated glomerular filtration rate (eGFR); (f) an increase in ejection fraction; and / or (g) Increased cardiac output The method of any one of claims 1 to 16, wherein the method is sufficient to result in one or more of:

18. 18. The method of any one of claims 1-17, wherein administration of the pharmaceutical composition, optionally at a biweekly dose of about 1 mg or about 1.1 mg, is sufficient to result in at least about a 1 percent increase in stroke volume (SV) in the subject compared to baseline levels.

19. 19. The method of any one of claims 1-18, wherein administration of the pharmaceutical composition, optionally at a biweekly dose of about 5.4 mg, is sufficient to result in at least about a 5 to 25 percent increase in SV in the subject compared to baseline levels.

20. 20. The method of any one of claims 1-19, wherein administration of the pharmaceutical composition, optionally at a biweekly dose of about 30 mg, is sufficient to result in at least about a 10 to 50 percent increase in SV in the subject compared to baseline levels.

21. 21. The method of any one of claims 1-20, wherein administration of the pharmaceutical composition, optionally at a biweekly dose of about 1 mg or about 1.1 mg, is sufficient to result in at least about an 8-20 percent decrease in systemic vascular resistance (SVR) in the subject compared to baseline levels.

22. 22. The method of any one of claims 1-21, wherein administration of the pharmaceutical composition, optionally at a biweekly dose of about 5.4 mg, is sufficient to result in at least about a 15 to 30 percent decrease in SVR in the subject compared to baseline levels.

23. 23. The method of any one of claims 1-22, wherein administration of the pharmaceutical composition, optionally at a biweekly dose of about 30 mg, is sufficient to result in at least about a 20-35 percent decrease in SVR in the subject compared to baseline levels.

24. 24. The method of any one of claims 17 to 23, wherein the change in PVR, mPAP, ePAD, SV, SVR, eGFR, ejection fraction, and / or cardiac output is measured after 1 to 24 weeks of treatment, optionally after 24 weeks of treatment.

25. 25. The method of any one of claims 1 to 24, wherein the heterodimeric fusion agonizes relaxin family peptide receptor 1 (RXFP1).

26. 26. The method of any one of claims 1 to 25, wherein the at least one relaxin A chain polypeptide or variant thereof and the at least one relaxin B chain polypeptide or variant thereof are (i) covalently linked by at least one interchain disulfide bond and / or (ii) not covalently linked to each other by an amino acid linker.

27. 27. The method of any one of claims 1 to 26, wherein the at least one relaxin A chain polypeptide is a relaxin-2 A chain polypeptide and the at least one relaxin B chain polypeptide is a relaxin-2 B chain polypeptide.

28. 28. The method of any one of claims 1 to 27, wherein the at least one relaxin-2 A chain polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and the at least one relaxin-2 B chain polypeptide comprises the amino acid sequence of SEQ ID NO:

2.

29. 29. The method of any one of claims 1 to 28, wherein the at least one relaxin A chain polypeptide or variant thereof is connected to the first heterodimerization domain via a connector, and the at least one relaxin B chain polypeptide or variant thereof is connected to the second heterodimerization domain via a connector, and optionally one or optionally both connectors are polypeptides.

30. 30. The method of any one of claims 26 to 29, wherein one or optionally both connectors have a length of 6 to 40 amino acids, optionally 21 amino acids.

31. 31. The method of claim 30, wherein one or optionally both connectors are G4S / G5S amino acid linkers.

32. 32. The method of claim 30 or 31, wherein one or optionally both connectors have the amino acid sequence of SEQ ID NO:

5.

33. 33. The method of any one of claims 1 to 32, wherein the first heterodimerization domain is derived from a first immunoglobulin Fc region and the second heterodimerization domain is derived from a second immunoglobulin Fc region, the first and second Fc regions comprising constant domains CH2 and CH3, optionally CH2 and CH3 domains derived from an IgG1 immunoglobulin, and further optionally wherein the CH2 and / or CH3 domains are mutated.

34. The method of claim 33, wherein the C-terminus of the first Fc region is connected to the N-terminus of the at least one relaxin A chain polypeptide and the C-terminus of the second Fc region is connected to the N-terminus of the at least one relaxin B chain polypeptide.

35. 35. The method of claim 33 or 34, wherein the first and second Fc regions comprise heterodimerization-promoting amino acid mutations, and optionally, the heterodimerization-promoting amino acid mutations are present in the CH3 domain.

36. 36. The method of claim 35, wherein the heterodimerization-promoting amino acid mutations in the first Fc region comprise S354C and T366W in the CH3 domain, and the heterodimerization-promoting amino acid mutations in the second Fc region comprise Y349C, T366S, L368A, and Y407V in the CH3 domain, wherein amino acid numbering is according to the EU index as in Kabat.

37. 37. The method of any one of claims 33 to 36, wherein the first and / or second Fc region further comprises the amino acid mutations L234F, L235E, and P331S, wherein amino acid numbering is according to the EU index as in Kabat.

38. 38. The method of any one of claims 33 to 37, wherein the first Fc region comprises the amino acid sequence of SEQ ID NO: 4 and the second Fc region comprises the amino acid sequence of SEQ ID NO:

3.

39. 39. The method of any one of claims 27 to 38, wherein the heterodimeric fusion comprises the amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO:

20.

40. 40. The method of any one of claims 33 to 39, wherein the heterodimeric fusion further comprises a second relaxin A chain polypeptide or variant thereof connected to the N-terminus of the first Fc region, and a second relaxin B chain polypeptide or variant thereof connected to the N-terminus of the second Fc region, optionally wherein the second relaxin A chain is connected to the first Fc region via a connector polypeptide having the amino acid sequence of SEQ ID NO: 5, and the second relaxin B chain is connected to the second Fc region via a connector polypeptide having the amino acid sequence of SEQ ID NO:

5.

41. 41. The method of any one of claims 1 to 40, wherein the heterodimeric fusion comprises or consists of AZD3427.

42. 42. The method of any one of claims 1 to 41, wherein the heterodimeric fusion is AZD3427 and is administered subcutaneously to the subject every other week at a dose of about 1 mg or about 1.1 mg.

43. 42. The method of any one of claims 1 to 41, wherein said heterodimeric fusion is AZD3427 and is administered subcutaneously to said subject every other week at a dose of about 5.4 mg.

44. 42. The method of any one of claims 1 to 41, wherein said heterodimeric fusion is AZD3427 and is administered subcutaneously to said subject every other week at a dose of about 30 mg.

45. 1. A method of treating a subject having heart failure, optionally heart failure accompanied by pulmonary hypertension, comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable excipient and about 1 mg to about 30 mg of a heterodimeric fusion, wherein the heterodimeric fusion: (i) an FcX-con-A fusion polypeptide; and (ii) an FcY-con-B fusion polypeptide; and Including, A is a relaxin A chain or variant thereof, e.g., a relaxin-2 A chain or variant thereof; B is a relaxin B chain or a variant thereof, e.g., a relaxin-2 B chain or a variant thereof; FcX is an Fc region comprising the constant domains CH2 and CH3 of a human IgG1 immunoglobulin having amino acid mutations, optionally the amino acid mutations S354C and T366W; FcY is an Fc region comprising the constant domains CH2 and CH3 of a human IgG1 immunoglobulin comprising amino acid mutations, optionally amino acid mutations Y349C, T366S, L368A, and Y407V; con is optionally a connector polypeptide having the sequence of SEQ ID NO: 5, wherein amino acid numbering is according to the EU index as in Kabat, and FcX heterodimerizes with FcY, and the heterodimeric fusion has relaxin activity.

46. 46. ​​The method of claim 45, wherein the heterodimeric fusion comprises or consists of AZD3427.

47. 47. The method of claim 45 or 46, wherein the amount of the heterodimeric fusion in the pharmaceutical composition is about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

48. 48. The method of any one of claims 45 to 47, wherein the pharmaceutical composition is administered to the subject every other week.

49. 49. The method of any one of claims 45 to 48, wherein the pharmaceutical composition is administered subcutaneously to the subject.

50. 50. The method of any one of claims 1 to 49, wherein the subject has heart failure accompanied by pulmonary hypertension group 2.

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