Dosing regimens using heterodimer relaxin fusion proteins

Abstract

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

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority under the following U.S. Provisional Patent Application Nos.:

[0003] 63 / 386,762, filed on December 9, 2022; 63 / 387,359, filed on December 14, 2022; and 63 / 497,169, filed on April 19, 2023, the content of each of these provisional patent applications being incorporated herein by reference. Technical Field

[0004] The present disclosure relates to dosing regimens and methods for treating a subject having heart failure with pulmonary hypertension, including administering a heterodimeric relaxin fusion protein. Background Art

[0005] Heart failure is associated with significant morbidity and mortality. It is characterized by complex tissue remodeling, including increased cardiomyocyte death and interstitial fibrosis. A significant number of heart failure patients have pulmonary hypertension. Statistically, approximately 50% of heart failure with preserved ejection fraction (HFpEF) patients also have pulmonary hypertension (PH), while approximately 60% of heart failure with reduced ejection fraction (HFrEF) patients also have co - existing PH (Guazzi, (2014) Circ Heart Fail., 7:367 - 377; Miller et al., (2013) JACC Heart Fail., 1(4):290 - 299). Studies have shown that compared to heart failure patients without pulmonary hypertension (PH), heart failure patients with PH (HF + PH) have a reduced survival rate (Barnett and DeMarco, (2012) Heart Fail. Clin. 8:447–459). In heart failure patients, for every 3 mmHg increase or decrease in the estimated pulmonary artery diastolic pressure (ePAD) (equivalent to approximately a 4 mmHg increase or decrease in mean pulmonary artery pressure (mPAP)), the cardiovascular mortality rate increases by 24% or decreases by 19%, respectively (Zile MR et al., (2017) Circ Heart Fail., 10:e003594). A 4 mmHg decrease in mPAP is also associated with improved dyspnea in patients with HF + PH (Solomonica A et al., (2013) CircHeart Fail., 6:53 - 60).

[0006] Group 2 pulmonary hypertension (PH) is caused by left ventricular dysfunction and is the most common cause of PH. Left ventricular dysfunction leads to increased pulmonary artery pressure, which in turn causes pulmonary edema, pulmonary vascular injury, and ultimately right ventricular failure. Group 2 PH mainly consists of HF patients in New York Heart Association (NYHA) functional class III and IV (i.e., HF patients with limited physical activity and accompanying fatigue, palpitations, or dyspnea). Among them, 40% to 75% of HFrEF patients and 36% to 83% of HFpEF patients are concomitantly complicated with PH. Group 2 PH is associated with decreased exercise tolerance and reduced survival. Despite receiving the best management plan guided by evidence-based guidelines, the physical activity and quality of life of Group 2 PH patients are still significantly restricted. There is currently no specific treatment for such patients.

[0007] Relaxin is a peptide hormone belonging to the insulin superfamily. In humans, the relaxin peptide family contains seven peptides with highly similar structures but low sequence similarity: relaxin 1, 2, 3, and insulin-like peptides INSL3, INSL4, INSL5, INSL6. Naturally occurring relaxin is composed of an A-chain and a B-chain polypeptide 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 B-C-A (the A-chain polypeptide and the B-chain polypeptide are linked by the C-peptide). The prohormone is cleaved to remove the C-peptide by endoproteolytic cleavage by the enzymes PC1 and PC2, and then the mature relaxin is secreted.

[0008] Without being limited by theory, relaxin is considered 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 gestation without causing cardiac injury; it is unique in its ability to synchronously reduce both systemic and renal vascular resistance (Conrad 2011). Without being bound by theory, if relaxin could recapitulate these hemodynamic adaptations in the setting of heart failure, it could reduce myocardial oxygen consumption demands and improve end-organ perfusion. Studies have also shown that relaxin has antifibrotic properties and beneficial effects in heart failure, including acute decompensated heart failure (ADHF). Relaxin activates many 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).

[0009] 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 hepatic 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 approximately 20 hours at a dose of 30 micrograms per kilogram per day to achieve (Ponikowski et al., (2014) European Heart Journal 35:431–441). Because serelaxin is rapidly cleared from the patient's bloodstream, its therapeutic effect is limited, and the therapeutic effect rapidly dissipates once the intravenous infusion is stopped. In addition, approximately one-third of patients experienced a significant decrease in blood pressure (>40 mm Hg) after intravenous infusion of serelaxin, necessitating a reduction in the infusion rate by half or more.

[0010] WO 2013 / 004607 and WO 2018 / 138170 describe recombinant relaxin polypeptides in which the relaxin A-chain and the relaxin B-chain are fused in a single-chain form via a linker peptide. WO 2013 / 004607 describes a recombinant relaxin in which the linker peptide comprises at least 5 amino acids and fewer than 15 amino acids. WO 2018 / 138170 describes a recombinant relaxin in which the linker peptide comprises at least 15 amino acids.

[0011] In contrast, WO 2021 / 255127 (incorporated herein by reference in its entirety) describes a heterodimeric fusion protein comprising a relaxin-chain polypeptide and a heterodimerization domain, and which exhibits relaxin activity without the need to fuse the relaxin A-chain polypeptide and the relaxin B-chain polypeptide in a single-chain form. WO 2021255127 describes that the heterodimerization of the heterodimerization domain induces the correct folding and heterodimerization of the relaxin A-chain polypeptide and the relaxin B-chain polypeptide. In addition, unlike the wild-type relaxin protein, the fusion protein described in WO 2021255127 has biological activity without endoproteolytic processing and exhibits an extended half-life.

[0012] Patients with HF+PH may require long-term treatment, which may affect patient compliance, quality of life, and may cause harmful side effects (such as in the case of serelaxin) when high doses and / or frequent (e.g., continuous) administrations are required. Accordingly, there remains a need to develop improved treatment regimens for HF+PH patients that allow for less frequent and / or lower doses of administration, thereby minimizing adverse reactions, improving patient compliance, quality of life, and / or providing other therapeutic benefits. SUMMARY OF THE INVENTION

[0013] Disclosed herein in part are low-dose and / or low-frequency dosing regimens for treating subjects with heart failure (optionally, HF+PH subjects) using a heterodimeric relaxin fusion protein. The dosing regimens provided herein can improve the quality of life of patients while maintaining efficacy and minimizing side effects.

[0014] In one aspect, the present disclosure provides a method of treating a subject with heart failure, optionally a subject with heart failure with pulmonary hypertension, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable excipient and from about 1 mg to about 30 mg of a heterodimeric fusion protein; wherein the heterodimeric fusion protein comprises:

[0015] (i) a first heterodimerization domain linked to at least one relaxin A-chain polypeptide or a variant thereof; and

[0016] (ii) a second heterodimerization domain linked to at least one relaxin B-chain polypeptide or a variant thereof,

[0017] wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and wherein the heterodimer fusion protein has relaxin activity.

[0018] In some embodiments, the amount of the heterodimer fusion protein in the pharmaceutical composition is about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

[0019] In some embodiments, the method comprises administering the heterodimer fusion protein to the subject once every two weeks (also referred to herein as "bi-weekly" administration). In some embodiments, the method comprises bi-weekly administration and the amount of the heterodimer fusion protein 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 bi-weekly administration and the amount of the heterodimer fusion protein in the pharmaceutical composition is about 1 mg. In some embodiments, the method comprises bi-weekly administration and the amount of the heterodimer fusion protein in the pharmaceutical composition is about 1.1 mg. In some embodiments, the method comprises bi-weekly administration and the amount of the heterodimer fusion protein in the pharmaceutical composition is about 5.4 mg. In some embodiments, the method comprises bi-weekly administration and the amount of the heterodimer fusion protein in the pharmaceutical composition is about 30 mg.

[0020] In some embodiments, the method comprises subcutaneous administration to the subject. In some embodiments, the method comprises subcutaneous administration to the subject and the amount of the heterodimer fusion protein 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 heterodimer fusion protein in the pharmaceutical composition is about 1 mg. In some embodiments, the method comprises subcutaneous administration and the amount of the heterodimer fusion protein in the pharmaceutical composition is about 1.1 mg. In some embodiments, the method comprises subcutaneous administration and the amount of the heterodimer fusion protein in the pharmaceutical composition is about 5.4 mg. In some embodiments, the method comprises subcutaneous administration and the amount of the heterodimer fusion protein in the pharmaceutical composition is about 30 mg.

[0021] In some embodiments, the method includes administering subcutaneously to a subject once every two weeks and the amount of the heterodimeric fusion protein in the pharmaceutical composition is about 1 mg. In some embodiments, the method includes administering subcutaneously to a subject once every two weeks and the amount of the heterodimeric fusion protein in the pharmaceutical composition is about 1.1 mg. In some embodiments, the method includes administering subcutaneously to a subject once every two weeks and the amount of the heterodimeric fusion protein in the pharmaceutical composition is about 5.4 mg. In some embodiments, the method includes administering subcutaneously to a subject once every two weeks and the amount of the heterodimeric fusion protein in the pharmaceutical composition is about 30 mg.

[0022] In some embodiments, the subject may have heart failure with reduced ejection fraction, heart failure with mid-range 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) 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 tightness; dyspnea at rest or with mild activity; N-terminal pro-brain natriuretic peptide (NT-proBNP) > 125 pg / mL, or brain natriuretic peptide (BNP) > 35 pg / mL; systolic blood pressure > 125 mmHg; mild to moderate renal insufficiency; body mass index (BMI) of at least 18 kg / m 2 ; and reduced ejection fraction (HFrEF), less than or equal to 40%.

[0023] In some embodiments, the endogenous relaxin plasma level of the subject is about one ten-thousandth of the endogenous relaxin plasma level in a pregnant subject in the first trimester of pregnancy. In some embodiments, the amount of the heterodimeric fusion protein in the pharmaceutical composition is sufficient to cause the relaxin exposure of the subject to correspond to up to about 0.5-fold, up to about 2.5-fold, or up to about 15-fold of the endogenous relaxin level in the plasma of a pregnant subject in the first trimester of pregnancy. In some embodiments, the amount of the heterodimeric fusion protein in the pharmaceutical composition is sufficient to cause the relaxin exposure of the subject to correspond to up to about 0.5-fold, up to about 3.5-fold, or up to about 7-fold of the endogenous relaxin level in the plasma of a pregnant subject in the first trimester of pregnancy. Relaxin levels, such as the endogenous relaxin level in the plasma of a pregnant subject in the first trimester of pregnancy, can be measured using a relaxin detection assay, such as by using 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 pregnant subject in the first trimester of pregnancy can be up to 0.2 ng / mL or about 0.2 ng / mL (e.g., as measured using a relaxin detection assay, such as the assay described in Example 10).

[0024] In some embodiments, administering the pharmaceutical composition is sufficient to cause a minimum steady-state plasma concentration of the heterodimeric fusion protein in a subject to reach about 0.026 μg / mL or at least 0.026 μg / mL. In some embodiments, administering the pharmaceutical composition is sufficient to cause a maximum steady-state plasma concentration of the heterodimeric fusion protein in a subject to reach about 1 to 2 μg / mL or at least 1 to 2 μg / mL. In some embodiments, administering the pharmaceutical composition is sufficient to cause a maximum steady-state plasma concentration of the heterodimeric fusion protein in a subject to reach about 1.6 μg / mL or at least 1.6 μg / mL. In some embodiments, administering the pharmaceutical composition is sufficient to cause the plasma concentration of the heterodimeric fusion protein in a subject to be from 0.026 μg / mL to 1.6 μg / mL (i.e., the plasma concentration in the subject is about 0.026 μg / mL, up to about 1.6 μg / mL and including about 1.6 μg / mL).

[0025] In some embodiments, compared to the baseline (pre-administration) level, administering the pharmaceutical composition is sufficient to cause at least a 2-fold dose-dependent increase in renin production. In some embodiments, compared to the baseline (pre-administration) level, administering the pharmaceutical composition is sufficient to cause at least a 2.5-fold dose-dependent increase in renin production. In some embodiments, compared to the baseline (pre-administration) level, administering the pharmaceutical composition is sufficient to cause at least a 3-fold dose-dependent increase in renin production.

[0026] In some embodiments, compared to the baseline (pre-administration) level, the hematocrit level in a subject after administering the pharmaceutical composition (e.g., after administering a dose of 1 mg to 30 mg of the heterodimeric fusion protein) is reduced by no more than 1%. In some embodiments, compared to the baseline (pre-administration) level, the hematocrit level in a subject after administering the pharmaceutical composition is reduced by no more than 2%. In some embodiments, compared to the baseline (pre-administration) level, the hematocrit level in a subject after administering the pharmaceutical composition is reduced by no more than 3%. In some embodiments, compared to the baseline (pre-administration) level, the hematocrit level in a subject after administering the pharmaceutical composition is reduced by no more than 4%. In some embodiments, compared to the baseline (pre-administration) level, the hematocrit level in a subject after administering the pharmaceutical composition is reduced by no more than 5%. In some embodiments, compared to the baseline (pre-administration) level, the hematocrit level in a subject after administering the pharmaceutical composition is reduced by no more than 6%. In some embodiments, compared to the baseline (pre-administration) level, the hematocrit level in a subject after administering the pharmaceutical composition is reduced by no more than 7%. In some embodiments, compared to the baseline (pre-administration) level, the hematocrit level in a subject after administering the pharmaceutical composition is reduced by no more than 8%.

[0027] In some embodiments, after administration of the pharmaceutical composition (e.g., after administration of a 1 mg to 30 mg dose of the heterodimeric fusion protein), the hemoglobin level in the subject does not decrease by more than 2% compared to the baseline (pre - administration) level. In some embodiments, after administration of the pharmaceutical composition, the hemoglobin level in the subject does not decrease by more than 3% compared to the baseline (pre - administration) level. In some embodiments, after administration of the pharmaceutical composition, the hemoglobin level in the subject does not decrease by more than 4% compared to the baseline (pre - administration) level. In some embodiments, after administration of the pharmaceutical composition, the hemoglobin level in the subject does not decrease by more than 5% compared to the baseline (pre - administration) level. In some embodiments, after administration of the pharmaceutical composition, the hemoglobin level in the subject does not decrease by more than 6% compared to the baseline (pre - administration) level. In some embodiments, the albumin level in the subject does not significantly decrease after administration of the pharmaceutical composition compared to the baseline level.

[0028] Administration of the pharmaceutical composition as described herein may be sufficient to cause one or more of the following compared to the baseline level:

[0029] (a) An increase in stroke volume (SV);

[0030] (b) A decrease in systemic vascular resistance (SVR) and / or an increase in estimated glomerular filtration rate (eGFR);

[0031] (c) An increase in ejection fraction;

[0032] (d) An increase in cardiac output;

[0033] Changes in SV, SVR, eGFR, ejection fraction, and / or cardiac output can each occur 1 to 24 weeks after treatment. In some embodiments, changes in SV, SVR, eGFR, ejection fraction, and / or cardiac output occur 24 weeks after treatment.

[0034] Administration of the pharmaceutical composition as described herein may also be sufficient to cause one or more of the following compared to the baseline level:

[0035] (a) A decrease in pulmonary vascular resistance (PVR);

[0036] (b) A decrease in mean pulmonary artery pressure (mPAP);

[0037] (c) A decrease in estimated pulmonary artery diastolic pressure (ePAD);

[0038] Changes in PVR, mPAP, and / or ePAD can each occur 1 to 24 weeks after treatment. In some embodiments, changes in PVR, mPAP, and / or ePAD occur 24 weeks after treatment.

[0039] Thus, compared to the baseline level, administration of a pharmaceutical composition as described herein may be sufficient to cause one or more of the following:

[0040] (a) a decrease in PVR;

[0041] (b) a decrease in mPAP;

[0042] (c) a decrease in ePAD;

[0043] (d) an increase in the stroke volume (SV) of the heart;

[0044] (e) a decrease in the systemic vascular resistance (SVR) and / or an increase in the estimated glomerular filtration rate (eGFR);

[0045] (f) an increase in the ejection fraction;

[0046] (g) an increase in the cardiac output;

[0047] Changes in PVR, mPAP, ePAD, SV, SVR, eGFR, ejection fraction, and / or cardiac output can 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.

[0048] In some embodiments, administration of the pharmaceutical composition is sufficient to cause an increase in the stroke volume (SV) of the subject, e.g., after 1 to 24 weeks of treatment, such as when administering a dose of 1 mg to 30 mg of the heterodimeric fusion protein. In some embodiments, compared to the baseline (pre - administration) level, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to 55%. In some embodiments, compared to the baseline (pre - administration) level, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to 50%. In some embodiments, compared to the baseline (pre - administration) level, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to 25%. In some embodiments, compared to the baseline (pre - administration) level, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to 20%. In some embodiments, compared to the baseline (pre - administration) level, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to 10%. In some embodiments, compared to the baseline (pre - administration) level, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to 5%. In some embodiments, the administration continues for at least 10 weeks. In some embodiments, the administration continues for at least 24 weeks.

[0049] In some embodiments, administration of the pharmaceutical composition is sufficient to cause an increase in stroke volume (SV) of up to 1%, optionally after 24 weeks of treatment, for example at a bi-weekly dose of about 1 mg or about 1.1 mg, compared to the baseline (pre-administration) level. In some embodiments, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to about 5%, optionally after 24 weeks of treatment, for example at a bi-weekly dose of about 5.4 mg, compared to the baseline (pre-administration) level. In some embodiments, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to about 10%, optionally after 24 weeks of treatment, for example at a bi-weekly dose of about 30 mg, compared to the baseline (pre-administration) level. In some embodiments, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to about 25%, optionally after 24 weeks of treatment, for example at a bi-weekly dose of about 5.4 mg, compared to the baseline (pre-administration) level. In some embodiments, administration of the pharmaceutical composition is sufficient to cause an increase in SV of up to about 60%, optionally after 24 weeks of treatment, for example at a bi-weekly dose of about 30 mg, compared to the baseline (pre-administration) level. In some embodiments, administration of the pharmaceutical composition is sufficient to cause an increase in SV of at least 10%, optionally after 24 weeks of treatment.

[0050] In some embodiments, administration of the pharmaceutical composition is sufficient to cause a decrease in systemic vascular resistance (SVR) in a subject, for example after 1 to 24 weeks of treatment. In some embodiments, compared to the baseline (pre-administration) level, administration of the pharmaceutical composition is sufficient to cause a decrease in SVR of up to about 35%, for example, after administration of a 1 mg to 30 mg dose of the heterodimeric fusion protein. In some embodiments, compared to the baseline (pre-administration) level, administration of the pharmaceutical composition is sufficient to cause a decrease in SVR of up to about 30%. In some embodiments, compared to the baseline (pre-administration) level, administration of the pharmaceutical composition is sufficient to cause a decrease in SVR of up to about 25%. In some embodiments, compared to the baseline (pre-administration) level, administration of the pharmaceutical composition is sufficient to cause a decrease in SVR of up to about 20%. In some embodiments, compared to the baseline (pre-administration) level, administration of the pharmaceutical composition is sufficient to cause a decrease in SVR of up to about 15%. In some embodiments, compared to the baseline (pre-administration) level, administration of the pharmaceutical composition is sufficient to cause a decrease in SVR of up to about 10%. In some embodiments, compared to the baseline (pre-administration) level, administration of the pharmaceutical composition is sufficient to cause a decrease in SVR of up to about 8%. In some embodiments, the administration continues for at least 10 weeks. In some embodiments, the administration continues for at least 24 weeks.

[0051] In some embodiments, administration of the pharmaceutical composition is sufficient to cause a reduction in SVR of up to about 20% compared to baseline (pre - administration) levels, optionally after 24 weeks of treatment, for example, at a bi - weekly dose of about 1 mg or about 1.1 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to cause a reduction in SVR of up to about 30% compared to baseline (pre - administration) levels, optionally after 24 weeks of treatment, for example, at a bi - weekly dose of about 5 mg or about 5.4 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to cause a reduction in SVR of up to about 30% compared to baseline (pre - administration) levels, optionally after 24 weeks of treatment, for example, at a bi - weekly dose of about 30 mg.

[0052] In some embodiments, administration of the pharmaceutical composition is sufficient to cause a reduction in systemic vascular resistance (SVR) in a subject, for example, after 1 to 10 weeks of treatment. In some embodiments, administration of the pharmaceutical composition is sufficient to cause a reduction in SVR of up to about 10% compared to baseline (pre - administration) levels, optionally after 10 weeks of treatment, for example, at a bi - weekly dose of about 5 mg or about 5.4 mg. In some embodiments, administration of the pharmaceutical composition is sufficient to cause a reduction in SVR of up to about 15% compared to baseline (pre - administration) levels, optionally after 10 weeks of treatment, for example, at a bi - weekly dose of about 15 mg to 30 mg.

[0053] In some embodiments, the heterodimeric fusion protein agonizes the relaxin family peptide receptor 1 (RXFP1). In some embodiments, at least one relaxin A - chain polypeptide or a variant thereof of the heterodimeric fusion protein and at least one relaxin B - chain polypeptide or a variant thereof of the heterodimeric fusion protein are covalently bound by at least one inter - chain disulfide bond. In some embodiments, at least one relaxin A - chain polypeptide or a variant thereof of the heterodimeric fusion protein and at least one relaxin B - chain polypeptide or a variant thereof of the heterodimeric fusion protein are not covalently linked to each other through 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 SEQ ID NO:1, and / or at least one relaxin - 2 B - chain polypeptide comprises the amino acid sequence SEQ ID NO:2. In some embodiments, at least one relaxin - 2 A - chain polypeptide comprises the amino acid sequence SEQ ID NO:1, and at least one relaxin - 2 B - chain polypeptide comprises the amino acid sequence SEQ ID NO:2.

[0054] In some embodiments, at least one relaxin A-chain polypeptide or a variant thereof is linked via a linker to the first heterodimerization domain of the heterodimeric fusion protein, and at least one relaxin B-chain polypeptide or a variant thereof is linked via a linker to the second heterodimerization domain. In some embodiments, at least one linker is a polypeptide. In some embodiments, both linkers are polypeptides. In some embodiments, at least one linker is a polypeptide having a length between 6 and 40 amino acids. In some embodiments, both linkers are polypeptides having a length between 6 and 40 amino acids. In some embodiments, at least one linker is a polypeptide having a length of 21 amino acids. In some embodiments, both linkers are polypeptides having a length of 21 amino acids.

[0055] In some embodiments, at least one linker is a G4S / G5S amino acid linker. In some embodiments, both linkers are G4S / G5S amino acid linkers. In some embodiments, at least one linker has the amino acid sequence SEQ ID NO:5. In some embodiments, both linkers have the amino acid sequence SEQ ID NO:5.

[0056] 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, wherein the first Fc region and the second Fc region comprise the constant domains CH2 and CH3. In some embodiments, the CH2 and CH3 domains are from an IgG1 immunoglobulin. In some embodiments, the CH2 and / or CH3 domains are mutated.

[0057] In some embodiments, the C-terminus of the first Fc region is linked to the N-terminus of at least one relaxin A-chain polypeptide, and the C-terminus of the second Fc region is linked to the N-terminus of at least one relaxin B-chain polypeptide.

[0058] In some embodiments, the first Fc region and the second Fc region comprise amino acid mutations that promote heterodimerization. In some embodiments, the amino acid mutations that promote heterodimerization are present in the CH3 domains of the first Fc region and the second Fc region.

[0059] In some embodiments, the amino acid mutations promoting heterodimerization in the first Fc region include S354C and T366W in the CH3 domain, and the amino acid mutations promoting heterodimerization in the second Fc region include Y349C, T366S, L368A, and Y407V in the CH3 domain, where the amino acid numbering is according to the EU index in Kabat. In some embodiments, the first Fc region and / or the second Fc region further comprise the amino acid mutations L234F, L235E, and P331S, where the amino acid numbering is according to the EU index in Kabat. In some embodiments, the first Fc region comprises the amino acid sequence SEQ ID NO:4. In some embodiments, the second Fc region comprises the amino acid sequence SEQ ID NO:3. In some embodiments, the first Fc region comprises the amino acid sequence SEQ ID NO:4, and the second Fc region comprises the amino acid sequence SEQ ID NO:3.

[0060] In some embodiments, the heterodimeric fusion protein comprises the amino acid sequences SEQ ID NO:11 (relaxin-2 A-chain polypeptide linked to the first Fc region via a linker) and SEQ ID NO:20 (relaxin-2 B-chain polypeptide linked to the second Fc region via a linker). In some embodiments, the heterodimeric fusion protein consists of the amino acid sequences SEQ ID NO:11 (relaxin-2 A-chain polypeptide linked to the first Fc region via a linker) and SEQ ID NO:20 (relaxin-2 B-chain polypeptide linked to the second Fc region via a linker), and may be referred to as "AZD3427".

[0061] In some embodiments, the heterodimeric fusion protein further comprises a second relaxin A-chain polypeptide or a variant thereof linked to the N-terminus of the first Fc region, and a second relaxin B-chain polypeptide or a variant thereof linked to the N-terminus of the second Fc region. In some embodiments, the second relaxin A-chain is linked to the first Fc region via a linker polypeptide. In some embodiments, the linker polypeptide has the amino acid sequence SEQ ID NO:5. In some embodiments, the second relaxin B-chain is linked to the second Fc region via a linker polypeptide. In some embodiments, the linker polypeptide has the amino acid sequence SEQ ID NO:5.

[0062] In some embodiments, the present disclosure provides a method of treating a subject suffering from heart failure with pulmonary hypertension, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable excipient and from about 1 mg to about 30 mg of a heterodimeric fusion protein; wherein the heterodimeric fusion protein comprises:

[0063] (i) an FcX-con-A fusion polypeptide; and

[0064] (ii) FcY-con-B fusion polypeptide,

[0065] Where:

[0066] A is a relaxin A-chain polypeptide or a variant thereof, such as a relaxin-2 A-chain polypeptide or a variant thereof;

[0067] B is a relaxin B-chain polypeptide or a variant thereof, such as a relaxin-2 B-chain polypeptide or a variant thereof;

[0068] FcX is an Fc region comprising CH2 and CH3 of human IgG1 immunoglobulin and comprising

[0069] amino acid mutations, optionally S354C and T366W;

[0070] FcY is an Fc region comprising CH2 and CH3 of human IgG1 immunoglobulin and comprising amino acid mutations, optionally Y349C, T366S, L368A, and Y407V; and

[0071] con is a linker polypeptide which optionally has the amino acid sequence SEQ ID NO:5,

[0072] where the amino acid numbering is according to the EU index in Kabat, where FcX and FcY heterodimerize, and where the heterodimer fusion protein has relaxin activity. In some embodiments, the heterodimer fusion protein comprises AZD3427. In some embodiments, the heterodimer fusion protein is AZD3427. In some embodiments, the amount of the heterodimer fusion protein (e.g., AZD3427) in the pharmaceutical composition is from about 1 mg to about 30 mg. In some embodiments, the amount of the heterodimer fusion protein (e.g., AZD3427) in the pharmaceutical composition is about 1 mg. In some embodiments, the amount of the heterodimer fusion protein (e.g., AZD3427) in the pharmaceutical composition is about 1.1 mg. In some embodiments, the amount of the heterodimer fusion protein (e.g., AZD3427) in the pharmaceutical composition is about 5.4 mg. In some embodiments, the amount of the heterodimer fusion protein (e.g., AZD3427) in the pharmaceutical composition is about 30 mg. In some embodiments, the pharmaceutical composition is administered to a subject via biweekly administration, e.g., at about 1 mg, about

[0073] 1.1 mg, about 5.4 mg, or about 30 mg of the heterodimer fusion protein (e.g., AZD3427)

[0074] Dosage. In some embodiments, the pharmaceutical composition is administered to a subject by subcutaneous administration once every two weeks, for example, at a dose of about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg of a heterodimeric fusion protein (such as AZD3427).

[0075] In some embodiments, AZD3427 is administered subcutaneously to a subject once every two weeks at a dose of about 1 mg. In some embodiments, AZD3427 is administered subcutaneously to a subject once every two weeks at a dose of about 1.1 mg. In some embodiments, AZD3427 is administered subcutaneously to a subject once every two weeks at a dose of about 5.4 mg. In some embodiments, AZD3427 is administered subcutaneously to a subject once every two weeks at a dose of about 30 mg.

[0076] In some embodiments, the heterodimeric fusion protein (such as AZD3427) is administered to a subject for a long term. In some embodiments, the heterodimeric fusion protein (such as AZD3427) is administered to a subject at least four times. In some embodiments, the heterodimeric fusion protein (such as AZD3427) is administered to a subject at least five times. In some embodiments, the heterodimeric fusion protein (such as AZD3427) is administered to a subject at least 12 times. Description of the Drawings

[0077] Figure 1 Shows an exemplary structure of the heterodimeric fusion protein provided herein. The structures of the respective fusion polypeptides in the heterodimeric fusion protein are represented by FcX, FcY, A, B, con, and L, where FcX and FcY are two Fc regions, containing amino acid mutations and / or modifications that promote heterodimerization; A (“Rlx A”) and B (“Rlx B”) are relaxin A-chain polypeptide and relaxin B-chain polypeptide; “con” is a linker polypeptide; L is a linker polypeptide, HC X and HC Y represent antibody heavy chains, and LC represents an antibody light chain.

[0078] Figure 2 Shows the pharmacokinetic (PK) characteristics of the exemplary heterodimeric fusion protein AZD3427 after intravenous (IV) and subcutaneous (SC) administration in cynomolgus monkeys. LLOQ = lowest limit of quantification.

[0079] Figures 3A to 3D Shows the cardiac function and systemic vascular resistance of non-human primates (NHP) after administration of AZD3427: Figure 3A Shows the ejection fraction (EF), Figure 3B Shows the cardiac output, Figure 3C Shows the systemic vascular resistance (SVR), Figure 3D Shows the percentage change in plasma renin concentration relative to that before dosing. Figure 3DThe dashed line in [ ] represents the last dose.

[0080] Figure 4 Shows the Phase 1 study design. SAD = Single Ascending Dose (SAD). MAD = Multiple Ascending Dose (MAD). HV = Healthy Volunteers. HF = Heart Failure patients. HF with EF≥41% = Heart Failure with an ejection fraction ≥ 41%. HFrEF = Heart Failure with reduced ejection fraction. JD = Japanese Descent. F / U = Follow - up. IV = Intravenous. SC = Subcutaneous.

[0081] Figure 5 Shows the trend of change over time (in hours (h)) in the geometric mean concentration (μg / mL) of serum AZD3427 after a single dose in the Phase 1 SAD cohort. SC = Subcutaneous.

[0082] Figures 6A to 6I Shows the interim clinical outcomes of cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 6A Shows the ejection fraction (EF) in HFpEF patients, Figure 6B Shows the EF in HFrEF patients, Figure 6C Shows the cardiac output of the pooled subjects, Figure 6D Shows the systemic vascular resistance (SVR) of the pooled subjects, Figure 6E Shows the stroke volume (SV) of the pooled subjects, Figure 6F Shows the estimated glomerular filtration rate (eGFR) of the pooled subjects, Figure 6G Shows the systolic blood pressure (SBP) in (i) patients with EF≤40% and (ii) patients with EF>40%, Figure 6H Shows the stroke volume in (i) patients with EF≤40% and (ii) patients with EF>40%, Figure 6I Shows the 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. Significantly different (p<0.1) compared to placebo. SE = Standard Error.

[0083] Figures 7A to 7C Shows the interim clinical outcomes of plasma renin, hematocrit, and hemoglobin levels in the MAD cohort subjects after administration of AZD3427: Figure 7A Shows the percent change in renin, Figure 7B Shows the percent change in hemoglobin, Figure 7C Shows the percent change in hematocrit. Observed data are presented as geometric means (90% confidence intervals), and the solid line represents the fitted Emax dose - response model ( Figure 7B and Figure 7C ).

[0084] Figure 8 Shows the Phase 2b study design.

[0085] Figure 9 Shows the comparison of AZD3427 dosing. QW = once weekly (QW). Q2W = once every two weeks. Q4W = once every four weeks. The shaded area shows the 90% prediction interval. The dashed line (0.08 μg / mL) represents the estimated concentration of AZD3427 equivalent to the early pregnancy relaxin reference gestational level (0.2 ng / mL; in-house assay).

[0086] Figure 10 Shows the concentration-response relationship using data from the NHP HFrEF model and the dose-response relationship of cardiac function and hemodynamic levels after once-every-two-week administration of AZD3427 extrapolated from human PK data based on mid-stage clinical data.

[0087] Figures 11A to 11C Shows mid-stage clinical data of hematocrit, hemoglobin, and albumin levels after administration of AZD3427: Figure 11A Shows the percentage change in hematocrit from baseline, Figure 11B Shows the percentage change in hemoglobin from baseline, Figure 11C Shows the percentage change in albumin from baseline. Observed data for subjects in the MAD cohort are presented as geometric means (90% confidence intervals), and the solid line represents the fitted Emax dose-response model.

[0088] Figures 12A to 12I Shows the final confirmed clinical data of cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after administration of AZD3427: Figure 12A Shows the ejection fraction (EF) in HFpEF patients, Figure 12B Shows the EF in HFrEF patients, Figure 12C Shows the cardiac output of the pooled subjects, Figure 12D Shows the systemic vascular resistance (SVR) of the pooled subjects, Figure 12E Shows the stroke volume (SV) of the pooled subjects, Figure 12F Shows the estimated glomerular filtration rate (eGFR) of the pooled subjects, Figure 12G Shows the systolic blood pressure (SBP) in (i) patients with EF ≤ 40% and (ii) patients with EF > 40%, Figure 12H Shows the stroke volume in (i) patients with EF ≤ 40% and (ii) patients with EF > 40%, Figure 12I Shows the 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 with placebo (p < 0.1). SE = standard error.

[0089] Figures 13A to 13C Shows the final clinical data on plasma renin, hematocrit, and hemoglobin levels in subjects of the MAD cohort after administration of AZD3427: Figure 13A Shows the percent change in renin, Figure 13B Shows the percent change in hemoglobin, Figure 13C Shows the percent change in hematocrit. Observed data are presented as geometric means (90% confidence intervals), and the solid line represents the fitted Emax dose-response model ( Figure 13B and Figure 13C ).

[0090] Figure 14 Shows the concentration-response relationship from the NHP HFrEF model and the dose-response relationship of cardiac function and hemodynamic levels after once-every-two-week administration of AZD3427 extrapolated from human PK data based on the final clinical data.

[0091] Figures 15A to 15C Shows the final clinical data on hematocrit, hemoglobin, and albumin levels after administration of AZD3427: Figure 15A Shows the percent change in hematocrit from baseline, Figure 15B Shows the percent change in hemoglobin from baseline, Figure 15C Shows the percent change in albumin from baseline. Observed data in subjects of the MAD cohort are presented as geometric means (90% confidence intervals), and the solid line represents the fitted Emax dose-response model. DETAILED DESCRIPTION

[0092] Disclosed herein are methods for treating subjects with heart failure, optionally HF+PH subjects, by administering a heterodimeric relaxin fusion protein at a low dose and / or low frequency.

[0093] Heterodimeric relaxin fusion protein

[0094] As used herein, a "heterodimeric fusion protein" (also referred to as a "heterodimeric fusant") refers to a heterodimer formed at least by a first fusion polypeptide and a second fusion polypeptide, wherein the first fusion polypeptide comprises a first heterodimerization domain linked 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 linked to a second subunit of the heterodimeric protein (e.g., a relaxin B-chain polypeptide or a variant thereof).

[0095] In some embodiments, the heterodimeric fusion protein of the present disclosure comprises a first heterodimerization domain linked to at least one relaxin A-chain polypeptide or a variant thereof; and a second heterodimerization domain linked to at least one relaxin B-chain polypeptide or a variant thereof, wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and wherein the heterodimeric fusion protein has relaxin activity. In some embodiments, the at least one relaxin A-chain polypeptide or a variant thereof is linked to the first heterodimerization domain via a linker (e.g., a linker polypeptide). In some embodiments, the at least one relaxin B-chain polypeptide or a variant thereof is linked to the first heterodimerization domain via a linker (e.g., a linker polypeptide).

[0096] Relaxin chain polypeptide

[0097] In some embodiments, the heterodimeric fusion protein of the present disclosure comprises a relaxin A-chain polypeptide and a relaxin B-chain polypeptide selected from relaxin-1, relaxin-2, 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.

[0098] As used herein, a "variant" of a relaxin chain polypeptide differs from the wild-type relaxin chain polypeptide while retaining relaxin activity.

[0099] In some embodiments, a relaxin A-chain polypeptide or a relaxin B-chain polypeptide variant that retains relaxin activity contains at least one conserved motif associated with relaxin activity. In some embodiments, the relaxin-2 B-chain polypeptide variant comprises the conserved motif Arg-X-X-X-Arg-X-X-Ile (Claasz AA et al., (2002) Eur. J. Biochem. 269(24):6287-6293) or Arg-X-X-X-Arg-X-X-Val (Bathgate RA et al., (2013) Physiol Rev. 93(1):405-480).

[0100] Variants may include one or more amino acid substitutions and / or insertions. In some embodiments, the relaxin-2 A-chain polypeptide variant includes 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, the relaxin-2 A-chain polypeptide variant includes the amino acid substitution K9H. In some embodiments, compared to SEQ ID NO:62, the relaxin-2 B-chain polypeptide variant includes one or more additional amino acids, such as K30, R31, and the N-terminal V-2, A-1, and M-1.

[0101] In some embodiments, the variant includes one or more amino acid derivatives. In some embodiments, the first amino acid of the relaxin-2 B-chain polypeptide variant is pyroglutamic acid.

[0102] In some embodiments, the relaxin-2 A-chain polypeptide includes the amino acid sequence SEQ ID NO:1. In some embodiments, the relaxin-2 B-chain polypeptide includes the amino acid sequence SEQ ID NO:2.

[0103] In some embodiments, the relaxin chain polypeptide of the heterodimeric fusion protein agonizes the relaxin family peptide receptor 1 (RXFP1). In some embodiments, the relaxin chain polypeptide is the relaxin chain polypeptide of AZD3427.

[0104] Relaxin activity

[0105] The heterodimeric fusions provided herein exhibit relaxin activity. As used herein, "relaxin activity" (which may be used interchangeably with "biological activity") refers to the ability of relaxin to bind to and / or activate a relaxin receptor and / or initiate an intracellular signaling cascade. In some embodiments, the relaxin activity is relaxin-2 activity, and the relaxin activity refers to the ability to bind to and / or activate receptor RXFP1 and / or receptor RXFP2. Relaxin activity can be determined in vitro and / or in vivo. In some embodiments, the relaxin activity is determined in vitro.

[0106] If the heterodimeric fusion protein of the present disclosure shows at least a portion of the activity of a reference relaxin protein, then it can be determined to have relaxin activity. For example, if the ratio of the activity of the heterodimeric fusion protein of the present disclosure to the activity of a reference relaxin protein is between about 10 -5 and about 1, between about 10 -4 and about 1, between about 10 -3 and about 1, between about 10 -2Between about 1 and about 1, between about 1 / 50 and about 1, between about 1 / 20 and about 1, between about 1 / 15 and about 1, between about 1 / 10 and about 1, between about 1 / 5 and about 1, or between about 1 / 2 and about 1, the heterodimeric fusion protein may have relaxin activity. In some embodiments, if the ratio of the activity of the heterodimeric fusion protein of the present disclosure to the activity of a reference relaxin protein is between about 1 and about 10 5 Between, between about 1 and about 10 4 Between, between about 1 and about 10 3 Between, between about 1 and about 100, between about 1 and about 50, between about 1 and about 20, between about 1 and about 15, between about 1 and about 10, between about 1 and about 5, or between about 1 and about 2, the heterodimeric fusion protein may have relaxin activity. In some embodiments, the activity of the heterodimeric fusion protein is at least half of the activity of the reference relaxin protein. In some embodiments, the activity of the heterodimeric fusion protein is at least three-quarters of the activity of the reference relaxin protein. In some embodiments, the activity level of the heterodimeric fusion protein is at least the same as that of the reference protein.

[0107] 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 arrangement structure of the relaxin A chain and the relaxin B chain of the mature relaxin protein. Recombinant relaxin having the arrangement structure of the relaxin A chain and the B chain of the mature relaxin protein is commercially available. For example, recombinant human relaxin-2, mouse relaxin-1, and INSL3 are purchased 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 B chain as the heterodimeric fusion protein of the present disclosure, or differs from the relaxin A chain and B chain of the heterodimeric fusion protein of the present disclosure by at most 10 amino acids, such as 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 protein. In some embodiments, the reference relaxin protein is a relaxin-2 protein having the arrangement structure of the relaxin-2A chain and the relaxin-2B chain of the mature relaxin-2 protein as disclosed in UniProtKB / Swiss-Prot accession number P04090.1. Relaxin activity can be determined by measuring the binding of relaxin to the relaxin receptor and / or by measuring downstream events after binding to the relaxin receptor. In some embodiments, relaxin activity is determined by measuring the amount and / or presence of downstream molecules after relaxin activates the receptor. In some embodiments, relaxin activity is determined by measuring the level of cAMP production after relaxin activates the receptor. Methods for detecting cAMP production induced by relaxin are known in the art. Such methods include cAMP ELISA, HTRF cAMP assay, and cAMP assay. In some embodiments, relaxin activity is determined by measuring the level of cAMP production induced by relaxin using the HTRF cAMP assay. In some embodiments, relaxin activity is determined by measuring the level of nitric oxide (NO) production after relaxin activates the receptor.

[0108] In some embodiments, relaxin activity is determined by measuring the activation level of downstream molecules after relaxin activates the receptor. In some embodiments, relaxin activity is determined by measuring the activation level of p42 / 44MAPK.

[0109] In some embodiments, relaxin activity is determined by measuring the activation level of a relaxin target gene. In some embodiments, relaxin activity is determined by measuring the transcriptional activation level of vascular endothelial growth factor (VEGF) (e.g., in THP-1 cells). Methods for determining gene transcriptional activation levels are known in the art and include quantitative PCR analysis of mRNA. In some embodiments, the relative expression level of VEGF mRNA is measured by detecting the induced expression level of VEGF transcripts by real-time quantitative PCR after incubation of THP-1 cells with relaxin, as described in Xiao et al., (2013) Nat Commun. 4:1953.

[0110] In some embodiments, relaxin activity is determined by measuring one or more downstream effects of relaxin. For example, according to standard methods, the alleviation of cardiac hypertrophy can be measured by echocardiography, the ratio of left ventricular weight to body weight, and / or tibial length. In some embodiments, relaxin activity is determined by measuring the alleviation of fibrosis by Masson's trichrome staining. In some embodiments, relaxin activity is determined by measuring the regulation of connective tissue metabolism, such as inhibiting profibrotic factors (such as transforming growth factor-β (TGF-β)), inhibiting fibroblast proliferation and differentiation, and / or activating matrix metalloproteinase (MMP)-mediated extracellular matrix degradation (Bathgate RA et al., (2013) Physiol Rev. 93(1):405-480).

[0111] Heterodimerization domain

[0112] The heterodimeric fusion proteins of the present disclosure comprise a first heterodimerization domain and a second heterodimerization domain. In some embodiments, the first heterodimerization domain and the second heterodimerization domain are derived from the immunoglobulin Fc region. The first Fc region and the second Fc region comprise the immunoglobulin domains CH2 and CH3. In some embodiments, either or both of the first Fc region and the second Fc region further comprise the CH4 domain. The Fc regions provided herein can be derived from immunoglobulins (e.g., IgG) of any species, optionally derived from humans (e.g., human IgG). In embodiments where the Fc region is derived from IgG, the Fc region can be derived from any IgG subtype (e.g., IgG1, IgG2, IgG3, IgG4). In some embodiments, the first Fc region and the second Fc region are derived from IgG1. In some embodiments, the first Fc region and the second Fc region are derived from human IgG1 immunoglobulin. In some embodiments, the first Fc region and the second Fc region are derived from IgG4. In some embodiments, the first Fc region and the second Fc region are derived from human IgG4 immunoglobulin.

[0113] In some embodiments, the first Fc region and the second Fc region comprise amino acid mutations that promote heterodimerization. In some embodiments, these mutations include asymmetric complementary modifications of the first Fc region and the second Fc region (e.g., generating Fc knob and Fc hole structures) such that the two chains are compatible with each other and thus able to form a heterodimer, but each chain is unable to homodimerize on its own. Such modifications can include insertions, deletions, conservative substitutions, non-conservative substitutions, and rearrangements.

[0114] In some embodiments, the amino acid mutations that promote heterodimerization are present in the CH3 domains of the first Fc region and the second Fc region.

[0115] In some embodiments, the first Fc region (FcX) and the second Fc region (FcY) are derived from human IgG1 immunoglobulin and comprise mutations in the CH3 domain, where the mutations are selected from the combinations listed in Table 1, or conservative substitutions thereof, where the positions are according to the EU index in Kabat.

[0116] Table 1: Exemplary combinations of FcX and FcY mutations

[0117]

[0118]

[0119] 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 the amino acid numbering is according to the EU index in Kabat. In some embodiments, the FcY mutations include Y349C, T366S, L368A, and Y407V, and the FcX mutations include S354C and T366W, where the amino acid numbering is according to the EU index as in Kabat.

[0120] It should be understood that the Fc region may also contain other amino acid modifications relative to the wild-type Fc region. The Fc region can be modified, for example, to increase the affinity of the IgG molecule for FcRn. WO 02 / 060919 discloses modified immunoglobulins comprising an Fc region having one or more amino acid modifications, and the entire content thereof is incorporated herein by reference. Methods for preparing an Fc region having one or more amino acid modifications are known in the art. In some embodiments, the first Fc region and / or the second Fc region provided in the present disclosure comprise one or more amino acid modifications that reduce or eliminate the effector functions of the Fc region. In some embodiments, the first Fc region and / or the second Fc region provided in the present disclosure comprise one or more amino acid modifications that reduce or circumvent 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, wherein the amino acid numbering is according to the EU index in Kabat. In some embodiments, the second Fc region further comprises the mutations L234F, L235E, and P331S or conservative substitutions thereof, wherein the amino acid numbering is according to the EU index in Kabat. In some embodiments, the first Fc region and the second Fc region further comprise the mutations L234F, L235E, and P331S or conservative substitutions thereof, wherein the amino acid numbering is according to the EU index in Kabat.

[0121] In some embodiments, the first Fc region comprises the amino acid sequence SEQ ID NO:4, and the second Fc region comprises the amino acid sequence SEQ ID NO:3. In some embodiments, the first Fc region consists of the amino acid sequence SEQ ID NO:4, and the second Fc region consists of the amino acid sequence SEQ ID NO:3.

[0122] In some embodiments, the cysteine at position 354 of the first Fc region (e.g., as shown in SEQ ID NO:4) forms a stable disulfide bond with the cysteine at position 349 of the second Fc region (e.g., as shown in SEQ ID NO:3).

[0123] Linker

[0124] One or both of the relaxin A-chain polypeptide and the B-chain polypeptide can be linked to their respective heterodimerization domains via a linker polypeptide. In some embodiments, the relaxin A-chain polypeptide is linked to the first heterodimerization domain (e.g., the first Fc region) via a linker polypeptide, and the relaxin B-chain polypeptide is linked to the second heterodimerization domain (e.g., the second Fc region) via a linker polypeptide.

[0125] In some embodiments, the heterodimeric fusion protein further comprises a second relaxin A-chain polypeptide or variant thereof linked to the N-terminus of the first Fc region, and a second relaxin B-chain polypeptide or variant thereof linked to the N-terminus of the second Fc region. In some embodiments, the second relaxin A-chain is linked to the first Fc region via a linker polypeptide.

[0126] The linker polypeptide can have any suitable length, such as a length between about 6 and 40 amino acids, optionally between about 6 and 21 amino acids. In some embodiments, the linker polypeptide is at least 6 amino acid residues in length, optionally at least 11 amino acids in length, and further optionally at least 16 amino acids in length. In some embodiments, the linker polypeptide is less than 40 amino acids in length. Linker polypeptides of different or the same lengths can be used for each arm of the heterodimeric fusion protein of the present disclosure. In some embodiments, at least one linker polypeptide has a length of 21 amino acids. In some embodiments, both linker polypeptides (i.e., the linker polypeptide linking the relaxin A-chain polypeptide to the first Fc region and the linker polypeptide linking the relaxin B-chain polypeptide to the second Fc region) have a length of 21 amino acids.

[0127] Linker polypeptides of different or the same amino acid composition can be used for each arm of the heterodimeric fusion protein of the present disclosure.

[0128] In some embodiments, one or optionally both linker polypeptides comprise glycine 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 linker polypeptides comprise the motif (GGGGS)n (SEQ ID NO:69), where n can be between 1 and 8, such as where n is 4. In some embodiments, one or more linker polypeptides consist of the 10-amino acid sequence GGGSGGGSGG (SEQ ID NO:60). In some embodiments, one or more linker polypeptides consist of the 21-amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:5). In some embodiments, both linker polypeptides (i.e., the linker polypeptide linking the relaxin A-chain polypeptide to the first Fc region and the linker polypeptide linking the relaxin B-chain polypeptide to the second Fc region) consist of the 21-amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:5).

[0129] In some embodiments, one or both of the relaxin A-chain and B-chain can be linked to their respective heterodimerization domains via a synthetic linker polypeptide, such as a polyethylene glycol (PEG) polymer chain. PEGylation can be carried out according to methods known in the art.

[0130] Exemplary heterodimeric fusion protein

[0131] In some embodiments, the heterodimeric fusion protein comprises (i) a relaxin A-chain polypeptide comprising the amino acid sequence SEQ ID NO:1, which is linked to a first Fc region comprising the amino acid sequence SEQ ID NO:4 via a linker polypeptide comprising the amino acid sequence SEQ ID NO:5, and (ii) a relaxin B-chain polypeptide comprising the amino acid sequence SEQ ID NO:2, which is linked to a second Fc region comprising the amino acid sequence SEQ ID NO:3 via a linker polypeptide comprising the amino acid sequence SEQ ID NO:5.

[0132] In some embodiments, the heterodimeric fusion protein comprises the amino acid sequences SEQ ID NO:11 and SEQ ID NO:20, wherein SEQ ID NO:11 is the amino acid sequence of a relaxin A-chain polypeptide linked to a first Fc region via a linker polypeptide, and SEQ ID NO:20 is the amino acid sequence of a relaxin B-chain polypeptide linked to a second Fc region via a linker polypeptide, wherein the first Fc region and the second Fc region heterodimerize, and the relaxin A-chain polypeptide and the relaxin B-chain polypeptide heterodimerize. In some embodiments, the heterodimeric fusion protein comprises a stabilizing disulfide bond between the cysteine at position 349 of the second Fc region and the cysteine at position 354 of the first Fc region, wherein the position numbering is according to the EU index in Kabat.

[0133] The exemplary heterodimeric fusion protein AZD3427 is composed of a relaxin A-chain-linker-Fc region fusion having the amino acid sequence SEQ ID NO:11 and a relaxin B-chain-linker-Fc region fusion having the amino acid sequence SEQ ID NO:20 bound by heterodimerization. In some embodiments, the heterodimeric fusion protein comprises AZD3427. In some embodiments, the heterodimeric fusion protein is AZD3427. The sequences used in AZD3427 are shown in Table 2.

[0134] Table 2. Sequences used in AZD3427. The upper hinge region is in italics, relaxin A is underlined, relaxin B is doubly underlined, and the Fc region is in bold.

[0135]

[0136]

[0137] Pharmaceutical composition

[0138] The heterodimeric fusion proteins of the present disclosure can be provided in the form of pharmaceutical compositions.

[0139] The pharmaceutical compositions of the present disclosure can comprise one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are known in the art, see, e.g., Remington's Pharmaceutical Sciences (Joseph P. Remington, 18th ed., Mack Publishing Co., Easton, PA), the entire content of which is incorporated herein by reference.

[0140] Kit

[0141] Kits comprising the pharmaceutical compositions of the present disclosure can be provided. The kit can include a package containing the pharmaceutical composition of the present disclosure and instructions for use. In some embodiments, the pharmaceutical composition of the present disclosure is dispensed in single-dose vials or container-sealing systems (e.g., prefilled syringes). Optionally, associated with such a container can be a label in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, the label reflecting the agency's approval of the manufacture, use, or sale of the pharmaceutical or biological product for human administration.

[0142] Methods of administration and methods of treatment

[0143] The present disclosure provides methods for treating a subject having heart failure, optionally heart failure with pulmonary hypertension, by administering to the subject a heterodimeric relaxin fusion protein as provided herein or a pharmaceutical composition comprising the heterodimeric relaxin fusion protein. It should be understood that any treatment method disclosed herein also provides the disclosure of the corresponding use of the heterodimeric relaxin fusion protein or the pharmaceutical composition comprising the heterodimeric relaxin fusion protein for treating a subject having heart failure, optionally heart failure with pulmonary hypertension. In the context of using the heterodimeric relaxin fusion protein or the pharmaceutical composition comprising the heterodimeric relaxin fusion protein to treat a subject having heart failure, optionally heart failure with pulmonary hypertension, the features of any treatment method described herein should be understood to be disclosed with necessary modifications. It should also be understood that any treatment method disclosed herein also provides the disclosure of the corresponding use of the heterodimeric relaxin fusion protein or the pharmaceutical composition comprising the heterodimeric relaxin fusion protein for manufacturing a medicament for treating a subject having heart failure, optionally heart failure with pulmonary hypertension. In the context of using the heterodimeric relaxin fusion protein or the pharmaceutical composition comprising the heterodimeric relaxin fusion protein to manufacture a medicament for treating a subject having heart failure, optionally heart failure with pulmonary hypertension, the features of any treatment method described herein should be understood to be disclosed with necessary modifications. It should also be understood that any treatment method disclosed herein also provides the disclosure of the corresponding use of the heterodimeric relaxin fusion protein or the pharmaceutical composition comprising the heterodimeric relaxin fusion protein for treating a subject having heart failure, optionally heart failure with pulmonary hypertension. In the context of using the heterodimeric relaxin fusion protein or the pharmaceutical composition comprising the heterodimeric relaxin fusion protein for treating a subject having heart failure, optionally heart failure with pulmonary hypertension, the features of any treatment method described herein should be understood to be disclosed with necessary modifications. The subject to be treated with the heterodimeric relaxin fusion protein or the pharmaceutical composition can be an animal, optionally a mammal, and optionally a human.

[0144] In some embodiments, the subject meets one or more of the following criteria: New York Heart Association (NYHA) 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 tightness; dyspnea at rest or with minor activity; N-terminal pro-brain natriuretic peptide (NT-proBNP) > 125 pg / mL, or brain natriuretic peptide (BNP) > 35 pg / mL; systolic blood pressure > 125 mmHg; mild to moderate renal insufficiency; body mass index (BMI) of at least 18 kg / m 2; and heart failure with reduced ejection fraction (HFrEF), less than or equal to 40%.

[0145] As used herein, a subject having "pulmonary hypertension" is a subject having a mean pulmonary artery pressure of at least 20 mmHg. In some embodiments, the pulmonary hypertension is classified as Group 2 pulmonary hypertension according to the World Health Organization's definition. 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 hypertension according to the World Health Organization's definition (see Ryan et al., 2012, Pulm. Circ. 2(1):107-121).

[0146] Parameters of pulmonary hypertension and heart failure can be measured or estimated using techniques known in the art. For example, such techniques include echocardiography, pulmonary artery catheterization, and implantable monitoring devices. In certain embodiments, the subject may have a blood pressure monitoring device installed, optionally a pulmonary artery pressure monitoring device known in the art. In some embodiments, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. Generally, the device is installed prior to treatment with the heterodimeric fusion protein of the present disclosure provided herein. The device is installed during or after the treatment period for the subject.

[0147] The heterodimeric relaxin fusion protein or pharmaceutical composition of the present disclosure can be administered to a subject by injection (such as by intravenous, subcutaneous, or intramuscular injection). In some embodiments, the heterodimeric fusion protein or pharmaceutical composition is administered subcutaneously. In some embodiments, the heterodimeric fusion is administered every two weeks.

[0148] In some embodiments, the heterodimeric relaxin fusion protein administered to the subject is administered to the subject chronically. In some embodiments, the heterodimeric fusion protein is administered to the subject at least four times. In some embodiments, the heterodimeric fusion protein is administered to the subject at least five times. In some embodiments, the heterodimeric fusion protein is administered to the subject at least 12 times. In some embodiments, the heterodimeric fusion protein is administered subcutaneously to the subject once every two weeks. In some embodiments, the heterodimeric fusion protein is administered subcutaneously to the subject once every two weeks in an amount of, for example, about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

[0149] In some embodiments, the heterodimeric fusion protein is composed of a relaxin A chain-linker-Fc region fusion having the amino acid sequence SEQ ID NO:11 and a relaxin B chain-linker-Fc region fusion having the amino acid sequence SEQ ID NO:20 bound by heterodimerization. In some embodiments, the heterodimeric fusion protein comprises AZD3427. In some embodiments, the heterodimeric fusion protein is AZD3427.

[0150] In some embodiments, AZD3427 is administered to a subject chronically. In some embodiments, AZD3427 is administered subcutaneously to a subject once every two weeks. In some embodiments, AZD3427 is administered subcutaneously to a subject once every two weeks in an amount of, for example, about 1 mg, about 1.1 mg, about 5.4 mg or about 30 mg.

[0151] In some embodiments, the amount of the heterodimeric fusion protein (such as AZD3427) administered to a subject or in a pharmaceutical composition administered to a subject is from about 1 mg to about 30 mg. In some embodiments, the amount of the heterodimeric fusion protein (such as 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 protein (such as 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 protein (such as AZD3427) administered to a subject or in a pharmaceutical composition administered to a subject is from 0.9 mg to 1.1 mg. In some embodiments, the amount of the heterodimeric fusion protein (such as 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 protein (such as AZD3427) administered to a subject or in a pharmaceutical composition administered to a subject is from 1.0 mg to 1.2 mg. In some embodiments, the amount of the heterodimeric fusion protein (such as 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 protein (such as AZD3427) administered to a subject or in a pharmaceutical composition administered to a subject is from 5.3 mg to 5.5 mg. In some embodiments, the amount of the heterodimeric fusion protein (such as 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 protein (such as AZD3427) administered to a subject or in a pharmaceutical composition administered to a subject is about 30 mg. In some embodiments, the amount of the heterodimeric fusion protein (such as AZD3427) administered to a subject or in a pharmaceutical composition administered to a subject is from 29.9 mg to 30.1 mg.

[0152] In some embodiments, the amount of the heterodimeric fusion protein (such as 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.

[0153] Without being bound by theory, subcutaneous administration (e.g., at a biweekly dose of about 1 mg to 30 mg) can provide convenience and compliance benefits to a subject. For example, administering the heterodimeric fusion protein (e.g., AZD3427) subcutaneously at a lower, less frequent dose once every two weeks can provide various advantages, such as better comfort for the subject or patient, improved patient compliance, and the opportunity to administer to the subject or patient outside of a hospital setting, thus providing a better quality of life.

[0154] In some embodiments, the dose of the heterodimeric fusion protein administered subcutaneously is sufficient to increase the relaxin level, e.g., at least 0.5-fold increase compared to the relaxin level measured in the first trimester of pregnancy, while minimizing adverse reactions (such as decreases in hemoglobin, hematocrit, and / or albumin levels). Unless otherwise specified, the relaxin level during pregnancy refers to the relaxin level in the first trimester of pregnancy. In some embodiments, the dose administered subcutaneously is sufficient to improve cardiac output. In some embodiments, the dose administered subcutaneously is sufficient to improve organ perfusion. In some embodiments, the dose administered subcutaneously is sufficient to increase the blood flow through the kidneys. In some embodiments, the dose administered subcutaneously is sufficient to increase the stroke volume, e.g., by at least 1%. In some embodiments, the dose administered subcutaneously is sufficient to reduce the systemic vascular resistance, e.g., by at least 8%. In some embodiments, the dose administered subcutaneously is sufficient to reduce the systemic vascular resistance, e.g., by at least 10%. In some embodiments, the dose administered subcutaneously is sufficient to reduce the systemic vascular resistance, e.g., by at least 15%.

[0155] In some embodiments, the dose of the heterodimeric fusion protein (e.g., AZD3427) is selected such that the relaxin exposure of the subject corresponds 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 pregnant subject in the first trimester of pregnancy. In some embodiments, the dose of the heterodimeric fusion protein (e.g., AZD3427) is selected such that the relaxin exposure of the subject corresponds to up to about 0.5-fold, up to about 3.5-fold, or up to about 7-fold the endogenous relaxin level in the plasma of a pregnant subject in the first trimester of pregnancy. Relaxin levels, such as the endogenous relaxin level in the plasma of a pregnant subject in the first trimester of pregnancy, can be measured using a relaxin detection assay, such as by using 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 pregnant subject in the first trimester of pregnancy can be up to 0.2 ng / mL or about 0.2 ng / mL (e.g., as measured using a relaxin detection assay, such as the assay described in Example 10).

[0156] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg causes a dose-dependent increase in renin production 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 the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg causes a dose-dependent increase in renin production of at least 2-fold. In some embodiments, the dose of the heterodimeric fusion protein (e.g., AZD3427) is selected to cause a dose-dependent increase in renin production of at least 2-fold.

[0157] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg increases the cardiac output of the subject by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%, for example, after 10 to 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg increases the cardiac output of the subject by about 10%, for example, after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1.1 mg increases the cardiac output of the subject by about 10%, for example, after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 5.4 mg increases the cardiac output of the subject by about 10%, for example, after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 30 mg increases the cardiac output of the subject by at least about 10%, for example, after 24 weeks of treatment.

[0158] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg causes an increase in the stroke volume (SV) of the subject of about 0.5%, 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15% or about 20%, for example after 10 weeks or 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg causes an increase in the stroke volume (SV) of the subject of about 5% to 60%, for example after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg causes an increase in the SV of the subject of about 1%, for example after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1.1 mg causes an increase in the SV of the subject of about 1%, for example after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 5.4 mg causes an increase in the SV of the subject of about 5%, for example after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 30 mg causes an increase in the SV of the subject of about 10%, for example after 24 weeks of treatment.

[0159] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg causes a decrease in the systemic vascular resistance (SVR) of the subject of about 5%, about 10% or about 15%, for example after 10 weeks of treatment.

[0160] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg causes a decrease in the systemic vascular resistance (SVR) of the subject of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40%, for example after 24 weeks of treatment.

[0161] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg causes a reduction in SVR of about 8% in a subject, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1.1 mg causes a reduction in SVR of about 8% in a subject, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg or 1.1 mg causes a reduction in SVR of up to about 20% in a subject, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 5.4 mg causes a reduction in SVR of about 15% in a subject, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 5.4 mg causes a reduction in SVR of up to about 30% in a subject, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 30 mg causes a reduction in SVR of about 20% in a subject, e.g., after 24 weeks of treatment.. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 30 mg causes a reduction in SVR of up to about 40% in a subject, e.g., after 24 weeks of treatment.

[0162] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) causes an increase in the estimated glomerular filtration rate (eGFR) of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% in a subject, e.g., after 24 weeks of treatment.

[0163] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (such as AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) causes at least a 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, or 1% to 50% or more reduction in the pulmonary vascular resistance (PVR) of the subject. In some embodiments, the reduction in PVR of the subject can be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%, for example, after 24 weeks of treatment.

[0164] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (such as AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) reduces the mean pulmonary artery pressure (mPAP) of the subject by at least 1 mmHg, for example, after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (such as AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) reduces the mean pulmonary artery pressure of the 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, for example, after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (such as AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) reduces the estimated pulmonary artery diastolic pressure (ePAD) of the subject by at least 1 mmHg to 15 mmHg, for example, after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (such as AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) reduces the estimated pulmonary artery diastolic pressure of the 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, for example, after 24 weeks of treatment.

[0165] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg increases the percentage of the ejection fraction (EF) of a subject by about 1% to about 10%, e.g., after 10 weeks of treatment, compared to placebo. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg increases the percentage of the ejection fraction (EF) of a subject by about 1% to about 5%, e.g., after 10 weeks of treatment, compared to placebo.

[0166] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg increases the percentage of the ejection fraction (EF) of a subject by about 1% to about 40%, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg increases the EF percentage of a subject by at least 5%, at least 10%, at least 20%, at least 30%, or at least 40%, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg increases the EF of a subject by at least 25%, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1.1 mg increases the EF of a subject by at least 25%, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 5.4 mg increases the EF of a subject by at least 30%, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 30 mg increases the EF of a subject by at least 35%, e.g., after 24 weeks of treatment.

[0167] In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) does not cause a change in the hematocrit, hemoglobin, and / or albumin levels of the subject. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg does not cause a significant decrease in the hematocrit, hemoglobin, and / or albumin levels of the subject. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) causes a decrease in the hematocrit level of the subject by no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, or no more than 8%. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) causes a decrease in the hematocrit level of the subject by no more than 5%. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) causes a decrease in the hematocrit level of the subject by no more than 7%. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) causes a decrease in the hematocrit level of the subject by no more than 8%. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) causes a decrease in the hemoglobin level of the subject by no more than 1%, no more than 2%, no more than 3%, no more than 4%, or no more than 5%. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) causes a decrease in the hemoglobin level of the subject by no more than 3%. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg causes a change in the albumin level by no more than 1%, no more than 2%, no more than 3%, no more than 4%, or no more than 5%.In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) results in a change in albumin level of no more than 2% compared to the baseline level. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) results in a decrease in albumin level of no more than 1%, no more than 2%, no more than 3%, no more than 4%, or no more than 5%. In some embodiments, subcutaneous administration of the heterodimeric fusion protein (e.g., AZD3427) at a biweekly dose of about 1 mg to 30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) results in an increase in albumin level of no more than 1%, no more than 2%, no more than 3%, no more than 4%, or no more than 5%.

[0168] In some embodiments, the change (e.g., increase or decrease) in biomarker level or patient outcome after administration of the heterodimeric fusion protein is relative to the baseline (pre - administration) measurement.

[0169] In some embodiments, administration of the heterodimeric fusion protein (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 (i.e., pre - administration) blood pressure and / or compared to a placebo group. In some embodiments, no significant change in the patient's blood pressure (e.g., systolic blood pressure) means a change of no more than 20% relative to the patient's baseline blood pressure (e.g., systolic blood pressure). This can be a decrease of no more than 20% relative to the patient's baseline blood pressure (e.g., systolic blood pressure).

[0170] Exemplary dosing regimens

[0171] In some embodiments, the heterodimeric fusion protein (e.g., AZD3427) is administered subcutaneously to a subject once every two weeks. In some embodiments, the heterodimeric fusion protein (e.g., AZD3427) is administered subcutaneously to a subject once every two weeks at a dose of about 1 mg to about 30 mg. In some embodiments, the heterodimeric fusion protein (e.g., AZD3427) is administered subcutaneously to a subject once every two weeks at a dose of about 1 mg. In some embodiments, the heterodimeric fusion protein (e.g., AZD3427) is administered subcutaneously to a subject once every two weeks at a dose of about 1.1 mg. In some embodiments, the heterodimeric fusion protein (e.g., AZD3427) is administered subcutaneously to a subject once every two weeks at a dose of about 5.4 mg. In some embodiments, the heterodimeric fusion protein (e.g., AZD3427) is administered subcutaneously to a subject once every two weeks at a dose of about 30 mg.

[0172] Preparation method

[0173] The heterodimeric fusion proteins of the present disclosure can be produced by any method known in the art. In some embodiments, the heterodimeric fusion proteins of the present disclosure are produced by recombinant expression in a host cell of a nucleic acid molecule encoding the heterodimeric fusion protein (or one or more fragments thereof). For example, a fragment of the heterodimeric fusion protein can be a relaxin A chain-linker-first Fc region fusion or a relaxin B chain-linker-second Fc region fusion.

[0174] Methods known to those of skill in the art can be used to construct an expression vector containing a nucleic acid molecule encoding the heterodimeric fusion protein of the present disclosure. Suitable vectors include, for example, plasmids, phagemids, phages, or viral vectors.

[0175] A vector containing a nucleic acid molecule encoding the heterodimeric fusion protein 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.

[0176] Transfected cells can be cultured by conventional techniques to produce the fusion polypeptide of the present disclosure.

[0177] Once the heterodimeric fusion protein or a fragment thereof of the present disclosure is produced (e.g., by recombinant expression), it can be purified by any method known in the art. Exemplary protein purification techniques include chromatography (e.g., ion exchange chromatography, affinity chromatography, and / or size exclusion chromatography), centrifugation, and solubility differences. The present disclosure provides an isolated heterodimeric fusion protein that has been isolated from a cell culture, optionally by at least one purification step.

[0178] Definition

[0179] As used herein, the articles "a" and "an" can refer to one or more than one (e.g., at least one) grammatical object of the article.

[0180] "About" and "approximately" are used interchangeably herein and mean that within the context of a pharmaceutical formulation, a measured value has an acceptable range of variation based on the nature of the measurement or the precision required. For example, for the dosage of a heterodimeric fusion protein as provided herein, "about" allows for a difference of ±0.1 from the specified value.

[0181] As used herein, the term "biweekly" administration means administration once every two weeks.

[0182] Embodiments provided herein that "comprise" one or more features can also be considered to disclose corresponding embodiments "consisting of" such features.

[0183] The term "EU index in Kabat" refers to the numbering system of human IgG1 EU antibodies 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 referred to in this application are EU index positions.

[0184] As used herein, the term "heart failure" includes acute heart failure, chronic heart failure (CHF), and acute 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% (EF ≥ 41% HF), mid-range ejection fraction heart failure, or heart failure with reduced ejection fraction (HFrEF). Also included is heart failure caused by hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0185] As used herein, the term "heart failure with pulmonary hypertension" refers to a subset of heart failure subjects with pulmonary hypertension (HF+PH subjects).

[0186] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal Government or a state government of the United States, or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopeias, for use in animals, and more specifically in humans.

[0187] The term "plasma concentration" refers to the mean plasma concentration at steady state.

[0188] The term "treatment" refers to ameliorating and / or eliminating one or more symptoms or causes of a target disease or disorder. In some embodiments, this involves modulating the level of one or more biomarkers or functions, e.g., compared to a diseased state, e.g., modulating to within a non-diseased range (compared to a healthy cohort).

[0189] Concentrations, amounts, volumes, percentages, and other numerical values may be presented herein in a range format. It should be understood that the use of such range format is for convenience and brevity only and that the interpretation thereof should be flexible, including not only the explicitly recited values defining the range limits but also all individual values or sub-ranges subsumed within that range as if each value and sub-range were explicitly recited. It should be understood that unless otherwise explicitly stated, ranges include their end values.

[0190] The above embodiments and the following examples are to be understood as illustrative and not restrictive. Additional embodiments are contemplated. It should be understood that any feature provided with respect to any one embodiment may be used alone or in combination with other provided features, and may also be used in combination with one or more features of any other embodiment, or with any combination of any other embodiments. Furthermore, equivalents and modifications not mentioned above may also be employed, provided they do not depart from the scope of the present disclosure as defined by the appended claims.

[0191] In the context of the present disclosure, other embodiments and variations of the fusion polypeptides and methods provided herein will be apparent to those skilled in the art.

[0192] Other embodiments and variations fall within the scope of the present disclosure as set forth in the appended claims. All documents cited herein are hereby incorporated by reference in their entirety, including all data, tables, figures, and textual matter presented in the cited documents.

[0193] Example

[0194] Example 1: Generation of recombinant heterodimeric Fc-relaxin-2 fusion protein

[0195] The A-chain and B-chain of relaxin-2 are genetically fused via a linker to two complementary Fc regions (at the N-terminus and / or C-terminus of the Fc region), as Figure 1 shown. CHO cells are then co-transfected with two expression vectors containing each single Fc-relaxin chain (A and / or B). The two complementary Fc moieties assemble within the CHO cells, thus facilitating the assembly and correct folding of relaxin-2.

[0196] The heterodimeric Fc-relaxin-2 fusion protein is secreted into the supernatant and then purified by affinity chromatography using an automated system, wherein the Fc region of the protein binds to the column matrix.

[0197] Example 2: PK characteristics of RELAX0023 (AZD3427) in cynomolgus monkeys

[0198] 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, which were randomly assigned to 4 experimental groups of 3 animals each. Animals in groups 1, 2, and 3 were administered 0.1 mg / kg, 1 mg / kg, and 10 mg / kg of RELAX0023 subcutaneously, respectively. Animals in group 4 were administered 10 mg / kg of RELAX0023 by intravenous bolus injection. 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.

[0199] The assay plates were coated with goat anti-human IgG antibody and incubated with cynomolgus monkey sera from animals in groups 1 to 4. RELAX0023 bound to the assay plates was detected by an anti-relaxin antibody conjugated to HRP. Cynomolgus monkey sera were diluted 1:10 and then added to the plates. In 100% serum, the lower limit of quantification was 0.010 μg / mL and the upper limit of quantification was 0.300 μg / mL.

[0200] Figure 2 The mean serum concentration-time curve of AZD3427 in cynomolgus monkeys after a single dose is shown. After a single subcutaneous dose, AZD3427 exhibited linear PK in the dose range of 0.01 mg / kg to 10 mg / kg. A dose-proportional increase in Cmax was observed. For the 0.1 mg / kg, 1 mg / kg, and 10 mg / kg subcutaneous dosing groups, the mean Cmax values were 0.400 μg / mL, 4.69 μg / mL, and 34.8 μg / mL, respectively. In the subcutaneous dosing groups from 0.1 mg / kg to 10 mg / kg, the AUC0-last values exhibited a dose-proportional increase. For the 0.1 mg / kg, 1 mg / kg, and 10 mg / kg subcutaneous dosing groups, the mean AUC0-last values were 2.01 μg·day / mL, 25.5 μg·day / mL, and 193 μg·day / mL, respectively. Overall, AZD3427 PK was linear in the range of 0.1 mg / kg to 10 mg / kg, with an average CL / F of 51.0 mL / day.

[0201] 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) (Macaca fascicularis)

[0202] Evaluate the long-term efficacy of AZD3427 on cardiac function in obese elderly cynomolgus monkeys (Macaca fascicularis). Cynomolgus monkeys were selected instead of other lower mammalian species as the experimental species because of their close phylogenetic and physiological relationships with humans. Elderly cynomolgus monkeys fed a high-fat diet for at least two years share common risk factors with human patients prone to cardiovascular diseases and develop metabolic syndrome, which may be characterized by a progressive development of heart failure accompanied by a decrease in left ventricular ejection fraction (LVEF). The effect of the drug on LVEF was evaluated by continuously administering different dose levels of AZD3427 subcutaneously (SC) for 20 weeks, with the first dose administered in week 1 of the study, followed by an 18-week observation period. From a population of approximately 100 obese elderly cynomolgus monkeys aged 12 to 20 years and weighing 6 to 15 kg that had been fed a high-fat diet for at least two years, 38 monkeys with an LVEF between 30% and 60% were confirmed by two-dimensional echocardiogram screening. The body weight of healthy monkeys in this age group is 5 to 8 kg and the LVEF is 70% to 75%, so when LVEF ≤ 60%, it meets the definition criteria of the HFrEF model. The animals confirmed by screening were randomly assigned to 3 treatment groups (8 monkeys per group) and 1 vehicle control group (14 monkeys). The dosing period included weekly subcutaneous administration of AZD3427, with 3 escalating dose gradients (0.015 mg / kg, 0.15 mg / kg, and 3 mg / kg).

[0203] Cardiac function was measured by two-dimensional echocardiogram, with a total of 9 determinations completed: at baseline week -2, and at weeks 5, 9, 13, 17, 21, 25, 29, 33 during the dosing period and the post-dosing observation period. Another two-dimensional echocardiogram examination was arranged at week 39 (study endpoint). Parameters including LVEF were measured based on the apical two-chamber and four-chamber views combined with the biplane method. Parameters including mean arterial pressure (MAP) and heart rate (HR) were measured using HDO (high-definition oscillometry).

[0204] Compared with the vehicle control group, AZD3427 significantly increased LVEF at weeks 5, 9, 13, 17, and 21 at all dose levels and had no effect on heart rate or blood pressure ( Figures 3A to 3D)。It is noteworthy that compared with week 0 (baseline value), the improvement effect of LVEF after treatment with AZD3427 persisted during the washout period after the end of treatment and was still observable until week 33 of the study. There was also a significant increase in stroke volume (SV) and a significant decrease in systemic vascular resistance (SVR). At the same time, a dose-dependent increase in renin was observed, which may be a compensatory response caused by the vasodilation induced by AZD3427 treatment. These remarkable results indicate a significant improvement in the hemodynamics of the treated animals and clearly demonstrate the efficacy of AZD3427 in treating heart failure in this model. In addition, based on the inventor's knowledge boundary judgment, the continuous pharmacodynamic intensity index presented after treatment has breakthrough innovation value among the known compounds targeting this mechanism of action pathway. The monkey population was continuously monitored until week 39 of the study.

[0205] Example 4: Phase 1 (Ph1) study in healthy volunteers and heart failure patients

[0206] Study D8330C00001 is a Phase Ia / b, randomized, single-blind, placebo-controlled, first-in-human (FTIH) study (ClinicalTrials.gov identifier NCT04630067). The primary objective of this study was to evaluate the safety and tolerability of single and multiple ascending doses of AZD3427; the secondary objectives were to evaluate the (i) pharmacokinetics (PK) and (ii) immunogenicity of single and multiple ascending doses of AZD3427.

[0207] 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 (non-fertile men and women), and Part B was a multiple ascending dose (MAD) study in HF participants (non-fertile men and women).

[0208] Part A included 56 healthy participants, divided into 7 cohorts (8 people per cohort), and all participants received a single dose of AZD3427 or placebo. In each cohort, 6 participants were randomly assigned to receive AZD3427 and 2 participants were randomly assigned to receive placebo. One of the cohorts consisted entirely of participants of Japanese descent (both parents and all grandparents were Japanese). The 7 cohorts were as follows:

[0209] · Cohorts 1a, 2a, 3a, and 4a: subcutaneous administration, with dose levels of 5 mg, 10 mg, 30 mg, and 90 mg respectively

[0210] · Cohort 5a: intravenous administration, with a dose level of 15 mg AZD3427

[0211] · Cohort 6a: Subcutaneous administration, dose level of 270 mg (Japanese-descent participants)

[0212] · Cohort 7a: Single subcutaneous administration of AZD3427, dose level of approximately 270 mg.

[0213] Part B included 48 patients, divided into 6 cohorts (8 participants per cohort). Among them, 3 cohorts consisted of HFrEF participants (Cohorts 1b, 3b, and 5b), and the other 3 cohorts consisted of HF participants with EF ≥ 41% (Cohorts 2b, 4b, and 6b). In the HFrEF participant cohorts and the HF participant cohorts with EF ≥ 41%, the dose levels were 5 mg (Cohorts 1b, 2b), 15 mg (Cohorts 3b, 4b), and 45 mg (Cohorts 5b, 6b), administered once a week (QW) for 5 weeks (i.e., a total of 5 doses).

[0214] A total of 48 patients were included in Part B, divided into 6 cohorts. The inclusion criteria included: (i) All cohorts: Known clinical diagnosis of Stage C HF (NYHA Class I to III), and received stable drug therapy for at least 12 weeks before screening, with no significant dose changes or new medications during that period; (ii) Cohorts 1b, 3b, 5b: Patients diagnosed with HFrEF (defined as EF ≤ 40%); (iii) Cohorts 2b, 4b, 6b: Patients diagnosed with HF with EF ≥ 41% (including patients diagnosed with HFpEF (defined as EF ≥ 50%)); (iv) All cohorts: BMI between 18 kg / m 2 and 40 kg / m 2 (including the end values), weight ≥ 55 kg and ≤ 120 kg (including the end values); (v) All cohorts: NT-proBNP > 125 pg / mL or BNP > 35 pg / mL in the previous records.

[0215] Example 5: Serum samples for determination of AZD3427 concentration

[0216] Serum samples for determining the concentration of AZD3427 were analyzed by a validated electrochemiluminescence (ECL) method, which accurately and precisely quantifies AZD3427 levels in the range of 0.10 μg / mL to 25.60 μg / mL in human serum samples.

[0217] AZD3427 was captured by a biotinylated antibody (clone number AB1510209) against the relaxin moiety of AZD3427, coated at a concentration of 2.0 μg / mL on a Meso Scale Discovery (MSD) streptavidin-coated standard binding plate. Calibrators, quality control (QC) samples, and specimens were diluted 1:400 at the method's minimum required dilution (MRD) in assay buffer and added to the plate at 50 μL / well, then incubated for approximately 1 hour at room temperature with shaking (600 rpm). After washing the assay plate, a ruthenium-labeled anti-AZD3427 antibody that binds to the Fc part of the AZD3427's mortar structure (a site different from the binding site of the capture antibody) was added. The assay plate was incubated for approximately 1 hour, and then the excess reagent was washed off the plate. MSD read buffer was added to the plate to initiate the ECL reaction, causing the bound ruthenium molecules to emit light, and the light emission was measured in relative light units. The concentration of AZD3427 in the specimens was determined by interpolation from a standard curve that used four-parameter curve fitting and related the light intensity to the AZD3427 concentration by the 1 / ECL 2 weighting method.

[0218] Pharmacokinetic data: After subcutaneous administration of 5 mg, 10 mg, 30 mg, 90 mg, and 270 mg of AZD3427, the serum concentration-time curves from the SAD cohort are shown in Figure 5 . After subcutaneous administration of the AZD3427 solution, AZD3427 was absorbed and Cmax was reached within 3 to 4 days. By non-compartmental analysis of the PK data from the SAD cohort, the terminal half-life (t 1 / 2 ) of AZD3427 was estimated to be 7 to 9 days.

[0219] In the MAD cohort, the Ctrough values showed that steady state had not been fully reached after 5 weekly doses. For dose levels of 5 mg, 15 mg, and 45 mg, the Ctrough values after the last dose were 329 ng / mL, 1005 ng / mL, and 2321 ng / mL, respectively.

[0220] Example 6: Phase 1 AZD3427 MAD study outcomes in HF patients

[0221] In part B of the MAD cohort, the data from HFpEF patients and HFrEF patients were first pooled. The trends indicated that AZD3427 improved cardiac function, including improving cardiac output and stroke volume (SV), reducing systemic vascular resistance (SVR), and improving organ perfusion (SVR and eGFR) ( Figures 6A to 6F) Additional analysis was performed, this time dividing the patients into the following two groups: (i) patients with baseline EF ≤ 40% and (ii) patients with baseline EF > 40%. The results showed that after treatment with AZD3427, the trends of improvement (increase) in stroke volume and eGFR observed in the pooled patients were also observed in the two groups of (i) patients with baseline EF ≤ 40% and (ii) patients with baseline EF > 40% (see Figures 6H to 6I ). These improvements were observed without any significant effect on blood pressure ( Figure 6G ). Administration of AZD3427 also caused a dose-dependent increase in plasma renin levels ( Figure 7A ). For test doses below 45 mg, the resulting hemoglobin and hematocrit levels were both within an acceptable safety range. ( Figure 7B , Figure 7C ). Therefore, it is beneficial that the AZD3427 doses of 1 mg to 30 mg, which have been selected based on the modeling and Ph1 data described herein as the predicted pharmacodynamic effective dose range for the Ph2 trial, also appear to be sufficient to maintain hemoglobin and hematocrit levels within acceptable safety limits.

[0222] Example 7: Phase 2B (Ph2b) study design of the AZD3427 dosing regimen

[0223] In the Ph2b study (design shown in Figure 8 ; study ID number: D8330C00003), the Q2W doses of approximately 1.0 mg (e.g., 1.1 mg), approximately 5.4 mg, and 30 mg of AZD3427 will be evaluated. From Day 1 to Day 155, participants will receive a single subcutaneous dose of AZD3427 or placebo (in a 1:1:1:1 ratio) every two weeks for 24 weeks.

[0224] The dose selection of AZD3427 is based on the SAD / MAD study (study D8330C00001 / NCT04630067) from healthy participants and HFrEF / HFpEF participants, as well as the PK data, pharmacodynamic data, and safety data from this study of NHP with reduced LVEF. The once-weekly, once-every-two-weeks, and once-every-four-weeks administrations were modeled using the said data to determine the once-every-two-weeks ("bi-weekly") administration, as Figure 9 shown. Figure 9 The dashed line (0.08 μg / mL) in represents the estimated concentration of AZD3427 equivalent to the average observed concentration of relaxin in the early pregnancy of pregnant subjects (0.2 ng / mL; in-house assay (see also Example 10)), and this dashed line is used as a reference level. This reference level of 0.08 μg / mL was calculated according to the following formula based on the 40-fold potency difference between AZD3427 and relaxin and the fact that the former has a molecular weight 10 times that of the latter:

[0225] 0.2 * 40 * 10 = 80 ng / mL = 0.08 μg / mL

[0226] As Figure 9 shown, the predicted Cmax / Cmin ratios for once-weekly and once-every-two-week administrations are similar, indicating that both dosing frequencies are suitable for maintaining the Ctrough level above the reference level. Once-every-two-week administration is preferred as it minimizes the number of administrations required to be given to the patient, thus helping to improve patient compliance.

[0227] It is predicted that the steady-state average concentrations (Css, ave) at these three dose levels will cover the sub-therapeutic to supra-therapeutic concentration range corresponding to relaxin levels during pregnancy, specifically 0.5-fold, 2.5-fold, and 15-fold of the relaxin level during pregnancy. This correlation between the AZD3427 level and the relaxin level stems from two differences: firstly, the in vitro potency difference (approximately 40-fold) between serelaxin and AZD3427 based on the cAMP production level in CHO cells expressing recombinant human RXFP1 receptor; secondly, the molecular weight difference between serelaxin and AZD3427 (AZD3427 is 10 times that of serelaxin). The above calculations use the relaxin concentration of 0.2 ng / mL (internal data) during pregnancy as the reference parameter. In addition, using the concentration-response relationships of ejection fraction, stroke volume, and systemic vascular resistance from the NHPHFrEF model, and the PK data from the SAD / MAD study (study number D8330C00001), it is predicted that the dose range selected for the Ph2b study will be able to characterize the dose-response relationships of these parameters ( Figure 10 , Figures 11A to 11C ).

[0228] The primary outcome measure will be the change in pulmonary vascular resistance (PVR) from baseline after 24 weeks of treatment. For participants with combined HF and Group 2 PH, the effect of AZD3427 on PVR parameters after 24 weeks of treatment will also be evaluated by right heart catheterization (RHC) measurement and compared with the placebo group.

[0229] Secondary outcome measures include:

[0230] · The change in mean pulmonary artery pressure (mPAP) from baseline

[0231] · The change in pulmonary artery wedge pressure (PAWP) from baseline

[0232] · The change in cardiac output from baseline

[0233] · The change in stroke volume (SV) from baseline

[0234] · Change in ejection fraction (EF) from baseline

[0235] · Change in left ventricular global longitudinal strain (LVGLS) from baseline

[0236] · Change in pulmonary artery systolic pressure (PASP) from baseline

[0237] · Change in right ventricle / left ventricle (RV / LV) ratio from baseline

[0238] · Change in right ventricular outflow tract acceleration time (RVOT AT) from baseline

[0239] · Change in tricuspid regurgitation velocity (TRV) from baseline

[0240] · Change in TAPSE / PASP [tricuspid annular plane systolic excursion / pulmonary artery systolic pressure] from baseline

[0241] · Change in right ventricular strain / pulmonary artery systolic pressure (RVS / PASP) from baseline

[0242] · Change in diameter of the inspiratory collapsible inferior vena cava (IVC) from baseline

[0243] · Change in systemic vascular resistance from baseline

[0244] · Change in 6-minute walk distance (6MWD) from baseline

[0245] · Change in Kansas City Cardiomyopathy Questionnaire total symptom score (KCCQ TSS) from baseline

[0246] · Change in New York Heart Association functional class (NYHA FC) from baseline

[0247] · Change in serum creatinine from baseline

[0248] · Change in N-terminal pro-brain natriuretic peptide (NT-proBNP) from baseline

[0249] · Change in cystatin C from baseline

[0250] · Change in eGFR (estimated glomerular filtration rate) from baseline

[0251] Inclusion Criteria: 1. The age of the participants must be ≥18 years old (inclusive). 2. The participants must meet the following conditions: having been diagnosed with HF, NYHA functional class (FC) II to IV, and having been diagnosed with PH-LHD according to the "2022 European Society of Cardiology / European Respiratory Society (ESC / ESR) Guidelines for Pulmonary Hypertension due to Left Heart Disease (PH-LHD)", or having a high likelihood of PH-LHD. The participants must be stably using standard HF medications (including diuretics). 3. The combination of echocardiogram parameters of the participants must meet the 2022 ESC / ERS guidelines, showing a moderate or high likelihood of PH. 4. At Screening Visit 2, the participants must perform RHC according to the RHC manual provided by the sponsor, and the study results must meet the following pulmonary artery pressure elevation criteria: (a) PAWP ≥ 15 mmHg; (b) mPAP ≥ 20 mmHg. 5. The minimum weight is 50 kg (inclusive). 6. Able to sign the informed consent form.

[0252] Exclusion Criteria: 1. Meeting the diagnostic criteria for Group 1, Group 3, Group 4, or Group 5 PH of the World Health Organization (WHO). 2. History or current evidence of clinically significant diseases or disorders. 3. Decompensated HF or any hospitalization. 4. Any contraindications to RHC. 5. Having a history of allergy to subcutaneous injection or related devices. 6. Having a history of allergy to any component of AZD3427 or drugs with a similar chemical structure or class, or having a current clinically significant allergy / hypersensitivity reaction to AZD3427. 7. Diagnosed with pulmonary disease and forced expiratory volume in one second / forced vital capacity (FEV1 / VC) < 30%. 8. Congenital long QT syndrome. 9. Cardiac ventricular arrhythmias requiring treatment. Participants with atrial fibrillation or atrial flutter with a controlled ventricular rate are allowed to be included. 10. Having previously undergone heart transplantation or implantation of a ventricular assist device, or planning to undergo such surgery. 11. Having any of the following known planned (scheduled) highly traumatic cardiovascular (CV) surgeries (e.g., coronary revascularization, atrial fibrillation / atrial flutter ablation, valve repair / replacement, aortic aneurysm surgery, etc.). 12. Participants who have previously received AZD3427.

[0253] Without being limited by theory, AZD3427 is expected to have vasodilatory, anti-inflammatory, and anti-fibrotic effects, and is expected to improve left ventricular function and prevent and / or reverse the remodeling of the diseased heart and pulmonary vascular system, which should in turn reduce PAP and PVR and improve cardiovascular outcomes.

[0254] Example 8: Final Phase 1 AZD3427 MAD study outcomes in HF patients

[0255] The Phase 1 data described in the above embodiments and the modeling derived therefrom were based on an interim dataset. The following Examples 8 and 9 are based on the finalized dataset available from the Phase 1 clinical trial, which has been verified and validated, and may include additional patient data in the analysis. The analysis of the final dataset of the Part B MAD cohort combining HFpEF and HFrEF patients fully supports the conclusions drawn from the interim data, particularly the trends observed in AZD3427 improving cardiac function (including improving cardiac output and stroke volume (SV)), reducing systemic vascular resistance (SVR), and improving organ perfusion (SVR and eGFR) compared to placebo ( Figures 12A to 12F ). Additional analyses were performed, this time dividing the patients into the following two groups: (i) patients with a baseline EF ≤ 40% and (ii) patients with a baseline EF > 40%. The results showed that the trends in improved (increased) stroke volume and eGFR observed in the pooled patients after treatment with AZD3427 were also observed in both groups: (i) patients with a baseline EF ≤ 40% and (ii) patients with a baseline EF > 40% (see Figures 12H to 12I ). There was no significant effect on blood pressure ( Figure 12G ). Administration of AZD3427 also caused a dose-dependent increase in plasma renin levels ( Figure 13A ). For the test doses below 45 mg, the resulting hemoglobin and hematocrit levels were within the acceptable safety range. ( Figure 13B , Figure 13C ). AZD3427 doses from 1 mg to 30 mg seemed to be able to maintain hemoglobin and hematocrit levels within the acceptable safety limits.

[0256] Example 9: Phase 2B (Ph2b) AZD3427 dosing regimen

[0257] The Ph2b study discussed in Example 7 (design shown in Figure 8 ; study ID number: D8330C00003) will be conducted to evaluate AZD3427. Based on the NHP HFrEF model and the PK data from the SAD / MAD study (study number D8330C00001) ( Figure 14 ), the dose range selected in the Ph2b study will be able to characterize the dose-response relationship of ejection fraction, stroke volume, renin, and systemic vascular resistance. This dose-response relationship will also characterize hematocrit, hemoglobin, and albumin levels ( Figures 15A to 15C ). Evaluate the primary and secondary outcomes described in Example 7. The inclusion and exclusion criteria in Example 7 also apply.

[0258] Example 10: Relaxin-2 detection assay

[0259] The relaxin-2 capture antibody (MAB2804; R&D Systems, USA) was used as the coating antibody. The relaxin-2 biotinylated antibody (R&D Systems, USA) was used as the detection antibody. The 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 made by diluting the standard from 36000 fg / mL to 10 fg / mL.

[0260] The single molecule array (Simoa TM ) technology was used according to the following steps: 0.1 mg / mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Thermo Fisher Scientific, USA) was added to the bead solution with a concentration of 1.4×10 6 homemade magnetic carboxylated beads (Quanterix, USA) / μL to activate the beads. After incubation at room temperature (RT) for 30 minutes (min), the beads were washed in a magnetic separator and then 0.3 mg / mL of ice-precooled capture antibody was added. The beads were placed on an oscillator (HulaMixer, Invitrogen, USA) and incubated at 4 °C for 2 hours (h). Then the beads were 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 later use. Human plasma samples and the relaxin-2 standard (370 μL) were detected in duplicate on a Simoa HD-X analyzer (Quanterix, Lexington, MA). The samples and standards were plated on a 96-well plate (NUNC, Thermo Fisher Scientific, USA). The conjugated beads were washed twice in the bead dilution buffer and then resuspended in the corresponding volume of bead diluent. An IgG blocker (MSD blocker D-M, Mesoscale Discovery, USA) was added to the samples at a ratio of 1:10 of the total volume. The detection antibody was diluted to a final concentration of 0.25 μg / mL in the assay diluent, and the enzyme SBG (streptavidin-β-galactosidase, Quanterix, USA) was diluted to a final concentration of 150 pM in the SBG diluent. The reagents, samples, and calibrators were all run on the HD-X analyzer using the two-step assay Neat 2.0 protocol, and 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) were added in sequence.

[0261] Table 3: Sequence listing. The upper hinge region is in italics, relaxin A is underlined, relaxin B is doubly underlined, and the Fc region is in bold.

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

Claims

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

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

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

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

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

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

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

8. The method according to any one of claims 1 to 7, wherein the subject has one or more of the following: (a) New York Heart Association (NYHA) class II-IV cardiac function; (b) mean pulmonary artery pressure (mPAP) greater than 20 mmHg; and (c) pulmonary artery wedge pressure (PAWP) greater than 15 mmHg.

9. The method according to any one of claims 1 to 8, wherein the subject's endogenous relaxin plasma level is about one ten-thousandth of the relaxin plasma level in a pregnant subject in the first trimester of pregnancy.

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

11. The method according to any one of claims 1 to 10, wherein administering the pharmaceutical composition is sufficient to cause the plasma concentration of the heterodimeric fusion protein in the subject to reach 0.026 μg / mL to 1.6 μg / mL.

12. The method according to any one of claims 1 to 11, wherein administering the pharmaceutical composition is sufficient to cause at least a 2-fold dose-dependent increase in renin production compared to the baseline level.

13. The method according to any one of claims 1 to 12, wherein, compared with the baseline level, after administration of the pharmaceutical composition, the hematocrit level in the subject is reduced by no more than 5% to 7%, such as no more than 5% or no more than 7%.

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

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

16. The method according to any one of claims 1 to 15, wherein, compared with the baseline blood pressure (such as systolic blood pressure), after administration of the pharmaceutical composition, the blood pressure (such as systolic blood pressure) of the subject does not change significantly.

17. The method according to any one of claims 1 to 16, wherein, compared with the baseline level, administration of the pharmaceutical composition is sufficient to cause one or more of the following: (a) A decrease in pulmonary vascular resistance (PVR); (b) A decrease in mean pulmonary artery pressure (mPAP); (c) A decrease in estimated pulmonary artery diastolic pressure (ePAD); (d) An increase in 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) An increase in cardiac output.

18. The method according to any one of claims 1 to 17, wherein, compared with the baseline level, administration of the pharmaceutical composition at an optional bi-weekly dose of about 1 mg or about 1.1 mg is sufficient to cause an increase in the stroke volume (SV) of the subject by at least about 1%.

19. The method according to any one of claims 1 to 18, wherein, compared with the baseline level, administration of the pharmaceutical composition at an optional bi-weekly dose of about 5.4 mg is sufficient to cause an increase in the SV of the subject by at least about 5% to 25%.

20. The method according to any one of claims 1 to 19, wherein, compared with the baseline level, administration of the pharmaceutical composition at an optional bi-weekly dose of about 30 mg is sufficient to cause an increase in the SV of the subject by at least about 10% to 50%.

21. The method according to any one of claims 1 to 20, wherein, compared with the baseline level, administration of the pharmaceutical composition at an optional bi-weekly dose of about 1 mg or about 1.1 mg is sufficient to cause a decrease in the systemic vascular resistance (SVR) of the subject by at least about 8% to 20%.

22. The method according to any one of claims 1 to 21, wherein, compared with the baseline level, administration of the pharmaceutical composition at an optional bi-weekly dose of about 5.4 mg is sufficient to cause a decrease in the SVR of the subject by at least about 15% to 30%.

23. The method according to any one of claims 1 to 22, wherein, compared with the baseline level, administration of the pharmaceutical composition at an optional bi-weekly dose of about 30 mg is sufficient to cause a decrease in the SVR of the subject by at least about 20% to 35%.

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

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

26. The method according to 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: (i) are covalently bound by at least one interchain disulfide bond; and / or (ii) are not covalently linked to each other by an amino acid linker.

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

2.

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

30. The method according to any one of claims 26 to 29, wherein the length of one linker or optionally both linkers is between 6 and 40 amino acids, optionally 21 amino acids in length.

31. The method according to claim 30, wherein one linker or optionally both linkers are G4S / G5S amino acid linkers.

32. The method according to claim 30 or claim 31, wherein one linker or optionally both linkers have the amino acid sequence SEQ ID NO:

5.

33. The method according to 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, wherein the first Fc region and the second Fc region comprise the constant domains CH2 and CH3, optionally the CH2 domain and CH3 domain from an IgG1 immunoglobulin, further optionally wherein the CH2 domain and / or the CH3 domain are mutated.

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

35. The method according to claim 33 or claim 34, wherein the first Fc region and the second Fc region comprise amino acid mutations that promote heterodimerization, optionally wherein the amino acid mutations that promote heterodimerization are present in the CH3 domain.

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

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

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

3.

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

20.

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

5.

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

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

43. The method according to any one of claims 1 to 41, wherein the heterodimeric fusion protein is AZD3427, and is administered subcutaneously to the subject once every two weeks at a dose of about 5.4 mg.

44. The method according to any one of claims 1 to 41, wherein the heterodimeric fusion protein is AZD3427, and is administered subcutaneously to the subject once every two weeks at a dose of about 30 mg.

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

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

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

48. The method according to any one of claims 45 to 47, wherein the pharmaceutical composition is administered to the subject once every two weeks.

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

50. The method according to any one of the preceding claims, wherein the subject suffers from heart failure with group 2 pulmonary hypertension.

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