EP4630036A1

EP4630036A1 - Dosing regimens using heterodimeric relaxin fusions

Info

Publication number: EP4630036A1
Application number: EP23821585.9A
Authority: EP
Inventors: Elin MATSSON; Magnus ALTHAGE; Magnus ÅSTRAND; Madeleine ANTONSSON; Marcin UFNAL
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 2022-12-09
Filing date: 2023-12-08
Publication date: 2025-10-15
Also published as: CN120322245A; IL321288A; AU2023389399A1; WO2024121362A1; TW202438534A; JP2025540294A; KR20250120357A

Abstract

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

Description

Dosing Regimens using Heterodimeric Relaxin Fusions

Cross-Reference to Related Applications

This application claims the benefit of priority of U.S. Provisional Patent Application Nos. 63/386,762 filed December 9, 2022, 63/387,359 filed December 14, 2022, and 63/497,169 filed April 19, 2023 the contents of each of which is incorporated herein by reference.

Field of the Disclosure

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

Background

Heart failure is associated with significant morbidity and mortality. It is characterized by complex tissue remodelling involving increased cardiomyocyte death and interstitial fibrosis. A significant number of heart failure patients suffer from pulmonary hypertension. It has been estimated that approximately 50% of heart failure patients with preserved ejection fraction (HFpEF) also suffer from pulmonary hypertension (PH), and that approximately 60% of heart failure patients with reduced ejection fraction (HFrEF) also suffer from PH (Guazzi, (2014) Circ Heart Fail., 7:367-377; Miller et al., (2013) JAGG Heart Fail., 1 (4):290-299). Patients suffering from heart failure with pulmonary hypertension (HF+PH) have been shown to have reduced survival as compared with heart failure patients without PH (Barnett and De Marco, (2012) Heart Fail. Clin. 8: 447-459). In heart failure patients, a 3 mmHg increase or decrease in Estimated Pulmonary Artery Diastolic Pressure (ePAD), equivalent to an approximately 4 mmHg increase or decrease in mean Pulmonary Arterial Pressure (mPAP), was associated with a 24% increase or a 19% decrease in cardiovascular mortality respectively (Zile MR, et al. (2017) Circ Heart Fail., 10:e003594). A 4 mmHg reduction in mPAP is also associated with dyspnea improvement in patients suffering from HF+PH (Solomonica A, et al. (2013) Circ Heart Fail., 6:53-60).

Pulmonary hypertension (PH) Group 2 results from dysfunction of the left heart and is the most common cause of PH. Left-sided dysfunction causes pulmonary pressure to rise, leading to lung oedema, damage of pulmonary vessels, and ultimately right ventricle failure. PH Group 2 is comprised mostly of HF New York Heart Association (NYHA) class III and IV patients (HF patients that have limited physical activity and experience fatigue, palpitation, or dyspnea), with 40% to 75% of HF patients with HFrEF and 36% to 83% of HF patients with heart failure HFpEF also having PH. PH Group 2 is associated with impaired exercise capacity and reduced survival. PH Group 2 patients live with significantly limited physical activity and quality of life despite optimal guideline-directed management. Currently, there is no dedicated treatment for this group of patients.

Relaxin is a peptide hormone that belongs to the insulin superfamily. In humans, the Relaxin peptide family includes seven peptides of high structural but low sequence similarity: Relaxin 1 , 2 and 3, and the insulin-like peptides INSL3, INSL4, INSL5 and INSL6. Naturally occurring Relaxins consist of A and B polypeptide chains covalently linked by two inter-chain disulphide bonds. The A chain has an additional intra-chain disulphide bond. The Relaxin genes encode prohormones with structure B-C-A (B and A polypeptide chains linked by a C peptide). The prohormone undergoes endoproteolytic cleavage with PC1 and PC2 enzymes to remove the C peptide before secretion of mature Relaxin.

Without being limited by theory, Relaxin is understood to be a pleiotropic hormone that mediates systemic haemodynamic and renal adaptive changes during pregnancy. In pregnancy, Relaxin mediates systemic hemodynamic and renal adaptive changes to meet the increased metabolic demands of gestation without causing cardiac damage and has a unique ability to reduce both systemic vascular resistance and renal resistance concomitantly (Conrad 2011). Without being bound by theory, if Relaxin were to replicate these hemodynamic adaptations in the setting of heart failure, it could reduce myocardial demand and improve end-organ perfusion. Relaxin has also been shown to have anti- fibrotic properties and to have beneficial effects in heart failure including acute decompensated heart failure (ADHF). Relaxin activates a number of signalling cascades that have been shown to be beneficial in the setting of ischemia-reperfusion and heart failure. These signalling pathways include activation of the phosphoinositide 3-kinase pathway and activation of the nitric oxide signalling 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).

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 damage 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 yielded improvements in pulmonary artery pressures, cardiac output, and systemic and pulmonary vascular resistance, however, these results required an approximate 20-hour continuous infusion at 30 pg/kg/day (Ponikowski et al., (2014) European Heart Journal 35:431-441). Due to the rapid clearance of serelaxin from the patients' circulation, its therapeutic effects were limited, and the therapeutic effects rapidly disappeared once intravenous infusion stopped. Additionally, approximately one third of the patients experienced a significant drop in blood pressure (> 40 mm Hg) after receiving serelaxin intravenously, with the consequence that the infusion rate had to be reduced by half or even more.

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

By contrast, WO 2021/255127, incorporated herein by reference in its entirety, describes heterodimeric fusions that comprise Relaxin chain polypeptides and heterodimerization domains and that exhibit Relaxin activity without requiring fusion of the Relaxin A chain polypeptide and the Relaxin B chain polypeptide in a single chain. WO 2021255127 describes that heterodimerisation of the heterodimerisation domains induces correct folding and heterodimerisation of the Relaxin A and Relaxin B chain polypeptides. In addition, unlike wild-type Relaxin proteins, the fusions of WO 2021255127 do not require endoproteolytic processing for biological activity, and exhibit an extended half-life.

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

Summary

Disclosed herein is, in part, low dose and/or low frequency dosing regimens for heart failure subjects, optionally HF+PH subjects, using a heterodimeric Relaxin fusion protein. The dosing regimen provided herein may improve quality of life for patients while retaining efficacy and minimizing side effects. In one aspect, the present disclosure provides a method of treating a subject having heart failure, optionally a subject having heart failure with pulmonary hypertension, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable excipient and about 1 mg to about 30 mg of a heterodimeric fusion; wherein the heterodimeric fusion comprises:

(i) a first heterodimerisation domain connected to at least one Relaxin A chain polypeptide or a variant thereof; and

(ii) a second heterodimerisation domain connected to at least one Relaxin B chain polypeptide or a variant thereof, wherein the first heterodimerisation domain heterodimerises with the second heterodimerisation domain, and wherein the heterodimeric fusion has Relaxin activity.

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

In some embodiments, the method comprises administration of the heterodimeric fusion to the subject once every two weeks (also termed herein as “biweekly” administration). In some embodiments, the method comprises biweekly administration and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg. In some embodiments, the method comprises biweekly administration and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1 mg. In some embodiments, the method comprises biweekly administration and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1.1 mg. In some embodiments, the method comprises biweekly administration and the amount of the heterodimeric fusion in the pharmaceutical composition is about 5.4 mg. In some embodiments, the method comprises biweekly administration and the amount of the heterodimeric fusion in the pharmaceutical composition is about 30 mg. 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 heterodimeric fusion in the pharmaceutical composition is about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg. In some embodiments, the method comprises subcutaneous administration and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1 mg. In some embodiments, the method comprises subcutaneous administration and the amount of the heterodimeric fusion in the pharmaceutical composition is about 1.1 mg. In some embodiments, the method comprises subcutaneous administration and the amount of the heterodimeric fusion in the pharmaceutical composition is about 5.4 mg. In some embodiments, the method comprises subcutaneous administration and the amount of the heterodimeric fusion in the pharmaceutical composition is about 30 mg.

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

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) Functional Class ll-IV; mean pulmonary arterial pressure (mPAP) greater than 20 mmHg; and pulmonary artery wedge pressure

(PAWP) greater than 15 mmHg. In some embodiments, the subject meets one or more of the following criteria: chest congestion; dyspnea at rest or with minimal exertion; greater than 125 pg/mL of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) or greater than 35 pg/mL of brain natriuretic peptide (BNP); systolic blood pressure >125 mm Hg; mild to moderate renal insufficiency; body mass index (BMI) of at least 18 kg/m²; and reduced ejection fraction (HFrEF) of less than or equal to 40 percent.

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

In some embodiments, administration of the pharmaceutical composition is sufficient to result in a minimum steady state plasma concentration of the heterodimeric fusion of about or at least 0.026 pg/mL in the subject. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a maximum steady state plasma concentration of the heterodimeric fusion of about or at least 1-2 pg/mL in the subject. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a maximum steady state plasma concentration of the heterodimeric fusion of about or at least 1 .6 pg/mL in the subject. In some embodiments, administration of the pharmaceutical composition is sufficient to result in a plasma concentration of the heterodimeric fusion of 0.026 pg/mL to 1 .6 pg/mL in the subject (i.e. from about 0.026 pg/mL up to, and including, about 1.6 pg/mL, in the subject).

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

In some embodiments, hematocrit levels in the subject are reduced by no more than 1 percent following administration of the pharmaceutical composition compared to baseline (pre-administration) levels, e.g., following administration of a dose of 1-30 mg of the heterodimeric fusion. In some embodiments, hematocrit levels in the subject are reduced by no more than 2 percent following administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, hematocrit levels in the subject are reduced by no more than 3 percent following administration of the pharmaceutical composition compared to baseline (pre- administration) levels. In some embodiments, hematocrit levels in the subject are reduced by no more than 4 percent following administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, hematocrit levels in the subject are reduced by no more than 5 percent following administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, hematocrit levels in the subject are reduced by no more than 6 percent following administration of the pharmaceutical composition compared to baseline (pre- administration) levels. In some embodiments, hematocrit levels in the subject are reduced by no more than 7 percent following administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, hematocrit levels in the subject are reduced by no more than 8 percent following administration of the pharmaceutical composition compared to baseline (pre-administration) levels.

In some embodiments, hemoglobin levels in the subject are reduced by no more than 2 percent following administration of the pharmaceutical composition compared to baseline (pre-administration) levels, e.g., following administration of a dose of 1-30 mg of the heterodimeric fusion. In some embodiments, hemoglobin levels in the subject are reduced by no more than 3 percent following administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, hemoglobin levels in the subject are reduced by no more than 4 percent following administration of the pharmaceutical composition compared to baseline (pre- administration) levels. In some embodiments, hemoglobin levels in the subject are reduced by no more than 5 percent following administration of the pharmaceutical composition compared to baseline (pre-administration) levels. In some embodiments, hemoglobin levels in the subject are reduced by no more than 6 percent following administration of the pharmaceutical composition compared to baseline (pre- administration) levels. In some embodiments, albumin levels are not significantly reduced in the subject following administration of the pharmaceutical composition compared to baseline levels. Administration of the pharmaceutical composition as described herein may be sufficient to result in one or more of:

(a) an increase in stroke volume (SV);

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

(c) an increase in ejection fraction;

(d) an increase in cardiac output; as compared to baseline levels. The change in SV, SVR, eGFR, ejection fraction, and/or cardiac output may each result after 1-24 weeks of treatment. In some embodiments, the change in SV, SVR, eGFR, ejection fraction, and/or cardiac output results after 24 weeks of treatment.

Administration of the pharmaceutical composition as described herein may also be sufficient to result in one or more of:

(a) a reduction in Pulmonary Vascular Resistance (PVR);

(b) a reduction in mean Pulmonary Artery Pressure (mPAP);

(c) a reduction in estimated Pulmonary Artery Diastolic Pressure (ePAD); as compared to baseline levels. The change in PVR, mPAP and/or ePAD may each result after 1-24 weeks of treatment. In some embodiments, the change in PVR, mPAP and/or ePAD results after 24 weeks of treatment.

Thus, administration of the pharmaceutical composition as described herein may be sufficient to result in one or more of: a) a reduction in PVR;

(b) a reduction in mPAP;

(c) a reduction in ePAD;

(d) an increase in stroke volume (SV) of the heart; (e) a reduction in systemic vascular resistance (SVR) and/or an increase in estimated glomerular filtration rate (eGFR);

(f) an increase in ejection fraction;

(g) an increase in cardiac output; as compared to baseline levels. The change in PVR, mPAP, ePAD, SV, SVR, eGFR, ejection fraction, and/or cardiac output may each result after 1-24 weeks of treatment. In some embodiments, the change in PVR, mPAP, ePAD, SV, SVR, eGFR, ejection fraction, and/or cardiac output results after 24 weeks of treatment.

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

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

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

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

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

In some embodiments, the heterodimeric fusion agonizes the Relaxin family peptide receptor 1 (RXFP1). In some embodiments, the at least one Relaxin A chain polypeptide or variant thereof of the heterodimeric fusion and the at least one Relaxin B chain polypeptide or variant thereof of the heterodimeric fusion are covalently bound by at least one inter-chain disulphide bond. In some embodiments, the at least one Relaxin A chain polypeptide or variant thereof of the heterodimeric fusion and the at least one Relaxin B chain polypeptide or variant thereof of the heterodimeric fusion are not covalently linked to each other by an amino acid linker. In some embodiments, the at least one Relaxin A chain polypeptide is a Relaxin-2 A chain polypeptide and/or the at least one Relaxin B chain polypeptide is a Relaxin-2 B chain polypeptide. In some embodiments, 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. In some embodiments, the at least one Relaxin-2 A chain polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and/or the at least one Relaxin-2 B chain polypeptide comprises the amino acids sequence of SEQ ID NO: 2. In some embodiments, the at least one Relaxin-2 A chain polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and the at least one Relaxin-2 B chain polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the at least one Relaxin A chain polypeptide or variant thereof is connected to the first heterodimerisation domain of the heterodimeric fusion via a connector and the at least one Relaxin B chain polypeptide or variant thereof is connected to the second heterodimerisation domain via a connector. In some embodiments, at least one connector is a polypeptide. In some embodiments, both connectors are polypeptides. In some embodiments, at least one connector is a polypeptide having a length of between 6 and 40 amino acids. In some embodiments, both connectors are polypeptides having a length of between 6 and 40 amino acids. In some embodiments, at least one connector is a polypeptide having a length of 21 amino acids. In some embodiments, both connectors are polypeptides having a length of 21 amino acids.

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

In some embodiments, the first heterodimerisation domain is derived from a first immunoglobulin Fc region and the second heterodimerisation domain is derived from a second immunoglobulin Fc region, wherein the first and second Fc regions comprise the constant domains CH2 and CH3. In some embodiments, the CH2 and CH3 domains are from an IgG 1 immunoglobulin. In some embodiments, the CH2 and/or CH3 domains are mutated.

In some embodiments, the C-terminus of the first Fc region is connected to the N- terminus of the at least one Relaxin A chain polypeptide and the C-terminus of the second Fc region is connected to the N-terminus of the at least one Relaxin B chain polypeptide. In some embodiments, the first and second Fc regions comprise heterodimerisation- promoting amino acid amino acid mutations. In some embodiments, the heterodimerisation-promoting amino acid mutations are present in the CH3 domains of the first and second Fc regions.

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

In some embodiments, the heterodimeric fusion comprises the amino acid sequences of SEQ ID NO: 11 (Relaxin-2 A chain polypeptide connected via a connector to the first Fc region) and SEQ ID NO: 20 (Relaxin-2 B chain polypeptide connected via a connector to the second Fc region). In some embodiments, the heterodimeric fusion consists of the amino acid sequences of SEQ ID NO: 11 (Relaxin-2 A chain polypeptide connected via a connector to the first Fc region) and SEQ ID NO: 20 (Relaxin-2 B chain polypeptide connected via a connector to the second Fc region), and may be termed “AZD3427”.

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

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

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

(ii) an FcY-con-B fusion polypeptide, wherein:

A is a Relaxin A chain polypeptide or variant thereof, e.g., a Relaxin-2 A chain polypeptide or variant thereof;

B is a Relaxin B chain polypeptide or variant thereof, e.g., a Relaxin-2 B chain polypeptide or variant thereof;

FcX is an Fc region comprising CH2 and CH3 of a human IgG 1 immunoglobulin and comprises amino acid mutations, optionally S354C and T366W;

FcY is an Fc region comprising CH2 and CH3 of a human IgG 1 immunoglobulin and comprises amino acid mutations, optionally Y349C, T366S, L368A, and Y407V; and con is a connector polypeptide, optionally having the amino acid sequence of SEQ ID NO: 5, wherein the amino acid numbering is according to the EU index as in Kabat, wherein FcX heterodimerises with FcY, and wherein the heterodimeric fusion has Relaxin activity.

In some embodiments, the heterodimeric fusion comprises AZD3427. In some embodiments, the heterodimeric fusion is AZD3427. In some embodiments, the amount of heterodimeric fusion (e.g., AZD3427) in the pharmaceutical composition is about 1 mg to about 30 mg. In some embodiments, the amount of heterodimeric fusion (e.g., AZD3427) in the pharmaceutical composition is about 1 mg. In some embodiments, the amount of heterodimeric fusion (e.g., AZD3427) in the pharmaceutical composition is about 1.1 mg. In some embodiments, the amount of heterodimeric fusion (e.g., AZD3427) in the pharmaceutical composition is about 5.4 mg. In some embodiments, the amount of heterodimeric fusion (e.g., AZD3427) in the pharmaceutical composition is about 30 mg. In some embodiments, the pharmaceutical composition is administered to the subject via biweekly administration, e.g., at a dose of the heterodimeric fusion (e.g., AZD3427) of about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg. In some embodiments, the pharmaceutical composition is administered to the subject via biweekly subcutaneous administration, e.g., at a dose of the heterodimeric fusion (e.g., AZD3427) of about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

In some embodiments, AZD3427 is administered biweekly to the subject subcutaneously at a dose of about 1 mg. In some embodiments, AZD3427 is administered biweekly to the subject subcutaneously at a dose of about 1.1 mg. In some embodiments, AZD3427 is administered biweekly to the subject subcutaneously at a dose of about 5.4 mg. In some embodiments, AZD3427 is administered biweekly to the subject subcutaneously at a dose of about 30 mg.

In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered to the subject chronically. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered to the subject at least four times. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered to the subject at least five times. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered to the subject at least 12 times. Brief Description of the Drawings

FIG. 1 shows exemplary formats of heterodimeric fusions provided herein. The format of each fusion polypeptide of the heterodimeric fusions is given in terms of FcX, FcY, A, B, con and L, wherein FcX and FcY are two Fc regions comprising heterodimerisation-promoting amino acid mutations and/or modifications; A (“Rix A”) and B (“Rix B”) are Relaxin A chain and Relaxin B chain polypeptides; “con” is a connector polypeptide; L is a linker polypeptide, HC X and HC Y denote heavy chains of an antibody, LC denotes a light chain of an antibody.

FIG. 2 shows the pharmacokinetic (PK) profile of an exemplary heterodimeric fusion, AZD3427, in cynomolgus monkeys following intravenous (IV) and subcutaneous (SC) administration. LLOQ = lowest level of quantitation.

FIGs. 3A-3D show cardiac function and systemic vascular resistance in nonhuman primates (NHP) following administration of AZD3427: FIG. 3A shows ejection fraction (EF), FIG. 3B shows cardiac output, FIG. 3C shows systemic vascular resistance (SVR), and FIG. 3D shows plasma renin concentration as a percentage relative to predose. The dotted line in FIG. 3D represents last dose.

FIG. 4 shows a 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 ejection fraction > 41 %. HFrEF = heart failure with reduced ejection fraction. JD = Japanese-descent. F/U = follow-up. IV = intravenous. SC = subcutaneous.

FIG. 5 shows geometric mean serum AZD3427 concentration (pg/mL) over time in hours (h) after a single dose in the Phase 1 SAD Cohorts. SC = subcutaneous.

FIGs. 6A-6I shows interim clinical results for cardiac function, systemic vascular resistance, and organ perfusion in the MAD Cohorts following administration of AZD3427: FIG. 6A shows ejection fraction (EF) in HFpEF subjects, FIG. 6B shows EF in HFrEF subjects, FIG. 6C shows cardiac output in pooled subjects, FIG. 6D shows systemic vascular resistance (SVR) in pooled subjects, FIG. 6E shows stroke volume (SV) in pooled subjects, FIG. 6F shows estimated glomerular filtration rate (eGFR) in pooled subjects, FIG. 6G shows systolic blood pressure (SBP) in (i) patients with EF < 40% and (ii) patients with EF > 40%, FIG. 6H shows stroke volume in (i) patients with EF < 40% and (ii) patients with EF > 40%, and FIG. 61 shows eGFR in (i) patients with EF < 40% and (ii) patients with EF > 40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant (p<0.1) compared to placebo. SE = standard error.

FIGs. 7A-7C shows interim clinical results for plasma renin, hematocrit, and hemoglobin levels in the MAD Cohorts following administration of AZD3427 administration in the MAD subjects: FIG. 7A shows percentage of renin, FIG. 7B shows percentage of hemoglobin, FIG. 7C shows percentage of hematocrit. Observed data are shown as geomean with 90% confidence interval and the solid line represents a fitted Emax dose response model (FIG. 7B and FIG. 7C).

FIG. 8 shows a phase 2b study design.

FIG. 9 shows a dosing comparison for AZD3427. QW = weekly (QW). Q2W = biweekly. Q4W = every four weeks. Shaded regions show 90 percent prediction intervals. Dashed line (0.08 pg/mL) represents the estimated concentration of AZD3427 that is equivalent to a reference pregnancy level of relaxin in the first trimester (0.2 ng/mL; inhouse assay).

FIG. 10 shows the dose-response for cardiac function and hemodynamic levels following biweekly AZD3427 administration utilizing the concentration-response relationship from the NHP HFrEF model and human PK data based on the interim clinical data. FIGs. 11A-11C show interim clinical data for hematocrit, hemoglobin, and albumin levels following AZD3427 administration: FIG. 11A shows percentage change from baseline of hematocrit, FIG. 11B shows percentage change from baseline of hemoglobin, FIG. 11C shows percentage change from baseline of albumin. Observed data for MAD subjects are shown as geomean with 90% confidence interval, the solid line represents a fitted Emax dose response model.

FIGs. 12A-12I shows finalised clinical data for cardiac function, systemic vascular resistance, and organ perfusion in the MAD Cohorts following administration of AZD3427: FIG. 12A shows ejection fraction (EF) in HFpEF subjects, FIG. 12B shows EF in HFrEF subjects, FIG. 12C shows cardiac output in pooled subjects, FIG. 12D shows systemic vascular resistance (SVR) in pooled subjects, FIG. 12E shows stroke volume (SV) in pooled subjects, FIG. 12F shows estimated glomerular filtration rate (eGFR) in pooled subjects, FIG. 12G shows systolic blood pressure (SBP) in (i) patients with EF < 40% and (ii) patients with EF > 40%, FIG. 12H shows stroke volume in (i) patients with EF < 40% and (ii) patients with EF > 40%, and FIG. 121 shows eGFR in (i) patients with EF < 40% and (ii) patients with EF > 40%. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant (p<0.1) compared to placebo. SE = standard error.

FIGs. 13A-13C shows finalised clinical data for plasma renin, hematocrit, and hemoglobin levels in the MAD Cohorts following administration of AZD3427 administration in the MAD subjects: FIG. 13A shows percentage of renin, FIG. 13B shows percentage of hemoglobin, FIG. 13C shows percentage of hematocrit. Observed data are shown as geomean with 90% confidence interval and the solid line represents a fitted Emax dose response model (FIG. 13B and FIG. 13C).

FIG. 14 shows the dose-response for cardiac function and hemodynamic levels following biweekly AZD3427 administration utilizing the concentration-response relationship from the NHP HFrEF model and human PK data based on the finalised clinical data.

FIGs. 15A-15C show finalised clinical data for hematocrit, hemoglobin, and albumin levels following AZD3427 administration: FIG. 15A shows percentage change from baseline of hematocrit, FIG. 15B shows percentage change from baseline of hemoglobin, FIG. 15C shows percentage change from baseline of albumin. Observed data for MAD subjects are shown as geomean with 90% confidence interval, the solid line represents a fitted Emax dose response model.

Detailed Description

Disclosed herein are methods involving low dose and/or low frequency of administration of a heterodimeric Relaxin fusion to treat heart failure subjects, optionally HF+PH subjects.

Heterodimeric Relaxin Fusions

As used herein, “heterodimeric fusion” (also referred to as a “heterodimeric fusion protein”) refers to a heterodimer of at least first and second fusion polypeptides, wherein the first fusion polypeptide comprises a first heterodimerisation domain connected to a first subunit of a heterodimeric protein (e.g., a Relaxin A chain polypeptide or a variant thereof), and the second fusion polypeptide comprises a second heterodimerisation domain connected to a second subunit of a heterodimeric protein (e.g., a Relaxin B chain polypeptide or a variant thereof).

In some embodiments, the heterodimeric fusions of the disclosure comprise a first heterodimerisation domain connected to at least one Relaxin A chain polypeptide or a variant thereof; and a second heterodimerisation domain connected to at least one Relaxin B chain polypeptide or a variant thereof, wherein the first heterodimerisation domain heterodimerises with the second heterodimerisation domain, and wherein the heterodimeric fusion has Relaxin activity. In some embodiments, the at least one Relaxin A chain polypeptide or variant thereof is connected to the first heterodimerisation domain via a connector, e.g., a connector polypeptide. In some embodiments, the at least one Relaxin B chain polypeptide or variant thereof is connected to the first heterodimerisation domain via a connector, e.g., a connector polypeptide.

Relaxin Chain Polypeptides

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

As used herein, a “variant” of a Relaxin chain polypeptide differs from a wild-type Relaxin chain polypeptide while retaining Relaxin activity.

In some embodiments, a variant of a Relaxin A or Relaxin B chain polypeptide that retains Relaxin activity contains at least one conserved motif associated with Relaxin activity. In some embodiments, a Relaxin-2 B chain polypeptide variant comprises the conserved motif Arg-X-X-X-Arg-X-X-lle (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).

A variant may comprise one or more amino acid substitutions and/or insertions. In some embodiments, a Relaxin-2 A chain polypeptide variant comprises 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, a Relaxin-2 A chain polypeptide variant comprises the amino acid substitution K9H. In some embodiments, a Relaxin-2 B chain polypeptide variant comprises one or more additional amino acids, for example K30 and R31 and N-terminal V-2, A-1 and M-1 , compared to

SEQ ID NO: 62.

In some embodiments, a variant comprises one or more amino acid derivatives. In some embodiments, the first amino acid of a Relaxin-2 B chain polypeptide variant is pyroglutamate.

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

In some embodiments, the Relaxin chain polypeptides of the heterodimeric fusion agonize the Relaxin family peptide receptor 1 (RXFP1). In some embodiments, the Relaxin chain polypeptides are the Relaxin chain polypeptides of AZD3427.

Relaxin Activity

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

The heterodimeric fusions of the disclosure may be determined to have Relaxin activity if they show at least a proportion of the activity of a reference Relaxin protein. For example, a heterodimeric fusion of the disclosure may have Relaxin activity if the ratio of the activity of the heterodimeric fusion over 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 10'² 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 and about 1. In some embodiments, a heterodimeric fusion of the disclosure may have Relaxin activity if the ratio of the activity of the heterodimeric fusion over the activity of a reference Relaxin protein is between about 1 and about 10⁵, between about 1 and about 10⁴, between about 1 and about 10³, between about 1 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. In some embodiments, the heterodimeric fusion has at least half the activity of the reference Relaxin protein. In some embodiments, the heterodimeric fusion has at least three quarters the activity of the reference Relaxin protein. In some embodiments, the heterodimeric fusion has at least the same level of activity as the reference protein.

In some embodiments, the reference Relaxin protein is a wild-type protein. In some embodiments, the reference Relaxin protein is a recombinant protein. In some embodiments, the reference Relaxin protein is a Relaxin protein having the Relaxin A chain and Relaxin B chain array of a mature Relaxin protein. Recombinant Relaxins having the Relaxin A chain and Relaxin B chain array of a mature Relaxin protein are commercially available. For example, recombinant human Relaxin-2, murine Relaxin-1 and INSL3 are available from R&D systems (catalogue numbers 6586-RN, 6637-RN and 4544-NS, respectively). In some embodiments, the reference Relaxin protein has the same Relaxin A and B chains as the heterodimeric fusion of the disclosure or differs from the Relaxin A and B chains of the heterodimeric fusion of the disclosure by up to 10 amino acids, for example by 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 from the first position of the Relaxin B chain of the heterodimeric fusion. In some embodiments, the reference Relaxin protein is a Relaxin-2 protein having the Relaxin-2 A chain and Relaxin-

2 B chain array of a mature Relaxin-2 protein as disclosed under UniProtKB/Swiss-Prot Accession Number P04090.1. Relaxin activity may be determined by measuring binding of a Relaxin to a Relaxin receptor and/or by measuring downstream events from binding to a Relaxin receptor. In some embodiments, Relaxin activity is determined by measuring the amount and/or presence of a molecule downstream from Relaxin activation of a receptor. In some embodiments, Relaxin activity is determined by measuring cAMP production following Relaxin activation of a receptor. Methods for the detection of Relaxin- induced cAMP generation are known in the art. Such methods include cAMP ELISA, HTRF cAMP assays, and the HitHunterOcAMP assay. In some embodiments, Relaxin activity is determined by measuring Relaxin-induced cAMP production in a HTRF cAMP assay. In some embodiments, Relaxin activity is determined by measuring nitric oxide (NO) production following Relaxin activation of a receptor.

In some embodiments, Relaxin activity is determined by measuring the activation of a molecule downstream from Relaxin activation of a receptor. In some embodiments, Relaxin activity is determined by measuring activation of p42/44 MAPK.

In some embodiments, Relaxin activity is determined by measuring the activation of a Relaxin target gene. In some embodiments, Relaxin activity is determined by measuring the activation of the transcription of vascular endothelial growth factor (VEGF), e.g., in THP-1 cells. Methods to determine activation of transcription of a gene are known in the art and include quantitative PCR analysis of mRNA. In some embodiments, relative expression of VEGF mRNA is measured by quantitative real-time PCR induction of VEGF transcripts following incubation of THP-1 cells with Relaxin, e.g., as described in Xiao et al. (2013) Nat Commun. 4: 1953.

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

Heterodimerisation Domains

The heterodimeric fusions of the disclosure comprise a first heterodimerisation domain and a second heterodimerisation domain. In some embodiments, the first and second heterodimerisation domains are derived from an immunoglobulin Fc region. The first and second Fc regions comprise the immunoglobulin domains CH2 and CH3. In some embodiments, either or both of the first and second Fc regions further comprise a CH4 domain. Fc regions provided herein may be derived from an immunoglobulin (e.g., IgG) from any species, optionally human (e.g., human IgG). In embodiments in which the Fc region is derived from IgG, the Fc region may be derived from an IgG of any subclass (e.g., I gG 1 , lgG2, I gG3, lgG4). In some embodiments, the first and second Fc regions are derived from lgG1. In some embodiments, the first and second Fc regions are derived from a human lgG1 immunoglobulin. In some embodiments, the first and second Fc regions are derived from lgG4. In some embodiments, the first and second Fc regions are derived from a human lgG4 immunoglobulin.

In some embodiments, the first and second Fc regions comprise heterodimerisation-promoting amino acid mutations. In some embodiments, the mutations comprise asymmetric complementary modification of the first and second Fc regions (e.g., generating Fc knob and Fc hole structures), such that both chains are compatible with each other and thus able to form a heterodimer, but each chain is not able to dimerize with itself. Such modifications may encompass insertions, deletions, conservative substitutions, non-conservative substitutions, and rearrangements.

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

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

Table 1 : Exemplary Combinations of FcX and FcY mutations

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

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

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

In some embodiments, a cysteine at position 354 of the first Fc region (e.g., of SEQ ID NO: 4) and a cysteine at position 349 of the second Fc region (e.g., of SEQ ID NO: 3) form a stabilising disulphide bond.

Connectors

One or both of the Relaxin A and B chain polypeptides may be connected to their respective heterodimerisation domains by a connector polypeptide. In some embodiments, the Relaxin A chain polypeptide is connected to the first heterodimerisation domain (e.g. first Fc region) via a connector polypeptide, and the Relaxin B chain polypeptide is connected to the second heterodimerisation domain (e.g. second Fc region) via a connector polypeptide. In some embodiments, the heterodimeric fusion further comprises a second

Relaxin A chain polypeptide or variant thereof connected to the N-terminus of the first Fc region and a second Relaxin B chain polypeptide or variant thereof connected to the N- terminus of the second Fc region. In some embodiments, the second Relaxin A chain is connected to the first Fc region via a connector polypeptide.

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

Connector polypeptides of different or the same amino acid compositions can be used for each arm of the heterodimeric fusions of the disclosure.

In some embodiments, one or optionally both connector 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 connector polypeptides comprise the motif (GGGGS)n (SEQ ID NO: 69), wherein n may be between 1 and 8, for instance wherein n is 4. In some embodiments, one or more connector polypeptide consists of the 10-amino acid sequence GGGSGGGSGG (SEQ ID NO: 60). In some embodiments, one or more connector polypeptide consists of the 21 -amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5). In some embodiments, both connector polypeptides (i.e., the connector polypeptides connecting the Relaxin A chain polypeptide with the first Fc region and connecting the Relaxin B chain polypeptide with the second Fc region) consist of the 21 -amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5).

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

Exemplary Heterodimeric Fusions

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

In some embodiments, the heterodimeric fusion comprises the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 20, wherein SEQ ID NO: 11 is the amino acid sequence of the Relaxin A chain polypeptide connected by a connector polypeptide to the first Fc region and SEQ ID NO: 20 is the amino acid sequence of the Relaxin B chain polypeptide connected by a connector polypeptide to the second Fc region, wherein the first and second Fc regions heterodimerise and the Relaxin A and Relaxin B chain polypeptides heterodimerise. In some embodiments, the heterodimeric fusion comprises a stabilising disulphide bond between a cysteine at position 349 of the second Fc region and a cysteine at position 354 of the first Fc region, wherein position numbering is according to the EU index as in Kabat. The exemplary heterodimeric fusion AZD3427 consists of the fusion Relaxin A- connector-Fc region of amino acid sequence SEQ ID NO: 11 heterodimerised with the fusion Relaxin B-connector-Fc region of amino acid sequence SEQ ID NO: 20. In some embodiments, the heterodimeric fusion comprises AZD3427. In some embodiments, the heterodimeric fusion is AZD3427. Sequences used in AZD3427 appear in Table 2.

Table 2. Sequences used in AZD3427. The upper hinge region is in Italics, Relaxin A is underlined, Relaxin B is double underlined, the Fc region is bold.

Pharmaceutical Compositions

The heterodimeric fusion of the disclosure may be provided in a pharmaceutical composition.

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

Kits

A kit comprising a pharmaceutical composition of the disclosure may be provided. The kit may comprise a package containing a pharmaceutical composition of the disclosure and instructions. In some embodiments, the pharmaceutical composition of the disclosure is formulated in single dose vials or a container closure system (e.g., pre-filled syringe). Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Dosing and Therapeutic Methods

The present disclosure provides methods of treating a subject with heart failure, optionally heart failure with pulmonary hypertension, by administering to the subject a heterodimeric Relaxin fusion or a pharmaceutical composition comprising a heterodimeric Relaxin fusion as provided herein. It is to be understood that any method of treatment disclosed herein also provides disclosure of a corresponding use of the heterodimeric Relaxin fusion or a pharmaceutical composition comprising the heterodimeric Relaxin fusion for treating a subject with heart failure, optionally heart failure with pulmonary hypertension. Any method of treatment features described herein should be understood to be disclosed mutatis mutandis in the context of use of the heterodimeric Relaxin fusion or a pharmaceutical composition comprising the heterodimeric Relaxin fusion for treating a subject with heart failure, optionally heart failure with pulmonary hypertension. It is also to be understood that any method of treatment disclosed herein also provides disclosure of a corresponding use of a heterodimeric Relaxin fusion or a pharmaceutical composition comprising a heterodimeric Relaxin fusion in the manufacture of a medicament for treating a subject with heart failure, optionally heart failure with pulmonary hypertension. Any method of treatment features described herein should be understood to be disclosed mutatis mutandis in the context of use of the heterodimeric Relaxin fusion or a pharmaceutical composition comprising the heterodimeric Relaxin fusion in the manufacture of a medicament for treating a subject with heart failure, optionally heart failure with pulmonary hypertension. It is also to be understood that any method of treatment disclosed herein also provides corresponding disclosure of a heterodimeric

Relaxin fusion or a pharmaceutical composition comprising a heterodimeric Relaxin fusion for use in treating a subject with heart failure, optionally heart failure with pulmonary hypertension. Any method of treatment features described herein should be understood to be disclosed mutatis mutandis in the context of the heterodimeric Relaxin fusion or a pharmaceutical composition comprising the heterodimeric Relaxin fusion for use in treating a subject with heart failure, optionally heart failure with pulmonary hypertension. The subject treated with the heterodimeric Relaxin fusion, or a pharmaceutical composition may be an animal, optionally a mammal, optionally a human.

In some embodiments, the subject meets one or more of the following criteria: New York Heart Association (NYHA) Functional Class ll-IV; mean pulmonary arterial pressure (mPAP) greater than 20 mmHg; and pulmonary artery wedge pressure (PAWP) greater than 15 mmHg. In some embodiments, the subject meets one or more of the following criteria: chest congestion; dyspnea at rest or with minimal exertion; greater than 125 pg/mL of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) or greater than 35 pg/mL of brain natriuretic peptide (BNP); systolic blood pressure >125 mm Hg; mild to moderate renal insufficiency; body mass index (BMI) of at least 18 kg/m²; and reduced ejection fraction (HFrEF) of less than or equal to 40 percent.

As used herein, a subject with “pulmonary hypertension” is a subject with a mean Pulmonary Arterial Pressure of at least 20 mmHg. In some embodiments, the pulmonary hypertension is classified as Group 2 pulmonary hypertension, as defined by the World Health Organisation. This may also be termed as “Heart Failure with Pulmonary Hypertension due to Left Heart Disease.” In some embodiments, the pulmonary hypertension is classified as Group 1 pulmonary arterial hypertension, as defined by the World Health Organisation (see Ryan et al., 2012, Pulm. Circ. 2(1):107-121 ).

Parameters of pulmonary hypertension and heart failure may be measured or estimated using techniques known in the art. For instance, these include echocardiography, pulmonary artery catheter and implantable monitoring device. In certain embodiments, the subject may have been fitted with a blood pressure monitoring device, optionally a pulmonary artery pressure monitoring device, as are known in the art.

In some embodiments, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. Typically, the device is fitted prior to treatment with a heterodimeric fusion of the disclosure as provided herein. The subject is fitted with the device during or after the period of treatment.

The heterodimeric Relaxin fusion or pharmaceutical composition of the disclosure may be administered by injection, such as by intravenous, subcutaneous, or intramuscular injection, to a subject. In some embodiments, the heterodimeric fusion or pharmaceutical composition is administered subcutaneously. In some embodiments, the heterodimeric fusion is administered biweekly.

In some embodiments, the heterodimeric Relaxin fusion administered to the subject is administered to the subject chronically. In some embodiments, the heterodimeric fusion is administered to the subject at least four times. In some embodiments, the heterodimeric fusion is administered to the subject at least five times. In some embodiments, the heterodimeric fusion is administered to the subject at least 12 times. In some embodiments, the heterodimeric fusion is administered biweekly to the subject subcutaneously. In some embodiments, the heterodimeric fusion is administered biweekly to the subject subcutaneously at, e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

In some embodiments, the heterodimeric fusion consists of the fusion Relaxin A- connector-Fc region of amino acid sequence SEQ ID NO: 11 heterodimerised with the fusion Relaxin B-connector-Fc region of amino acid sequence SEQ ID NO: 20. In some embodiments, the heterodimeric fusion comprises AZD3427. In some embodiments, the heterodimeric fusion is AZD3427. In some embodiments, AZD3427 is administered to the subject chronically. In some embodiments, AZD3427 is administered biweekly to the subject subcutaneously. In some embodiments, AZD3427 is administered biweekly to the subject subcutaneously at, e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg.

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

In some embodiments, the amount of the heterodimeric fusion, e.g., AZD3427, administered to the subject or in the pharmaceutical composition administered to the 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.

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

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

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

In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg, results in a dose dependent increase in renin of at least 0.5-fold, at least 1-fold, at least 1 .5-fold, at least 1 .7-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, or at least 4-fold. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg, results in a dose dependent increase in renin of at least 2-fold. In some embodiments, the dose of the heterodimeric fusion, e.g., AZD3427, is selected to result in a dose dependent increase in renin of at least 2-fold.

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

In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg, results in an increase in stroke volume (SV) in the subject of about 0.5 percent, 1 percent, about 2 percent, about 3 percent, about 4 percent, about 5 percent, about 6 percent, about 7 percent, about 8 percent, about 9 percent, about 10 percent, about 11 percent, about 12 percent, about 13 percent, about 14 percent, about 15 percent, or about 20 percent, e.g., after 10 or 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg, results in an increase in stroke volume (SV) in the subject of about 5-60 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 mg, results in an increase in SV in the subject of about 1 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1.1 mg, results in an increase in SV in the subject of about 1 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 5.4 mg, results in an increase in SV in the subject of about 5 percent, e.g., after24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 30 mg, results in an increase in SV in the subject by about 10 percent, e.g., after 24 weeks of treatment.

In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg results a decrease in systemic vascular resistance (SVR) in the subject by about 5 percent, about 10 percent, or about 15 percent, e.g., after 10 weeks of treatment.

In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg results a decrease in systemic vascular resistance (SVR) in the subject by about 1 percent, about 2 percent, about 3 percent, about 4 percent, about 5 percent, about 6 percent, about 7 percent, about 8 percent, about 9 percent, about 10 percent, about 11 percent, about 12 percent, about 13 percent, about 14 percent, about 15 percent, about 16 percent, about 17 percent, about 18 percent, about 19 percent, about 20 percent, about 21 percent, about 22 percent, about

23 percent, about 24 percent, about 25 percent, about 26 percent, about 27 percent, about

28 percent, about 29 percent, about 30 percent, about 31 percent, about 32 percent, about

33 percent, about 34 percent, about 35 percent, about 36 percent, about 37 percent, about

38 percent, about 39 percent, or about 40 percent, e.g., after 24 weeks of treatment.

In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 mg, results a decrease in SVR in the subject by about 8 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1.1 mg, results a decrease in SVR in the subject by about 8 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1 or 1 .1 mg, results in a decrease in SVR in the subject of up to about 20 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 5.4 mg, results a decrease in SVR in the subject by about 15 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 5.4 mg, results in a decrease in SVR in the subject of up to about 30 percent, e.g., after 24 weeks of treatment. In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 30 mg, results a decrease in SVR in the subject by about 20 percent, e.g., after 24 weeks of treatment. . In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 30 mg, results in a decrease in SVR in the subject of up to about 40 percent, e.g., after 24 weeks of treatment In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) results an increase in estimated glomerular filtration rate (eGFR) in the subject by about 1 percent, about 2 percent, about 3 percent, about 4 percent, about 5 percent, about 6 percent, about 7 percent, about 8 percent, about 9 percent, about 10 percent, about 11 percent, about 12 percent, about 13 percent, about 14 percent, about 15 percent, about 16 percent, about 17 percent, about 18 percent, about 19 percent, about 20 percent, about 21 percent, about 22 percent, about 23 percent about 24 percent, or about 25 percent, e.g., after 24 weeks of treatment.

In some embodiments, subcutaneous administration of the heterodimeric fusion, e.g., AZD3427, at a biweekly dose of about 1-30 mg (e.g., about 1 mg, about 1.1 mg, about 5.4 mg, or about 30 mg) results in a decrease in Pulmonary Vascular Resistance (PVR) in the subject by at least 1-10 percent, 1-20 percent, 1-30 percent, 1-40 percent or 1-50 percent or greater. In some embodiments, the reduction in PVR in the subject may be about 1 percent, about 2 percent, about 3 percent, about 4 percent, about 5 percent, about 6 percent, about 7 percent, about 8 percent, about 9 percent, about 10 percent, about 11 percent, about 12 percent, about 13 percent, about 14 percent, about 15 percent, about 16 percent, about 17 percent, about 18 percent, about 19 percent, about 20 percent, about 21 percent, about 22 percent, about 23 percent, about 24 percent, or about 25 percent, e.g., after 24 weeks of treatment.

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

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

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

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

In some embodiments, changes (e.g., increases or decreases) in biomarker levels or patient outcomes following administration of the heterodimeric fusion are relative to baseline (pre-administration) measures.

In some embodiments, administration to the patient of the heterodimeric fusion, e.g., AZD3427, causes no significant change in blood pressure (e.g. systolic blood pressure) of the patient. This may be no significant change (e.g. percentage change) relative to baseline blood pressure of the patient (i.e. pre-administration) and/or as compared to placebo. In some embodiments, no significant change in blood pressure (e.g. systolic blood pressure) of the patient means no more than a 20% change relative to baseline blood pressure (e.g. systolic blood pressure) of the patient. This may be no more than a 20% decrease relative to baseline blood pressure (e.g. systolic blood pressure) of the patient.

Exemplary Dosing Regimens

In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered biweekly to the subject subcutaneously. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered biweekly to the subject subcutaneously at a dose of about 1 mg to about 30 mg. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered biweekly to the subject subcutaneously at a dose of about 1 mg. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered biweekly to the subject subcutaneously at a dose of about 1.1 mg. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered biweekly to the subject subcutaneously at a dose of about 5.4 mg. In some embodiments, the heterodimeric fusion, e.g., AZD3427, is administered biweekly to the subject subcutaneously at a dose of about 30 mg.

Methods of Making

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

Methods that are known to those skilled in the art can be used to construct expression vectors containing the nucleic acid molecules encoding the heterodimeric fusions of the disclosure. Suitable vectors include, for example, plasmids, phagemids, phages or viral vectors.

Vectors containing the nucleic acid molecules encoding the heterodimeric fusions of the disclosure may be transferred to a host cell by conventional techniques. Suitable host cells are known in the art. The host cells may be mammalian cells such as HEK293 cells or CHO cells.

The transfected cells may be cultured by conventional techniques to produce the fusion polypeptides of the disclosure.

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

Definitions

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

"About" and “approximately” are used interchangeably herein and mean an acceptable degree of variation for the quantity measured given the nature or precision of the measurements based on pharmaceutical formulation. For example, for doses of a heterodimeric fusion as provided herein, “about” permits variation of ± 0.1 from the stated value.

As used herein, the term “biweekly” administration refers to administration once every two weeks.

Embodiments provided herein as "comprising" one or more features may also be considered as disclosure of the corresponding embodiments "consisting of such features.

The term "EU index as in Kabat" refers to the numbering system of the human IgG 1 EU antibody described in Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service. National Institutes of Health. Bethesda, MD. All amino acid positions referenced in the present application refer to EU index positions.

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 ejection fraction > 41 % (HF with EF > 41 %), heart failure with mid-range ejection fraction, or heart failure with reduced ejection fraction (HFrEF). This also includes heart failure due to hypertrophic cardiomyopathy or dilated cardiomyopathy. As used herein, the term “heart failure with pulmonary hypertension” refers to the subset of heart failure subjects who simultaneously suffer from pulmonary hypertension (HF+PH subjects).

The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term “plasma concentration” refers to average plasma concentration at steady state.

The term “treatment” refers to the amelioration and/or elimination of one or more symptoms or causes of the target disease or condition. In some embodiments, this involves modulating the levels of one or more biological markers or functions, e.g., as compared to a diseased state, e.g., to within a non-diseased range (as compared against a healthy cohort).

Concentrations, amounts, volumes, percentages, and other numerical values may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. It is to be understood that ranges include the range endpoints unless explicitly stated otherwise.

The above embodiments and below Examples are to be understood as illustrative and nonlimiting. Further embodiments are envisaged. It is to be understood that any feature provided in relation to any one embodiment may be used alone, or in combination with other features provided, and may also be used in combination with one or more features of any other of the embodiments, or any combination of any other of the embodiments. Furthermore, equivalents and modifications not provided above may also be employed without departing from the scope of the disclosure, which is defined in the accompanying claims.

In the context of the present disclosure other examples and variations of the fusion polypeptides and methods provided herein will be apparent to a person of skill in the art.

Other examples and variations are within the scope of the disclosure, as set out in the appended claims. All documents cited herein are each entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited documents.

Examples

Example 1: Generation of recombinant heterodimeric Fc Relaxin-2 fusion proteins

Relaxin-2 chains A and B were genetically fused to two complementary Fes (at the N- and/or C-terminus of the Fc) via connectors, as illustrated in FIG. 1. CHO cells were then co-transfected with two expression vectors comprising each of the single Fc-Relaxin chains (A and/or B). The two complementary Fc moieties assemble within the CHO cells and, thus, facilitate the assembly and correct folding of Relaxin-2.

The heterodimeric Fc Relaxin-2 fusion proteins were secreted in the supernatant, then purified using an automated system by affinity chromatography, wherein the Fc region of the protein binds to the column matrix.

Example 2: PK profile of RELAX0023 (AZD3427) in cynomolgus monkeys

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

Assay plates were coated with goat anti-human IgG antibody and were incubated with cynomolgus monkey sera from group 1-4 animals. RELAX0023 bound to the plates was detected by an anti-relaxin antibody conjugated with HRP. Cynomolgus serum was diluted 1 :10 prior to addition to plates. The lower limit of quantitation is 0.010 pg/mL and upper limit of quantitation is 0.300 pg/mL in 100% serum.

FIG. 2 shows the mean serum concentration-time profiles of AZD3427 in cynomolgus monkeys following a single dose. Following a single dose administered SC, AZD3427 exhibited linear PK in a dose range of 0.01 to 10 mg/kg. A dose-proportional increase in Cmax was observed. Mean Cmax values were 0.400, 4.69, 34.8 pg/mL for 0.1 , 1 , and 10 mg/kg SC dose groups, respectively. A dose-proportional increase in AUC0- last values were also observed from 0.1 mg/kg to 10 mg/kg SC group. Mean AUCO-last values were 2.01 , 25.5, 193 pg day/mL for 0.1 , 1 , and 10 mg/kg SC dose groups, respectively. Overall, AZD3427 PK is linear in the range of 0.1 mg/kg to 10 mg/kg with the mean CL/F of 51.0 mL/day.

Example 3: Evaluation of chronic efficacy of AZD3427 in cynomolgus monkey (Macaca fascicularis) with heart failure and reduced left ventricular ejection fraction (LVEF)

The chronic efficacy of AZD3427 on cardiac function was evaluated in obese and aged cynomolgus monkeys (Macaca fascicularis). The cynomolgus monkey was selected as the test species over other lower mammalian species because of its close relationship to humans, both phylogenetically and physiologically. Old cynomolgus monkeys fed with a high fat diet for at least 2 years share risk factors with human patients susceptible to cardiovascular disease and develop metabolic syndrome that can characteristically progress to heart failure and reduced left ventricular ejection fraction (LVEF). The effects of AZD3427 on LVEF were evaluated when administered by subcutaneous (SC) injection at different dose levels for 20 weeks, with the first dose administered at week 1 of the study, followed by an 18-week observational period. From a pool of approximately 100 obese and aged cynomolgus monkeys aged 12-20 years with a body weight of 6-15 kg that had been fed a high-fat diet for at least 2 years, 38 monkeys were identified by 2D echocardiographic screening as having an LVEF between 30%-60%. Healthy monkeys of this age weigh 5-8 kg and have an LVEF of 70%-75%, and therefore LVEF of 60% or below represents an HFrEF model. Identified animals were selected and randomly assigned to 3 treatment groups with 8 monkeys each, and a vehicle group with 14 monkeys. The dosing period consisted of once weekly (QW) SC administrations of AZD3427 at 3 ascending dose levels (0.015 mg/kg, 0.15 mg/kg, and 3 mg/kg).

Cardiac functional measurements by 2D echocardiography were determined 9 times, at baseline week -2 and at week 5, 9, 13, 17, 21 , 25, 29, 33 of the dosing and postdose observation periods. A further 2D echocardiography was scheduled for week 39 (study end). Parameters, including LVEF, were based on apical two- and four-chamber views and the biplane method. HDO (High Definition Oscillometry) was used to measure parameters including mean arterial pressure (MAP) and heart rate (HR).

AZD3427 greatly improved LVEF at weeks 5, 9, 13, 17 and 21 at all AZD3427 dose levels compared with vehicle control, without affecting heart rate or blood pressure (FIGs. 3A-3D). Remarkably, improved LVEF following treatment with AZD3427 as compared with week 0 (baseline) was observed throughout the washout period after the end of treatment to week 33 of the study. There was also a clear increase in stroke volume (SV) and decrease in systemic vascular resistance (SVR). A dose dependent increase in renin was also observed which is likely to be a compensatory response to vasodilation after

AZD3427 treatment. These striking results indicate a remarkable improvement in hemodynamics in the treated animals and clearly demonstrate efficacy of AZD3427 in treating heart failure in this model. Moreover, the magnitude of the sustained response after treatment is something which, to the best of the inventors’ knowledge, has not been achieved previously by any other known compound targeting this mechanism of action pathway. Monkeys continued to be monitored until week 39 of the study.

Example 4: Phase 1 (Ph1) Study in Healthy Volunteers and Heart Failure Patients

Study D8330C00001 was a Phase la/b, randomized, single-blinded, placebo- controlled, first-time-in-human (FTIH) study (ClinicalTrials.gov identifier NCT04630067). The primary objective of the study was to assess the safety and tolerability of single and multiple ascending doses of AZD3427, and the secondary objectives were to evaluate (i) the pharmacokinetics (PK) and (ii) the immunogenicity of single and multiple ascending doses of AZD3427.

The study was performed in 2 parts, Part A and Part B (FIG. 4). Part A was a single ascending dose (SAD) study in healthy participants (males and females of nonchildbearing potential), and Part B was a multiple ascending dose (MAD) study in participants with HF (males and females of non-childbearing potential).

Part A included 56 healthy participants across 7 cohorts (8 participants in each cohort) who received a single dose of AZD3427 or placebo. Within each cohort, 6 participants were randomized to receive AZD3427 and 2 participants were randomized to receive placebo. One cohort was exclusively made up of participants of Japanese descent (both parents and all grandparents are Japanese). The 7 cohorts were as follows:

• Cohorts 1a, 2a, 3a, and 4a: SC dose at dose levels of 5, 10, 30, and 90 mg respectively

• Cohort 5a: IV dose at dose level of 15 mg AZD3427

• Cohort 6a: SC dose at dose level of 270 mg (participants of Japanese descent)

Cohort 7a: single SC AZD3427 dose of approximately 270 mg. Part B included 48 patients across 6 cohorts (8 participants in each cohort). Of these, 3 cohorts were comprised of participants with HFrEF (Cohorts 1b, 3b, and 5b) and 3 cohorts were comprised of participants with HF with EF > 41 % (Cohorts 2b, 4b, and 6b). The dose levels in HFrEF and HF with EF > 41 % cohorts were 5 mg (Cohorts 1 b, 2b), 15 mg (Cohorts 3b, 4b), and 45 mg (Cohorts 5b, 6b) administered once weekly (QW) for 5 weeks (i.e. , a total of 5 doses).

For Part B, which included 48 patients across 6 cohorts, the inclusion criteria included: (i) All Cohorts: Have a known clinical diagnosis of Stage C HF (NYHA Class I to III) and be on stable medical therapy for at least 12 weeks prior to screening with no significant dose change or new medications added during that period, (ii) Cohorts 1 b, 3b, 5b: Patients with a diagnosis of HFrEF defined as EF < 40%, (iii) Cohorts 2b, 4b, 6b: Patients with a diagnosis of HF with EF > 41 % (including patients with a diagnosis of HFpEF defined as EF > 50%), (iv) All Cohorts: Have a BMI between 18 and 40 kg/m² (inclusive) and weigh at least 55 kg and no more than 120 kg (inclusive), and (v) All Cohorts: Prior recording of either NT-proBNP > 125 pg/mL or BNP > 35 pg/mL.

Example 5: Serum samples for determination of AZD3427 concentrations

Serum samples for determination of AZD3427 concentrations were analyzed by an electrochemiluminescent (ECL) method validated to accurately and precisely quantify AZD3427 levels ranging from 0.10 pg/mL to 25.60 pg/mL in human serum samples.

AZD3427 is captured by a biotin-labeled antibody directed against the relaxin part of AZD3427 (clone AB1510209) coated on an Meso Scale Discovery (MSD) streptavidin- coated standard bind plate at 2.0 pg/mL. Calibrators, quality controls (QCs), and samples are diluted to the method minimum required dilution (MRD) of 1 :400 in assay buffer and 50 pL/well is incubated on the plate for approximately 1 hour at room temperature with shaking (600 rpm). The assay plate was washed before addition of a ruthenium-labeled anti AZD3427 antibody that binds the knob into hole Fc portion, a site on AZD3427 distinct from the site bound by the capture antibody. The assay plate was incubated for approximately 1 hour before excess of reagents was washed away from the plate. Addition of MSD read buffer to the plate resulted in an ECL reaction causing bound ruthenium molecules to emit light which was measured as relative light units. The AZD3427 concentration in a sample was determined by interpolation from a standard curve using a 4-parameter curve fit with 1/ECL² weighting relating the light intensity to the concentration of AZD3427.

Pharmacokinetic data: AZD3427 serum concentration versus time profiles from the SAD cohorts after SC dosing of 5 mg, 10 mg, 30 mg, 90 mg and 270 mg are shown in FIG. 5. Following SC administration of AZD3427 solution, AZD3427 was absorbed with Cmax being reached within 3-4 days. With non-compartmental analysis of the PK data from the SAD cohorts, the terminal ti/2 of AZD3427 was estimated to 7-9 days.

In the MAD cohorts, Ctrough values showed that steady state had not completely been reached after the 5 once weekly doses. The Ctrough value after the last dose was 329 ng/mL, 1005 ng/mL, and 2321 ng/mL for the dose levels 5 mg, 15 mg, and 45 mg, respectively.

Example 6: Ph1 AZD3427 MAD study outcomes in HF patients

In the Part B MAD cohort, data were first pooled for HFpEF and HFrEF patients. Trends suggest AZD3427 improved cardiac function, including improved cardiac output and stroke volume (SV), reduced systemic vascular resistance (SVR), and improved organ perfusion (SVR and eGFR) (FIGs. 6A - 6F). Additional analysis, this time parsing patients into the following groups: (i) patients with baseline EF < 40% and (ii) patients with baseline EF > 40%, showed that the observed trends in improved (increased) stroke volume and eGFR after treatment with AZD3427 in pooled patients were also observed in both (i) patients with baseline EF < 40% and (ii) patients with baseline EF > 40% (FIGs. 6H-I). These were observed without any apparent effect on blood pressure (FIG. 6G). AZD3427 administration also resulted in a dose dependent increase in plasma renin levels

(FIG. 7A). For tested doses lower than 45 mg, resulting hemoglobin and hematocrit levels were within an acceptable safety profile. (FIGs. 7B, 7C). Advantageously therefore, doses of 1 mg to 30 mg of AZD3427, which have been selected as the range of predicted pharmacodynamically efficacious doses for Ph2 trials based on the modelling and Ph1 data described herein, appear to also be sufficient to enable hemoglobin and hematocrit levels to remain within acceptable safety limits.

Example 7: Phase 2B (Ph2b) study design for AZD3427 dosing regimen

In the Ph2b study (design shown in FIG. 8; Study ID Number: D8330C00003), about 1.0 mg (e.g., 1.1 mg), about 5.4 mg, and about 30 mg Q2W doses of AZD3427 will be evaluated. Participants will receive a single, subcutaneous dose of AZD3427 or placebo (in a 1 :1 :1 :1 ratio) once every 2 weeks for 24 weeks from Day 1 to Day 155.

The dose selection of AZD3427 was based on PK, pharmacodynamic, and safety data from the SAD/MAD study in healthy participants and HFrEF/HFpEF participants (study D8330C00001/NCT04630067) and from the study in NHPs with reduced LVEF. Dosing of once every 2 weeks (“biweekly”) was determined by using said data to model once weekly, once every 2 weeks, and once every 4 weeks dosing, as shown in FIG. 9. The dashed line in FIG. 9 (0.08 ug/mL) represents the estimated concentration of AZD3427 that is equivalent to the average observed concentration of relaxin in pregnant subjects in the first trimester (0.2 ng/mL; in-house assay (see also Example 10)) and acts as a reference level. This reference level of 0.08 pg/mL was calculated as follows based on a 40-fold potency difference of AZD3427 versus relaxin and 10-fold greater molecular weight:

0.2*40*10 = 80 ng/mL = 0.08 pg/mL

Similar Cmax/Cmin ratios were predicted for once weekly and once every 2 weeks dosing, as shown in FIG. 9, indicating that both would be suitable for maintaining Ctrough levels above the reference level. Once every 2 weeks dosing is preferred as this minimises the doses needed to be administered to the patient thereby facilitating greater patient compliance.

The average concentration at steady state (Css.ave) at the three dose levels are predicted to range from sublevels to supra levels of corresponding relaxin levels in pregnancy: 0.5-fold, 2.5-fold, and 15-fold of relaxin pregnancy levels, respectively. This correlation between levels of AZD3427 and relaxin accounts for the difference in in vitro potency between serelaxin and AZD3427 (approximately 40-fold) based on cAMP production in CHO cells expressing the recombinant human RXFP1 receptor and the difference in molecular weight between serelaxin and AZD3427 (AZD3427 being 10-fold greater) and assumes a relaxin concentration of 0.2 ng/mL in pregnancy (in-house data). In addition, using concentration-response relationships for ejection fraction, stroke volume, and systemic vascular resistance from the NHP HFrEF model and PK data from the SAD/MAD study (study D8330C00001), it is predicted that the dose range selected in the Ph2b study will enable characterization of the dose-response for these parameters (FIG. 10, FIGs. 11A-11C).

The Primary Outcome measure will be the change from baseline in Pulmonary Vascular Resistance (PVR) after 24 weeks of treatment. The effect of AZD3427 on PVR parameter will also be evaluated as compared with placebo as measured by right heart catheterization (RHC) after 24 weeks of treatment in participants with HF and PH Group 2.

Secondary Outcome measures include:

• Change from baseline in Mean pulmonary arterial pressure (mPAP)

• Change from baseline in Pulmonary artery wedge pressure (PAWP)

• Change from baseline in cardiac output

• Change from baseline in Stroke Volume (SV)

• Change from baseline in Ejection fraction (EF) Change from baseline in left ventricular global longitudinal strain (LVGLS)

• Change from baseline in pulmonary arterial systolic pressure (PASP)

• Change from baseline in right ventricle/left ventricle (RV/LV) ratio

• Change from baseline in right ventricular outflow tract acceleration time (RVOT AT)

• Change from baseline in Tricuspid regurgitation velocity (TRV)

• Change from baseline in TAPSE/PASP [Tricuspid annular plane systolic excursion/ Pulmonary arterial systolic pressure]

• Change from baseline in right ventricular strain/pulmonary arterial systolic pressure (RVS/PASP)

• Change from baseline in inferior vena cava (I VC) diameter with inspiratory collapse

• Change from baseline in systemic vascular resistance

• Change from baseline in 6-minute walking distance (6MWD)

• Change from baseline in Kansas City Cardiomyopathy Questionnaire total symptom score (KCCQ TSS)

• Change from baseline in New York Heart Association Functional Class (NYHA FC)

• Change from baseline in serum creatinine

• Change from baseline in N-terminal prohormone of brain natriuretic peptide (NT- proBNP)

• Change from baseline in cystatin C

• Change from baseline in eGFR (estimated glomerular filtration rate)

Inclusion Criteria: 1 . Participant must be > 18 years of age inclusive. 2. Participants must have a pre-existing diagnosis of HF, NYHA function class (FC) II to IV, and a pre-existing diagnosis of PH-LHD or likely or intermediate probability of Pulmonary hypertension due to left heart disease (PH-LHD) as per 2022 Pulmonary hypertension due to left heart disease European Society of Cardiology/European Respiratory Society (ESC/ESR) guidelines. Participants must be on stable HF standard of care medication, including diuretics. 3. Participants must have a combination of echocardiographic parameters that show intermediate or high probability of PH as per 2022 ESC/ERS guidelines. 4. Participants must have an on-study elevated pulmonary artery pressure from RHC performed as per RHC manual provided by the Sponsor, at Screening Visit 2: (a) PAWP > 15 mmHg (b) mPAP > 20 mmHg 5. Minimum body weight of 50 kg (inclusive). 6. Capable of giving signed informed consent.

Exclusion Criteria: 1. Diagnosis of PH in World Health Organization (WHO) Group 1 , WHO Group 3, WHO Group 4, or WHO Group 5. 2. Historical or current evidence of a clinically significant disease or disorder. 3. Decompensated HF or any hospitalisation. 4. Any contraindications to RHC. 5. History of hypersensitivity to SC injections or devices. 6. History of hypersensitivity to drugs with a similar chemical structure or class to AZD3427 or any component of AZD3427 drug product, or ongoing clinically important allergy/hypersensitivity. 7. Known lung disease with Forced expiratory volume in the first second/Vital capacity (FEV1/VC) < 30%. 8. Congenital long QT syndrome. 9. Cardiac ventricular arrhythmia which requires treatment. Participants with atrial fibrillation or flutter and controlled ventricular rate are permitted. 10. History of or anticipated heart transplant or ventricular assist device implantation. 11. Any known planned (scheduled) highly invasive Cardiovascular (CV) procedure (eg, coronary revascularisation, ablation of atrial fibrillation/flutter, valve repair/replacement, aortic aneurysm surgery, etc). 12. Participants who have previously received AZD3427.

Without being limited by theory, AZD3427 is expected to have vasodilatory, antiinflammatory, and anti-fibrotic effects and is expected to improve left ventricle function and stop and/or reverse remodelling of the diseased heart and pulmonary vasculature, which in turn should decrease PAP and PVR and improve cardiovascular outcomes. Example 8: Final Ph1 AZD3427 MAD study outcomes in HF patients

The Phase 1 data described in the above Examples, and modelling derived therefrom, was based on an interim data set. The following Examples 8 and 9 are based on the finalised data set available from the Ph1 trial, whereby the data was checked and verified and additional patient data since available may be included in the analysis. Analysis of the final data set for the Part B MAD cohort, pooled for HFpEF and HFrEF patients, fully supports the conclusions drawn from the interim data, in particular the observed trend for AZD3427 to improve cardiac function, including improved cardiac output and stroke volume (SV), reduced systemic vascular resistance (SVR), and improved organ perfusion (SVR and eGFR) (FIGs. 12A-F) compared to placebo. Additional analysis, this time parsing patients into the following groups: (i) patients with baseline EF < 40% and (ii) patients with baseline EF > 40%, showed that the observed trends in improved (increased) stroke volume and eGFR after treatment with AZD3427 in pooled patients were also observed in both (i) patients with baseline EF < 40% and (ii) patients with baseline EF > 40% (FIGs. 12H-I). There was no apparent effect on blood pressure (FIG. 12G). AZD3427 administration also resulted in a dose dependent increase in plasma renin levels (FIG. 13A). For tested doses lower than 45 mg, resulting hemoglobin and hematocrit levels were within an acceptable safety profile. (FIGs. 13B, 13C). Doses of 1 mg to 30 mg of AZD3427 appeared to maintain acceptable safety limits for hemoglobin and hematocrit levels.

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

The Ph2b study (design shown in FIG. 8; Study ID Number: D8330C00003) discussed in Example 7, will be conducted to evaluate AZD3427. The dose range selected in the Ph2b study will enable characterization of the dose-response for ejection fraction, stroke volume, renin, and systemic vascular resistance based on the NHP HFrEF model and PK data from the SAD/MAD study (study D8330C00001) (FIG. 14). The dose response will also characterize hematocrit, hemoglobin, and albumin levels (FIG. 15A- 150). Primary and secondary outcomes described in Example 7 will be evaluated. Inclusion and exclusion criteria from Example 7 will also be applied.

Example 10: Relaxin-2 detection assay

Relaxin-2 capture antibody (MAB2804; R&D Systems, USA) was used as a coating antibody. Relaxin-2 biotinylated antibody (R&D Systems, USA) was used as a detection antibody. Relaxin-2 standard (3596-RN-025/CF; R&D Systems, USA) was used as a calibrator. The calibrator was prepared by resuspending in 200 pL Assay Diluent. A dilution curve was made by diluting the standard from 36000 fg/mL down to 10 fg/mL.

Single molecule array (Simoa™) technology was used according to the following steps: Magnetic homebrew carboxylated beads (Quanterix, USA) were activated by adding 0.1 mg/mL of 1 -ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (EDO, Thermo Fisher Scientific, USA) to a bead solution with 1.4x10⁶ beads/pL. After incubating at room temperature (RT) for 30 minutes (min), the beads were washed in a magnetic separator and 0.3 mg/mL of ice-cold capture antibody was added. The beads were incubated for 2 hours (hrs) on an agitator (HulaMixer, Invitrogen, USA) at 4°C. The beads were then washed, and a blocking solution was added. After additional washes, the conjugated beads were re-suspended in corresponding bead diluent and stored at 4°C for future use. Human plasma samples and Relaxin-2 standard (370 pL) were analyzed in duplicates in the Simoa HD-X Analyzer (Quanterix, Lexington, MA). Samples and standard were plated on a 96-well plate (NUNC, Thermo Fisher Scientific, USA). The conjugated beads were washed 2 times in bead diluent 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 in a ratio of 1 :10 of the total volume. The detector antibody was diluted in Assay Diluent to a final concentration of 0.25 pg/mL and the enzyme SBG (streptavidin [3-galactosidase, Quanterix, USA) was diluted in SBG Diluent to a final concentration of 150 pM. Reagents, samples, and calibrators were run in the HD-X Analyzer using a 2-step Assay Neat 2.0 protocol with 25 pL conjugated beads, 20 pL biotinylated antibody, 100 pL SBG, and 50 pL Resorufin [3-D-galactopyranoside (RGP, Quanterix, Lexington, MA).

Table 3: Sequence Listing. The upper hinge region is in Italics, Relaxin A is underlined,

Relaxin B is double underlined, the Fc region is bold.

Claims

1 . A method of treating a subject having heart failure, optionally heart failure with pulmonary hypertension, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable excipient and about 1 mg to about 30 mg of a heterodimeric fusion; wherein the heterodimeric fusion comprises:

(ii) a second heterodimerisation domain connected to at least one Relaxin B chain polypeptide or a variant thereof; wherein the first heterodimerisation domain heterodimerises with the second heterodimerisation domain; and wherein the heterodimeric fusion has Relaxin activity.

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

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

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

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

6. The method of any one of claims 1-5, wherein the pharmaceutical composition is administered to the subject biweekly.

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

8. The method of any one of claims 1-7, wherein the subject has one or more of:

(a) New York Heart Association (NYHA) Functional Class ll-IV;

(b) mean pulmonary arterial pressure (mPAP) greater than 20 mmHg; and

(c) pulmonary artery wedge pressure (PAWP) greater than 15 mmHg.

9. The method of any one of claims 1-8, wherein endogenous relaxin plasma levels in the subject are about 10,000-fold lower than relaxin plasma levels in pregnant subjects in their first trimester.

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

11. The method of any one of claims 1-10, wherein administration of the pharmaceutical composition is sufficient to result in a plasma concentration of the heterodimeric fusion of 0.026 pg/mL to 1 .6 pg/mL in the subject.

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

13. The method of any one of claims 1-12, wherein, following administration of the pharmaceutical composition, hematocrit levels in the subject are reduced by no more than 5-7 percent, e.g., by no more than 5 percent or no more than 7 percent, compared to baseline levels.

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

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

16. The method of any one of claims 1-15, wherein blood pressure (e.g. systolic blood pressure) of the subject is not significantly changed, compared to baseline blood pressure (e.g. systolic blood pressure), following administration of the pharmaceutical composition.

17. The method of any one of claims 1-16, wherein administration of the pharmaceutical composition is sufficient to result in one or more of:

(a) a reduction in pulmonary vascular resistance (PVR);

(b) reduction in a reduction in mean Pulmonary Artery Pressure (mPAP);

(c) a reduction 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; as compared to baseline levels.

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

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

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

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

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

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

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

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

26. The method of any one of claims 1-25, 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 inter-chain disulphide bond; and/or (ii) are not covalently linked to each other by an amino acid linker.

27. The method of any one of claims 1-26, 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 of any one of claims 1-27, wherein the at least one Relaxin-2 A chain polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and the at least one Relaxin-2 B chain polypeptide comprises the amino acids sequence of SEQ ID NO: 2.

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

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

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

32. The method of claim 30 or claim 31 , wherein one or optionally both connectors have the amino acid sequence of SEQ ID NO: 5.

33. The method of any one of claims 1-32, wherein the first heterodimerisation domain is derived from a first immunoglobulin Fc region and the second heterodimerisation domain is derived from a second immunoglobulin Fc region, wherein the first and second Fc regions comprise the constant domains CH2 and CH3, optionally CH2 and CH3 domains from an lgG1 immunoglobulin, further optionally wherein the CH2 and/or CH3 domains are mutated.

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

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

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

37. The method of any one of claims 33-36, wherein the first and/or second Fc region further comprises the amino acid mutations L234F, L235E, and P331 S, wherein the amino acid numbering is according to the EU index as in Kabat.

38. The method of any one of claims 33-37, wherein the first Fc region comprises the amino acid sequence of SEQ ID NO: 4 and the second Fc region comprises the amino acid sequence of SEQ ID NO: 3.

39. The method of any one of claims 27-38, wherein the heterodimeric fusion comprises the amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 20.

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

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

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

43. The method of any of one of claims 1-41 , wherein the heterodimeric fusion is AZD3427 and is administered biweekly to the subject subcutaneously at a dose of about

5.4 mg.

44. The method of any one of claims 1-41 , wherein the heterodimeric fusion is AZD3427 and is administered biweekly to the subject subcutaneously at a dose of about 30 mg.

45. A method of treating a subject having heart failure, optionally heart failure with pulmonary hypertension, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable excipient and about 1 mg to about 30 mg of a heterodimeric fusion; wherein the heterodimeric fusion comprises:

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

(ii) an FcY-con-B fusion polypeptide, wherein:

A is a Relaxin A chain or variant thereof, e.g., a Relaxin-2 A chain or variant thereof; B is a Relaxin B chain or variant thereof, e.g., a Relaxin-2 B chain or variant thereof; FcX is an Fc region comprising the constant domains CH2 and CH3 of a human lgG1 immunoglobulin with amino acid mutations, optionally the amino acid mutations S354C and T366W;

FcY is an Fc region comprising the constant domains CH2 and CH3 of a human lgG1 immunoglobulin comprising amino acid mutations, optionally the amino acid mutations Y349C, T366S, L368A, and Y407V; and con is a connector polypeptide, optionally having the sequence SEQ ID NO: 5, wherein the amino acid numbering is according to the EU index as in Kabat, wherein FcX heterodimerises with FcY, and wherein the heterodimeric fusion has Relaxin activity.

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

AZD3427.

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

48. The method of any one of claims 45-47, wherein the pharmaceutical composition is administered to the subject biweekly.

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

50. The method of any one of the preceding claims, wherein the subject has heart failure with pulmonary hypertension Group 2.