CN118434437A - Treatment with heterodimeric relaxin fusion - Google Patents

Abstract

The present invention relates to the use of heterodimeric relaxin fusion polypeptides, particularly for the treatment of heart failure with pulmonary hypertension.

Description

Treatment with heterodimeric relaxin fusions

Technical Field

The present invention relates to therapeutic methods using heterodimeric relaxin fusions. In particular, the present invention relates to therapeutic methods using relaxin-2 fusions.

Background technique

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

Relaxin is a pleiotropic hormone known to mediate systemic hemodynamic changes and renal adaptive changes during pregnancy. Relaxin has also been shown to have anti-fibrotic properties and to have beneficial effects in heart failure, such as acute decompensated heart failure (ADHF). Heart failure is associated with significant morbidity and mortality. It is characterized by complex tissue remodeling involving increased cardiomyocyte death and interstitial fibrosis. Relaxin activates a number of signaling cascades that have been shown to be beneficial in the setting of ischemia-reperfusion and heart failure. These signaling pathways include activation of the phosphoinositide 3-kinase pathway and activation of the nitric oxide signaling pathway (Bathgate RA et al. (2013) Physiol. Rev. 93(1):405-480; Mentz RJ et al. (2013) Am. Heart J. 165(2):193-199; Tietjens J et al. (2016) Heart 102:95-99; Wilson SS et al. (2015) Pharmacology 35:315-327).

A large proportion of heart failure patients also suffer from pulmonary hypertension (HF+PH patients). It is estimated that approximately 50% of heart failure patients with preserved ejection fraction also suffer from pulmonary hypertension, and this proportion increases to 60% of heart failure patients with reduced ejection fraction (Guazzi, (2014) Circ Heart Fail. [Circulation: Heart Failure], 7:367-377; Miller et al., (2013) JACC Heart Fail. [Journal of the American College of Cardiology: Heart Failure], 1(4):290-299). Patients with heart failure and pulmonary hypertension have been shown to have decreased survival compared with heart failure patients without pulmonary hypertension (Barnett and De Marco, (2012) Heart Fail. Clin. [Heart Failure Clinical] 8:447–459). In patients with heart failure, an increase or decrease of 3 mmHg in estimated pulmonary artery diastolic pressure (ePAD) (equivalent to an increase or decrease of approximately 4 mmHg in mean pulmonary artery pressure (mPAP)) is associated with a 24% increase or 19% decrease in cardiovascular mortality, respectively (Zile MR et al. (2017) Circ Heart Fail. 10:e003594). A 4 mmHg decrease in mPAP is also associated with improved dyspnea in patients with heart failure and pulmonary hypertension (Solomonica A et al. (2013) Circ Heart Fail. 6:53-60).

Clinical trials have been conducted using unmodified recombinant human relaxin-2, serelaxin. Continuous intravenous administration of serelaxin to hospitalized patients improved markers of cardiac, renal, and liver damage and congestion (Felker GM et al. (2014) J. Am. Coll. Cardiol. [Journal of the American College of Cardiology] 64(15):1591-1598; Metra M et al. (2013) J. Am. Coll. Cardiol. [Journal of the American College of Cardiology] 61(2):196-206; Teerlink JR et al. (2013) Lancet [Lancet] 381(9860):29-39). Serelaxin also showed improvements in pulmonary artery pressure, cardiac output, and systemic and pulmonary vascular resistance in these patients with approximately 20 hours of continuous infusion (Ponikowski et al., (2014) European Heart Journal [European Heart Journal] 35:431–441). However, the therapeutic effect is limited due to the rapid clearance of cedrelatin from the patient's circulation, and the positive effects disappear rapidly once the intravenous injection is stopped. In addition, approximately one-third of patients experience a significant decrease in blood pressure (>40 mmHg) after receiving cedrelatin intravenously, necessitating a dose reduction of half or even more.

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

In view of the promising clinical studies currently being conducted with unmodified recombinant relaxin, there remains a need for additional recombinant relaxins that retain the biological activity of relaxin and provide advantages such as extended half-life, convenient routes of administration, and ease of dosing.

Summary of the invention

The present invention relates to the use of heterodimeric fusions with relaxin activity in treating subjects with heart failure combined with pulmonary hypertension (HF+PH). In particular, there is still a significant unmet need for the treatment of HF+PH subjects.

Thus, in one aspect, the present invention provides a method of treating a subject having heart failure combined with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

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

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

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

Similarly, the present invention provides a heterodimeric fusion for use in treating a subject suffering from heart failure combined with pulmonary hypertension, the heterodimeric fusion comprising:

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

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

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

Similarly, the present invention provides a use of a heterodimeric fusion in the manufacture of a medicament for treating a subject suffering from heart failure combined with pulmonary hypertension, the heterodimeric fusion comprising:

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

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

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

In some embodiments, the relaxin A chain and relaxin B chain of the heterodimer fusion are covalently bound by one or more (e.g., two) interchain bonds, preferably one or more (e.g., two) interchain disulfide bonds. In some embodiments, the relaxin A chain and relaxin B chain are not covalently linked to each other by an amino acid linker.

In some embodiments, the relaxin A chain is relaxin-2A chain and the relaxin B chain is relaxin-2B chain.

In a preferred embodiment, the first and second heterodimerization domains are derived from immunoglobulin Fc regions, such as immunoglobulin G (IgG) Fc regions ("first Fc region" and "second Fc region"). The first and second Fc regions may comprise constant domains CH2 and/or CH3. Preferably, the first and second Fc regions comprise CH2 and CH3.

In alternative embodiments, the first and second heterodimerization domains are derived from immunoglobulin Fab regions.

In yet further alternative embodiments, the first and second heterodimerization domains heterodimerize to form a parallel coiled coil.

In some embodiments, the relaxin A chain is linked to a first heterodimerization domain (e.g., a first Fc region) via a linker, and the relaxin B chain is linked to a second heterodimerization domain (e.g., a second Fc region) via a linker. In preferred embodiments, one or preferably both linkers are polypeptides.

In some embodiments, at least one linker is a polypeptide of 6 to 40 amino acids in length. Preferably, both linkers are polypeptides of 6 to 40 amino acids in length. In a preferred embodiment, at least one linker is a polypeptide of 21 amino acids in length. In a particularly preferred embodiment, both linkers are polypeptides of 21 amino acids in length. In certain embodiments, both linkers have the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5].

In a preferred embodiment, the C-terminus of the first heterodimerization domain (e.g., the first Fc region) is linked to the N-terminus of the relaxin A chain, and the C-terminus of the second heterodimerization domain (e.g., the second Fc region) is linked to the N-terminus of the relaxin B chain. In an alternative embodiment, the N-terminus of the first heterodimerization domain (e.g., the first Fc region) is linked to the C-terminus of the relaxin A chain, and the N-terminus of the second heterodimerization domain (e.g., the second Fc region) is linked to the C-terminus of the relaxin B chain.

In some embodiments, the first and second heterodimerization domains (e.g., the first and second Fc regions) comprise amino acid mutations and/or modifications that promote heterodimerization, preferably, amino acid mutations and/or modifications that promote asymmetric heterodimerization. In a preferred embodiment, the amino acid mutations that promote heterodimerization are "Fc knob" and "Fc hole" mutations. In a particularly preferred embodiment, the "Fc knob" and "Fc hole" mutations are present in the CH3 domain. In a preferred embodiment, the first Fc region comprises an "Fc knob" mutation and the second Fc region comprises an "Fc hole" mutation. Alternatively, the first Fc region has an "Fc hole" mutation and the second Fc region has an "Fc knob" mutation. Preferably, the amino acid mutations promoting heterodimerization comprise "Fc hole" mutations Y349C, T366S, L368A and Y407V or conservative substitutions thereof in one CH3 domain; and "Fc knob" mutations S354C and T366W or conservative substitutions thereof in the other CH3 domain, wherein the amino acid numbering is according to the EU index as in Kabat.

In embodiments of any aspect of the invention, the relaxin-2A chain polypeptide comprises the sequence as set forth in SEQ ID NO: 1 or a variant thereof, and the relaxin-2B chain polypeptide comprises the sequence as set forth in SEQ ID NO: 2 or a variant thereof. In some embodiments, the relaxin-2A chain polypeptide comprises the amino acid mutations K9H, K17M or K17I, preferably K9H.

The present invention also provides a method for treating a subject suffering from heart failure combined with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimer fusion, the heterodimer fusion comprising:

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

(ii) Fcγ-con-B fusion polypeptide,

in:

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof;

Fcγ is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation S354C:T366W or a conservative substitution thereof; and

con is a linker, for example a linker polypeptide preferably having the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5],

wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX and FcY heterodimerize, and wherein the heterodimeric fusion has relaxin activity.

Similarly, the present invention provides a heterodimeric fusion for use in a method of treating a subject having heart failure with pulmonary hypertension, the heterodimeric fusion comprising:

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

(ii) Fcγ-con-B fusion polypeptide,

in:

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof;

Fcγ is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation S354C:T366W or a conservative substitution thereof; and

con is a linker, for example a linker polypeptide preferably having the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5],

wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX and FcY heterodimerize, and wherein the heterodimeric fusion has relaxin activity.

Similarly, the present invention provides a use of a heterodimeric fusion in the manufacture of a medicament for treating a subject suffering from heart failure combined with pulmonary hypertension, the heterodimeric fusion comprising:

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

(ii) Fcγ-con-B fusion polypeptide,

in:

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof;

Fcγ is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation S354C:T366W or a conservative substitution thereof; and

con is a linker, for example a linker polypeptide preferably having the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5],

wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX and FcY heterodimerize, and wherein the heterodimeric fusion has relaxin activity.

In a particularly preferred embodiment, the heterodimeric fusion comprises a fusion polypeptide having the amino acid sequence of SEQ ID NO:11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO:20.

In some embodiments of any aspect of the invention, the heterodimeric fusion further comprises one or more Fabs, optionally wherein the heterodimeric fusion comprises one Fab linked to the N-terminus of a first heterodimerization domain (e.g., a first Fc region) and a second Fab linked to the N-terminus of a second heterodimerization domain (e.g., a second Fc region).

In some embodiments of any of the aspects of the invention, the heterodimeric fusion further comprises a second relaxin A chain polypeptide or variant thereof linked to the N-terminus of the first heterodimerization domain (e.g., the first Fc region) and a second relaxin B chain polypeptide or variant thereof linked to the N-terminus of the second heterodimerization domain (e.g., the second Fc region), optionally wherein the second relaxin A chain is linked to the first heterodimerization domain (e.g., the first Fc region) via a linker polypeptide, and the second relaxin B chain is linked to the second heterodimerization domain (e.g., the second Fc region) via a linker polypeptide.

In another aspect, the present invention provides a method of treating a subject having heart failure combined with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

(i) FcX-B-L-A and FcY, optionally FcY-B-L-A; or

(ii) FcY-B-L-A and FcX, optionally FcX-B-L-A;

in:

Fcγ is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation S354C:T366W or a conservative substitution thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof;

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60],

wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX heterodimerizes with FcY, and wherein the heterodimeric fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerization domains as described herein. In some embodiments, the relaxin B chain is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

Similarly, the present invention provides a heterodimeric fusion for use in a method of treating a subject having heart failure with pulmonary hypertension, the heterodimeric fusion comprising:

(i) FcX-B-L-A and FcY, optionally FcY-B-L-A; or

(ii) FcY-B-L-A and FcX, optionally FcX-B-L-A;

in:

Fcγ is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation S354C:T366W or a conservative substitution thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof;

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60],

wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX heterodimerizes with FcY, and wherein the heterodimeric fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerization domains as described herein. In some embodiments, the relaxin A chain is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

Similarly, the present invention provides a use of a heterodimeric fusion in the manufacture of a medicament for treating a subject suffering from heart failure combined with pulmonary hypertension, the heterodimeric fusion comprising:

(i) FcX-B-L-A and FcY, optionally FcY-B-L-A; or

(ii) FcY-B-L-A and FcX, optionally FcX-B-L-A;

in:

Fcγ is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation S354C:T366W or a conservative substitution thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof;

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60],

wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX heterodimerizes with FcY, and wherein the heterodimeric fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerization domains as described herein. In some embodiments, the relaxin A chain is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

In yet another aspect, the present invention provides a method of treating a subject having heart failure combined with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

(i) FcX-A-L-B and FcY, optionally FcY-A-L-B; or

(ii) FcY-A-L-B and FcX, optionally FcX-A-L-B;

in:

Fcγ is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation S354C:T366W or a conservative substitution thereof;

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60],

wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX heterodimerizes with FcY, and wherein the heterodimeric fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerization domains as described herein. In some embodiments, the relaxin A chain is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

Similarly, the present invention provides a heterodimeric fusion for use in a method of treating a subject having heart failure with pulmonary hypertension, the heterodimeric fusion comprising:

(i) FcX-A-L-B and FcY, optionally FcY-A-L-B; or

(ii) FcY-A-L-B and FcX, optionally FcX-A-L-B;

in:

Fcγ is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation S354C:T366W or a conservative substitution thereof;

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60],

wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX heterodimerizes with FcY, and wherein the heterodimeric fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerization domains as described herein. In some embodiments, the relaxin A chain is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

Similarly, the present invention provides a use of a heterodimeric fusion in the manufacture of a medicament for treating a subject suffering from heart failure combined with pulmonary hypertension, the heterodimeric fusion comprising:

(i) FcX-A-L-B and FcY, optionally FcY-A-L-B; or

(ii) FcY-A-L-B and FcX, optionally FcX-A-L-B;

in:

Fcγ is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation S354C:T366W or a conservative substitution thereof;

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60],

wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX heterodimerizes with FcY, and wherein the heterodimeric fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerization domains as described herein. In some embodiments, the relaxin A chain is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

According to all aspects of the invention, the heart failure may be heart failure with reduced ejection fraction, heart failure with intermediate ejection fraction or heart failure with preserved ejection fraction.

According to all aspects of the invention, the subject may have a mean pulmonary artery pressure of about 25 mmHg or more and/or a right ventricular systolic pressure of about 40 mmHg or more. Typically, this is prior to treatment with the heterodimeric fusion of the invention.

According to all aspects of the invention, the subject may have a pulmonary vascular resistance of less than 3.0 Wood units. Alternatively, the subject may have a pulmonary vascular resistance of 3.0 or more Wood units. Typically, this is prior to treatment with the heterodimer fusion of the invention.

According to all aspects of the invention, the subject may have been fitted with a blood pressure monitoring device, preferably a pulmonary artery pressure monitoring device.Preferably, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device.

In some embodiments of any of the aspects of the invention, the ratio of relaxin activity of the heterodimeric fusion to the relaxin activity of a reference relaxin protein is about 0.001 to about 10.

According to all aspects of the invention, the heterodimeric fusion may be administered as a pharmaceutical composition comprising the heterodimeric fusion of the invention.

Also described herein are nucleic acid molecules (e.g., DNA molecules) encoding the heterodimeric fusions of the invention, vectors comprising the nucleic acid molecules, host cells comprising the vectors or nucleic acids, and methods of producing the heterodimeric fusions of the invention by culturing the host cells and collecting the fusion protein.

Various aspects and embodiments of the invention are set out in the accompanying claims.These and other aspects and embodiments of the invention are also described herein.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

Figure 1 shows an exemplary format of a heterodimer fusion according to some embodiments of the present invention. The format of each fusion polypeptide of the heterodimer fusion is given as FcX, FcY, A, B, con and L, wherein FcX ("Fc knob") and FcY ("Fc hole") are two Fc regions containing amino acid mutations and/or modifications that promote heterodimerization; A ("Rlx A") and B ("RlxB") are relaxin A chain and relaxin B chain polypeptides; "con" is a linker polypeptide; L is a linker polypeptide, HC X and HC Y-heavy chains of antibodies, LC-light chains of antibodies, hinge-hinge regions of antibodies, and Fab is a Fab fragment of antibodies.

Figure 2 shows LC-MS analysis of RELAX0019 and RELAX0023. A) Deglycosylated and non-reduced analysis of RELAX0019 and RELAX0023 showing the masses of intact molecules; B) Deglycosylated and reduced analysis of RELAX0019 and RELAX0023 showing the masses of individual Fc fusion chains (knob-Relaxin A chain and hole-Relaxin B chain).

Figure 3 shows the analysis of the C-terminal peptides of RELAX0019 and RELAX0023 by non-reducing peptide mapping using LC-MS. The amino acid sequence of the C-terminal peptide with predicted disulfide bonds represented by lines is shown in the top panel. Panels A and E - extracted ion chromatograms of the C-terminal peptide in the absence of a reducing agent (-DTT). Panels C and G - deconvoluted mass spectra of the C-terminal peptide in the absence of a reducing agent. Panels B and F - extracted ion chromatograms in the presence of a reducing agent (+DTT), and panels D and H - deconvoluted mass spectra in the presence of a reducing agent. Figure 3 discloses SEQ ID NOs 75, 77 and 76, respectively, in order of appearance.

FIG. 4 shows the in vitro biological activity of some heterodimeric fusions of the invention measured by cAMP induction in cells expressing recombinant human RXFP1.

Figure 5 shows in vivo pharmacokinetic (PK) curves from a series of ELISA experiments in which heterodimeric fusions of the invention were administered intravenously to mice. Data were normalized to % cMax at the 5 min time point (T1).

Figure 6 shows the reversal of isoproterenol-induced cardiac fibrosis and hypertrophy in mice treated with RELAX0019 and RELAX0023. The following levels of fibrosis and hypertrophy are shown: (1) vehicle (baseline), (2) isoproterenol, (3) isoproterenol + relaxin-2, (4) isoproterenol + RELAX0019 and (5) isoproterenol + RELAX0023.

FIG. 7 shows in vitro nonspecific binding of heterodimeric fusions of the invention in a baculovirus (BV) ELISA assay.

FIG. 8 shows the percentage of purity loss, aggregation and fragmentation of RELAX0023, RELAX0127 and RELAX0128 in solution after storage.

Figure 9 shows the stability of RELAX0023, RELAX0127 and RELAX0128 in solution over time as assessed by reduced LC-MS analysis. A) Total ion chromatogram B) Mass spectra of reduced molecules

FIG. 10 shows the PK profile of RELAX0023 in cynomolgus monkeys after intravenous and subcutaneous injections.

FIG. 11 shows nucleotide sequences encoding some of the polypeptides of the present invention (SEQ ID NOs 80-140, respectively, in order of appearance).

Figure 12 shows the results of a long-term efficacy study of RELAX0023 in cynomolgus monkeys (Macaca fascicularis) with heart failure and reduced left ventricular ejection fraction (LVEF). The effects of RELAX0023 on LVEF, heart rate (HR) and mean arterial pressure (MAP) are shown in (A), (B) and (C), respectively, in monkeys treated with relatively low, medium or high doses of RELAX0023 or vehicle control for 20 weeks, followed by an observation period. Each data point represents the mean of the group (treatment group n = 8, vehicle group n = 14). The x-axis of each of (A), (B) and (C) represents the number of weeks since the start of the treatment period.

Figures 13A-13F show cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after RELAX0023 administration: Figure 6A shows ejection fraction (EF) in HFpEF subjects, Figure 6B shows EF in HFrEF subjects, Figure 6C shows cardiac output in the combined subjects, Figure 6D shows systemic vascular resistance (SVR) in the combined subjects, Figure 6E shows stroke volume (SV) in the combined subjects, and Figure 6F shows estimated glomerular filtration rate (eGFR) in the combined subjects. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant compared to placebo (p<0.1).

Table 1: In the sequence listing, the hinge region is indicated in italics, Relaxin A is indicated in underline, Relaxin B is indicated in double underline, and the FC region is indicated in bold.

Detailed ways

Relaxin

The present invention is based, at least in part, on the discovery that the heterodimer fusions described herein can exhibit relaxin activity when the relaxin A chain and the relaxin B chain are not covalently linked to each other by an amino acid linker. This is surprising based on the disclosures of WO 2013/004607 and WO 2018/138170, which describe recombinant relaxins in which relaxin A and relaxin B single chains are fused. The present inventors have also discovered that heterodimerization of the heterodimerization domain induces correct folding and heterodimerization of the relaxin A chain and the relaxin B chain (see Example 2). Furthermore, unlike the wild-type relaxin protein, the fusion polypeptides of the present invention do not require endoproteolytic processing for biological activity.

As used herein, the term "heterodimer fusion" refers to a heterodimer of fusion polypeptides, wherein one fusion polypeptide comprises a first heterodimerization domain linked to a first subunit of a heterodimeric protein (e.g., relaxin A chain), and the other fusion polypeptide comprises a second heterodimerization domain linked to a second subunit of a heterodimeric protein (e.g., relaxin B chain).

The heterodimeric fusion of the present invention may comprise a relaxin A chain polypeptide and a relaxin B chain polypeptide from the relaxin group selected from the group consisting of relaxin-1, relaxin-2, and relaxin-3. In a preferred embodiment, the relaxin A chain polypeptide of the present invention is a relaxin-2A chain polypeptide or a variant thereof; and the relaxin B chain polypeptide of the present invention is a relaxin-2B chain polypeptide or a variant thereof. In a specific embodiment, the relaxin A chain polypeptide comprises a human relaxin-2A chain polypeptide or a variant thereof and a human relaxin-2B chain polypeptide or a variant thereof.

The terms "chain," "polypeptide," and "peptide" are used interchangeably herein to refer to a chain of two or more amino acids connected by peptide bonds.

In some embodiments, the relaxin-2A chain polypeptide has a sequence as set forth in SEQ ID NO: 1 or a variant thereof, and the relaxin-2B chain polypeptide has a sequence as set forth in SEQ ID NO: 2 or a variant thereof. The variant may comprise one or more amino acid substitutions, deletions and/or insertions. In some embodiments, the relaxin-2A chain polypeptide comprises one or more amino acid mutations selected from the group consisting of K9E, K9H, K9L, K9M, R18E, R18H, R22A, R22I, R22M, R22Q, R22S, R22Y, F23E, F23A and F23I. In a preferred embodiment, the relaxin-2A chain comprises the amino acid mutation K9H.

Relaxin A chain variants and relaxin B chain variants are known in the art. In addition, the skilled artisan can obtain guidance for the design of relaxin A chain variants and relaxin B chain variants. For example, it will be appreciated that the variants may retain those amino acids essential for relaxin function. For example, a relaxin-2 B chain variant may contain 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). Variants may contain one or more amino acid substitutions and/or insertions. For example, compared to SEQ ID NO: 62, a relaxin-2B chain variant may have one or more additional amino acids, such as K30 and R31 and N-terminal V-2, A-1 and M-1. Alternatively or in addition, the variant may comprise one or more amino acid derivatives. For example, the first amino acid of a relaxin-2B chain variant may be pyroglutamic acid.

In a preferred embodiment, the relaxin A chain and the relaxin B chain are covalently bound via two interchain disulfide bonds (see Example 2).

Relaxin family peptides mediate their biological effects at least in part by activating G protein-coupled receptors (GPCRs) and subsequently stimulating or inhibiting cAMP signaling pathways through Gs or Gi protein subunits, respectively. Relaxin-2 is known to activate GPCRRXFP1 (also known as LGR7) and to a lesser extent GPCRRXFP2 (also known as LGR8), thus stimulating the Gs-cAMP-dependent signaling pathway, leading to an increase in the second messenger molecule cAMP.

As used herein, the term "relaxin activity" refers to the ability of a relaxin molecule to bind to a relaxin receptor, and/or activate said relaxin receptor and/or initiate an intracellular signaling cascade. In embodiments where the relaxin activity is the activity of relaxin-2, relaxin activity may refer to the ability to bind to and/or activate receptors RXFP1 and/or RXFP2. The term "relaxin activity" may be used interchangeably with "biological activity".

Relaxin activity can be determined by measuring the binding of the relaxin molecule to a relaxin receptor, and/or by measuring downstream events from binding to a relaxin receptor.

Relaxin activity can be determined in vitro and/or in vivo. In some embodiments, relaxin activity is determined in vitro.

Relaxin activity can be determined by measuring the amount and/or presence of molecules downstream from receptor activation by relaxin. For example, relaxin activity can be determined by measuring cAMP production followed by measuring receptor activation by relaxin. Methods for detecting relaxin-induced cAMP production are known in the art. Such methods include cAMP ELISA, HTRF cAMP assay, and cAMP Assay. In some embodiments, relaxin activity is determined by measuring relaxin-induced cAMP production via the HTRF cAMP assay, for example, as performed in Example 3. Relaxin activity can also be determined by measuring nitric oxide (NO) production followed by receptor activation by relaxin. Relaxin activity can also be determined by measuring activation of molecules downstream of receptor activation by relaxin. For example, relaxin activity can be determined by measuring activation of p42/44 MAPK.

Alternatively or in addition, relaxin activity can be determined by measuring activation of known relaxin target genes. For example, relaxin activity can be determined by measuring activation of transcription of a known relaxin target gene (i.e., VEGF) in THP-1 cells. Methods for determining activation of gene transcription are known in the art and include quantitative PCR analysis of mRNA. Relative expression of VEGF mRNA can be measured by quantitative real-time PCR induction of VEGF transcripts followed by incubation of THP-1 cells with relaxin, as described in Xiao et al. (2013) Nat Commun. 4:1953.

Alternatively or in addition, relaxin activity can be 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 another example, relaxin activity can be determined by measuring reduction of fibrosis using Masson's Trichrome stain. In another example, relaxin activity can be determined by measuring modulation of connective tissue metabolism, for example, inhibition of profibrotic factors (such as TGF-β), inhibition of fibroblast proliferation and differentiation, and/or activation of MMP-mediated degradation of the extracellular matrix (Bathgate RA et al. (2013) Physiol Rev. 93(1):405-480).

In some embodiments, relaxin activity is determined by measuring reversal of isoproterenol-induced cardiac hypertrophy (measured as heart weight relative to tibia length) and fibrosis (measured as collagen content relative to heart weight), e.g., as performed in Example 7.

The activity of the heterodimeric fusions of the invention can be determined relative to a reference relaxin protein. In some embodiments, the reference relaxin protein is a recombinant protein. In a preferred embodiment, the reference relaxin protein is a relaxin protein having a relaxin A chain and a relaxin B chain array of a mature relaxin protein. Recombinant relaxins having a relaxin A chain and a 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 (Catalog Nos. 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 fusions of the invention, or differs from the relaxin A and B chains of the heterodimeric fusions of the invention by up to 10 amino acids, such as 1 or 2 amino acids. In some embodiments, the first amino acid of the B chain of the reference relaxin-2 is D, and this amino acid is missing in the relaxin B chain of the heterodimeric fusions of the invention.

The reference relaxin protein may be selected from:

(i) recombinant human relaxin-2 (referred to herein as RELAX0013); and

(ii) recombinant murine relaxin-1 (referred to herein as RELAX0014); and

(iii) recombinant Fc fusion relaxin-2, wherein relaxin A and relaxin B are single chain fused, and wherein the Fc is a half-life extending Fc region (referred to herein as RELAX0010 and described in WO2018/138170); and

(iv) recombinant Fc fusion relaxin-2, wherein relaxin A and relaxin B are single chain fused, and wherein the Fc is a half-life extending Fc region (referred to herein as RELAX0009 and described in WO 2018/138170); and

(v) recombinant Fc fusion relaxin-2, in which relaxin A and relaxin B are single chain fused (referred to herein as RELAX0126 and described in WO 2013/004607); and

(vi) recombinant Fc fusion relaxin-2, in which relaxin A and relaxin B are single chain fused (referred to herein as RELAX0127 and described in WO 2013/004607); and

(vii) Recombinant Fc fusion relaxin in which relaxin A and relaxin B are single chain fused (referred to herein as RELAX0128 and described in WO 2013/004607).

In a particularly preferred embodiment, the reference relaxin protein is a relaxin-2 protein having the relaxin-2A chain and relaxin-2B chain array of a mature relaxin-2 protein, as disclosed under UniProtKB/Swiss-Prot Accession No. P04090.1.

If the heterodimeric fusions of the invention exhibit at least a portion of the activity of a reference relaxin protein, they can be considered to have relaxin activity. For example, if the fusion polypeptide has at least about half the activity of a reference relaxin protein, it can be considered to have relaxin activity. If the ratio of the activity of the fusion polypeptide to the activity of the reference relaxin protein is between about ^10-5 to about 1, about ^10-4 to about 1, about ^10-3 to about 1, about ^10-2 to about 1, about 1/50 to about 1, about 1/20 to about 1, about 1/15 to about 1, about 1/10 to about 1, about 1/5 to about 1, or about 1/2 to about 1, then the heterodimeric fusions of the invention can be considered to have relaxin activity. Alternatively, a heterodimeric fusion of the invention can be considered to have relaxin activity if the ratio of the activity of the fusion polypeptide to the activity of a reference relaxin protein is between about 1 to about 10 ⁵ , about 1 to about 10 ⁴ , about 1 to about 10 ³ , about 1 to about 100, about 1 to about 50, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 2.

In some embodiments, the relaxin activity of the heterodimeric fusion is about 0.001 to about 10 of the relaxin activity of the reference relaxin protein.

Relaxin activity can be measured as an EC50 value. As used herein, the term "EC50" (half maximal effective concentration) refers to the effective concentration of a therapeutic compound that induces a response halfway between baseline and maximum after a specified exposure time.

Heterodimerization domain

The heterodimer fusion of the present invention comprises a first heterodimerization domain and a second heterodimerization domain. In a preferred embodiment, the first and second heterodimerization domains are derived from immunoglobulin Fc regions.

The term "Fc region" defines the C-terminal region of an immunoglobulin heavy chain, which is produced by papain digestion of intact antibodies. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain.

The first and second Fc regions may comprise immunoglobulin domains CH2 and/or CH3. In a preferred embodiment, the first and second Fc regions comprise immunoglobulin domains CH2 and CH3.

The Fc region can be derived from an immunoglobulin (e.g., IgG) from any species, preferably a human immunoglobulin (e.g., human IgG). In embodiments where the Fc region is derived from IgG, the Fc region can be derived from any subclass of IgG (e.g., IgG1, IgG2, IgG3, IgG4), preferably IgG1. Preferably, the first and second Fc regions are derived from human IgG1 immunoglobulins. In other embodiments, the first and second Fc regions are derived from human IgG4 immunoglobulins.

In a preferred embodiment, the first and second Fc regions contain amino acid mutations and/or modifications that promote heterodimerization. Such modifications may include introducing an asymmetric complementary modification into each of the first and second Fc regions, such that the two chains are compatible with each other and are therefore capable of forming heterodimers, but each chain cannot dimerize with itself. Such modifications may encompass insertions, deletions, conservative and non-conservative substitutions, and rearrangements. Incorporation of such modifications provides a method that increases the yield of heterodimers produced by recombinant cell culture relative to other unwanted end products (such as homodimers).

The first and second Fc regions may comprise any amino acid mutations and/or modifications known in the art that promote heterodimerization. Combinations of modifications may be used to maximize assembly efficiency while minimizing the impact on antibody stability.

In the "knob in hole" approach, heterodimerization can be promoted by introducing steric hindrance between contacting residues. A "knob" is created by replacing one or more small amino acid side chains from the interface of one Fc region ("Fc knob") with a larger side chain (e.g., tyrosine or tryptophan). A compensating "cavity" of the same or similar size to the one or more large side chains is created at the interface of the other Fc region ("Fc hole") by replacing an amino acid with a large side chain with an amino acid with a smaller side chain (e.g., alanine or valine). The "knob in hole" modification is described in detail in the following literature, for example, Ridgway JB et al. (1996) Protein Eng. 9(7) 617-621; Merchant AM et al. (1998) Nat. Biotechnol. 16(7): 677-681.

Other modifications that can be used to generate heterodimers include, but are not limited to, those that generate favorable electrostatic interactions between the two Fc regions. For example, one or more positively charged amino acids can be introduced into one Fc region, and one or more negatively charged amino acids can be introduced into the corresponding positions in the other Fc region. Alternatively or in addition, the Fc region can be modified to include mutations that introduce cysteine residues capable of forming disulfide bonds. Alternatively or in addition, the Fc region may include one or more modifications to hydrophilic and hydrophobic residues at the interface between the chains, so that the formation of heterodimers is more favorable in terms of entropy and enthalpy compared to the formation of homodimers.

Thus, in some embodiments, amino acid mutations and/or modifications that promote heterodimerization generate steric hindrance between contact residues (e.g., by a "knob-in-hole" approach), generate favorable electrostatic interactions between the two Fc regions, introduce cysteine residues capable of forming disulfide bonds, and/or modify the hydrophilic and hydrophobic residues at the interface between the two Fc regions.

In a preferred embodiment, the amino acid mutations that promote heterodimerization are "Fc knob" and "Fc hole" mutations. In a preferred embodiment, the "Fc knob" and "Fc hole" mutations are present in the CH3 domain.

In some embodiments, the first and second Fc regions are derived from human IgG1 immunoglobulin, and comprise "Fc X" and "Fc Y" having mutations in the CH3 domain, wherein the "Fc X" and "Fc Y" mutations are selected from the combinations shown in Table 2 (or conservative substitutions thereof).

Table 2: "FcX" and "Fc Y" mutations

*wherein the amino acid numbering is according to the EU index as in Kabat.

In a preferred embodiment, "Fc Y" is an "Fc hole" having mutations Y349C, T366S, L368A and Y407V or conservative substitutions thereof, and "Fc X" is an "Fc knob" having mutations S354C and T366W or conservative substitutions thereof, wherein the amino acid numbering is according to the EU index as in Kabat.

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

In some embodiments, the first Fc region has an "Fc hole" mutation and the second Fc region has an "Fc knob" mutation. In alternative and preferred embodiments, the first Fc region has an "Fc knob" mutation and the second Fc region has an "Fc hole" mutation.

It should be understood that, relative to the wild-type Fc region, these Fc regions may further comprise other amino acid modifications. The Fc region may be modified, for example, to increase the affinity of the IgG molecule to FcRn. WO 02/060919 discloses modified immunoglobulins comprising Fc regions having one or more amino acid modifications, and is incorporated herein by reference in its entirety. Methods for preparing Fc regions having one or more amino acid modifications are known in the art.

In some embodiments, the first and/or second Fc region may comprise one or more amino acid modifications, thereby reducing or eliminating the effector functions of the Fc region. In some embodiments, the amino acid modifications reduce or circumvent cytotoxicity, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

In some embodiments, the first and/or second Fc region may comprise one or more amino acid modifications to increase the half-life of the heterodimeric fusion.

In some embodiments, the first and/or second Fc region comprises at least one of the following combinations of amino acid mutations:

(i) M252Y, S254T and T256E or conservative substitutions thereof;

(ii) L234F, L235Q and K322Q or conservative substitutions thereof;

(iii) L234F, L235E and P331S or conservative substitutions thereof;

(iv) M252Y, S254T, T256E, L234F, L235Q and K322Q or conservative substitutions thereof; or

(v) M252Y, S254T, T256E, L234F, L235E and P331S or conservative substitutions thereof,

Therein the amino acid numbering is according to the EU index as in Kabat.

In some embodiments, the first and/or second Fc region may comprise the amino acid 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 Fc region comprising an "Fc hole" mutation has the sequence shown in SEQ ID NO:3 or a variant thereof, and the Fc region comprising an "Fc knob" mutation has the sequence shown in SEQ ID NO:4 or a variant thereof.

In some embodiments, the Fc region comprises a SEQ ID NO: 3 variant having an amino acid mutation Y349C reverted to Y349, and a SEQ ID NO: 4 variant having an amino acid mutation S354C reverted to S354, such that the Fc region cannot form a stable disulfide bond.

In some embodiments, the Fc region comprises a SEQ ID NO:3 variant and/or a SEQ ID NO:4 variant in which the first five residues DKTHTCPPC (SEQ ID NO:69) are modified. In some embodiments, this region is replaced with the sequence DKTHTACPPC (SEQ ID NO:70). In an alternative embodiment, this region is replaced with the sequence GGAGGACPPC (SEQ ID NO:71). In an alternative embodiment, this region is replaced with the sequence ACPPC (SEQ ID NO:72).

In alternative embodiments, the first and second heterodimerization domains are derived from immunoglobulin Fab regions. In some embodiments, the heterodimerization domain comprises CH1 and CL regions. It has been found that Fab regions comprising L and Fd chains mediate effective heterodimerization (Schoonjans R et al. (2000) J. Immunol. [Journal of Immunology] 165 (12): 7050-7057). Therefore, in alternative embodiments, the heterodimerization domain comprises L and Fd chains. In some embodiments, L and Fd chains heterodimerize to form disulfide bridge-stabilized heterodimers.

In yet another alternative embodiment, the first and second heterodimerization domains heterodimerize to form parallel coiled coils. For example, heterodimeric coiled coils are described in Aronsson et al. (2015) Sci. Rep. [Science Report] 5: 14063. In some embodiments, the heterodimerization domain comprises amino acid mutations and/or modifications to prevent the formation of an undesirable folded assembly and/or promote the formation of parallel coiled coils.

The first and second heterodimerization domains (e.g., first and second Fc regions) can form a half-life extending moiety. Thus, in some embodiments, the heterodimeric fusions of the invention have an extended half-life compared to a reference relaxin.

As used herein, the term "half-life" is used to refer to the time taken for the concentration of the fusion protein in the plasma to decrease to 50% of its original level. The "half-life" of a protein in plasma may depend on different factors, e.g., the size of the protein, its stability, its clearance rate, turnover rate, in vivo proteolytic degradation, absorption rate by the body or a specific tissue, etc. Methods for determining the half-life of a protein are known in the art and are described in the Examples below.

The inventors have demonstrated that heterodimeric fusions of the invention having first and second heterodimerization domains derived from immunoglobulin Fc have a half-life of at least 5 hours in a mouse model (see Example 6). In contrast, the half-life of human relaxin-2 in humans after IV administration is approximately 0.09+/-0.04 hours, i.e., 5.4+/-2.4 minutes (Chen SA et al. (1993) Pharm. Res. [Drug Research] 10(6):834-838).

It will be appreciated that the extended half-life is advantageous because it allows the therapeutic protein to be administered according to a safe and convenient dosing regimen (e.g., a lower dosage that can be administered less frequently). In addition, the realization of a lower dosage can provide further advantages, e.g., provide improved safety characteristics and/or activate multiple mechanisms of action in vivo.

Linker

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

The linker polypeptide can be any suitable length, for example, a length of about 6 to 40 amino acids, preferably a length of about 6 to 21 amino acids. In some embodiments, the linker polypeptide is at least 6 amino acid residues in length, preferably at least 11 amino acids in length, preferably at least 16 amino acids in length. In some embodiments, the linker polypeptide is less than 40 amino acids in length. Linker polypeptides with different or identical lengths can be used for each arm of the heterodimer fusion of the present invention. In some embodiments, at least one linker polypeptide is 21 amino acids in length. In a preferred embodiment, the length of both linker polypeptides is 21 amino acids. The linker polypeptide can have any amino acid sequence. Linker polypeptides with different or identical amino acid compositions can be used for each arm of the heterodimer fusion of the present invention.

In some embodiments, one or preferably two linker polypeptides comprise the proline and alanine repeat sequence (PA)x (SEQ ID NO:73), preferably wherein x is 3 to 15, preferably wherein the linker polypeptide is greater than 16 amino acids in length, preferably wherein the linker polypeptide consists of: the 21 amino acid sequence PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO:6).

In some embodiments, one or preferably both linker polypeptides comprise glycine and serine repeats, such as those described in Chen X et al. (2013) Adv. Drug. Deliv. Rev. 65(10): 1357-1369. In some embodiments, one or both linker polypeptides comprise the motif (GGGGS)n (SEQ ID NO: 74), wherein n may be between 1 and 8, such as wherein n is 4. In some embodiments, one or more linker polypeptides consist of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5). In certain embodiments, two linker polypeptides consist of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5).

In some embodiments, one linker polypeptide comprises a proline and alanine repeating sequence as described herein, and the other linker polypeptide comprises a glycine and serine repeating sequence as described herein.

Alternatively, one or both of the relaxin A and B chains can be linked to their respective heterodimerization domains via a synthetic linker polypeptide, such as a polyethylene glycol (PEG) polymer chain. Thus, the relaxin A chain can be linked to a first heterodimerization domain (e.g., a first Fc region) via a synthetic linker, such as a polyethylene glycol (PEG) polymer chain, and the relaxin B chain can be linked to a second heterodimerization domain (e.g., a second Fc region) via a synthetic linker, such as a polyethylene glycol (PEG) polymer chain, wherein the synthetic linker can be covalently or non-covalently attached to the heterodimerization domain (e.g., Fc region). Pegylation, which is the process of attaching a PEG polymer chain to a molecule, can be performed according to methods known in the art.

stability

The inventors of the present invention have demonstrated that the heterodimeric fusions of the present invention have unexpectedly superior physical and chemical stability. Thus, in some embodiments, the heterodimeric fusions of the present invention have superior physical and/or chemical stability compared to a reference relaxin protein.

The physical stability of relaxin can be determined by measuring purity and aggregation, for example, by HP-SEC as in Example 9. The chemical stability of relaxin can be determined by measuring fragmentation and modification of the molecule, for example, by LC-MS as in Example 9.

Surprisingly, the present inventors have demonstrated that the heterodimeric fusions of the present invention have superior physical and chemical stability compared to recombinant Fc fusion relaxins in which relaxin A and relaxin B are single chain fused (as opposed to relaxin A and B being in separate fusion polypeptides). WO 2013/004607 describes recombinant single chain relaxin fusion polypeptides fused to an immunoglobulin Fc region, e.g., fusion polypeptides referred to herein as RELAX0127 and RELAX0128. Thus, in some embodiments, the heterodimeric fusions of the present invention have superior physical and/or chemical stability compared to RELAX0127 and RELAX0128.

In addition to the first and second heterodimerization domains, the heterodimer fusion may also include a half-life extending moiety. In some embodiments, the half-life extending moiety is a protein half-life extending moiety. The protein half-life extending moiety may be selected from the group consisting of: an Fc region of an immunoglobulin, an albumin binding domain, and serum albumin. In further embodiments, the half-life extending moiety is a chemical entity other than a protein or peptide, such as a polyethylene glycol (PEG) polymer chain.

The half-life extension portion can be attached to the N-terminus or C-terminus of the first or second heterodimerization domain. In some embodiments, the half-life extension portion is attached to the N-terminus of the first or second heterodimerization domain. In other embodiments, the half-life extension portion is attached to the C-terminus of the first or second heterodimerization domain. Methods for attaching the half-life extension portion to heterodimer fusions are known in the art. For example, the half-life extension portion can be attached by chemical conjugation or recombinant technology. The half-life extension portion can be attached to the heterodimer fusion directly or through a linker (e.g., a linker polypeptide). When the fusion polypeptide comprises a protein half-life extension portion such as an Fc region, the use of a linker polypeptide may be particularly suitable.

Exemplary Embodiments

The heterodimeric fusions of the invention can have a variety of formats and/or sequences.

The terms "fusion polypeptide of the invention and fusion polypeptides of the invention" may be used to refer to a first heterodimerization domain fused to the relaxin A chain, and/or a second heterodimerization domain fused to the relaxin B chain. The fusion polypeptide of the invention may be a recombinant fusion polypeptide, i.e., it has been produced by recombinant DNA technology.

In a preferred embodiment, the C-terminus of the first heterodimerization domain (e.g., the first Fc region) is linked to the N-terminus of the relaxin A chain, and the C-terminus of the second heterodimerization domain (e.g., the second Fc region) is linked to the N-terminus of the relaxin B chain. In some embodiments, the relaxin A chain polypeptide and/or the relaxin B chain polypeptide have a free C-terminus.

In alternative embodiments, the N-terminus of the first heterodimerization domain (e.g., the first Fc region) is linked to the C-terminus of the relaxin A chain, and the N-terminus of the second heterodimerization domain (e.g., the second Fc region) is linked to the C-terminus of the relaxin B chain. In some embodiments, the relaxin A chain polypeptide and/or the relaxin B chain polypeptide have a free N-terminus.

The heterodimer fusion of the present invention may further comprise one or more Fabs. In some embodiments, the heterodimer fusion comprises one Fab connected to the N-terminus of the first heterodimerization domain (e.g., the first Fc region) and a second Fab connected to the N-terminus of the second heterodimerization domain (e.g., the second Fc region).

The heterodimeric fusions of the present invention may further comprise a second relaxin A chain polypeptide or variant thereof and a second relaxin B chain polypeptide or variant thereof. In some embodiments, the second relaxin A chain polypeptide or variant thereof is linked to the N-terminus of the first heterodimerization domain (e.g., the first Fc region), and the second relaxin B chain polypeptide or variant thereof is linked to the N-terminus of the second heterodimerization domain (e.g., the second Fc region), optionally wherein the second relaxin A chain is linked to the first heterodimerization domain (e.g., the first Fc region) via a linker (e.g., a linker polypeptide), and the second relaxin B chain is linked to the second heterodimerization domain (e.g., the second Fc region) via a linker (e.g., a linker polypeptide).

Thus, in some embodiments, the format of the heterodimer fusion is selected from:

(i) FcX-con-A/FcY-con-B (see, for example, FIG1 );

(ii) FcX-con-B/FcY-con-A (see, for example, Figure 1 );

(iii) A-con-FcX/B-con-FcY (see, for example, Figure 1 );

(iv) B-con-FcX/A-con-FcY (see, for example, Figure 1 );

(v) Fab-FcX-con-A/Fab-FcY-con-B (see, for example, FIG. 1 );

(vi)Fab-FcX-con-B/Fab-FcY-con-A;

(vii) A-con-FcX-con-A/B-con-FcY-con-B (see, for example, Figure 1 );

(viii)B-con-FcX-con-B/A-con-FcY-con-A;

(ix) FcX-con-B-L-A and FcY, optionally FcY-con-B-L-A (see, e.g., Figure 1 );

(x) FcY-con-B-L-A and FcX, optionally FcX-con-B-L-A;

(xi) FcX-con-A-L-B and FcY, optionally FcY-con-A-L-B; and

(xii) FcY-con-A-L-B and FcX, optionally FcX-con-A-L-B,

in:

Fcγ is an immunoglobulin Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having an amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof;

FcX is an Fc region with an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain with an amino acid mutation S354C:T366W or a conservative substitution thereof;

“con” is the linker polypeptide;

B is relaxin B chain or a variant thereof;

A is relaxin A chain or a variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60).

In another aspect, the invention provides a heterodimer fusion comprising:

(i) X-B-L-A and Y, optionally Y-B-L-A; or

(ii) Y-B-L-A and X, optionally X-B-L-A;

in:

X and Y are heterodimerization domains as described herein;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof;

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60),

wherein X heterodimerizes with Y, and wherein the heterodimeric fusion has relaxin activity.

In yet another aspect, the present invention provides a heterodimeric fusion comprising:

(i) X-A-L-B and Y, optionally Y-A-L-B or

(ii) Y-A-L-B and X, optionally X-A-L-B,

in:

X and Y are heterodimerization domains as described herein;

A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof;

B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60),

wherein X heterodimerizes with Y, and wherein the heterodimeric fusion has relaxin activity.

In a particularly preferred embodiment according to all aspects of the present invention, the heterodimeric fusion comprises the fusion polypeptide Rlx011DD as shown in SEQ ID NO: 11 and Rlx014DD as shown in SEQ ID NO: 20. This heterodimeric fusion may also be referred to as "RELAX0023" or "AZD3427".

In an alternative preferred embodiment, the heterodimeric fusion comprises the fusion polypeptide Rlx013DD as shown in SEQ ID NO:17 and Rlx012DD as shown in SEQ ID NO:14.

In one aspect of the present invention, a heterodimeric fusion is provided, which comprises a fusion polypeptide combination selected from the combination of FcX and FcY shown in Table 3.

Table 3: Combinations of fusion polypeptides in the heterodimer fusions of the present invention

*Table 1 shows the sequences of the listed fusion polypeptides.

**In this particular embodiment, the heterodimeric fusion is an IgG and comprises an additional polypeptide corresponding to the light chain set forth in SEQ ID NO:54.

According to all aspects of the present invention, there is provided a heterodimeric fusion comprising the fusion polypeptides shown as SEQ ID NO: 11 and SEQ ID NO: 20 for use in a method of treating a subject with heart failure and pulmonary hypertension, as described herein.

Alternatively, according to all aspects of the present invention, there is provided a heterodimeric fusion comprising the fusion polypeptides shown as SEQ ID NO: 17 and SEQ ID NO: 14 for use in a method of treating a subject with heart failure and pulmonary hypertension, as described herein.

The fusion polypeptides of the present invention can be produced by any method known in the art. In some embodiments, the fusion polypeptides of the present invention are produced by recombinantly expressing a nucleic acid molecule encoding the fusion polypeptide in a host cell.

Methods known to those skilled in the art can be used to construct expression vectors containing nucleic acid molecules encoding the fusion polypeptide of the present invention. Suitable vectors include, for example, plasmids, phagemids, phages or viral vectors.

The vector containing the nucleic acid molecule encoding the fusion polypeptide of the present invention can be transferred to the host cell by conventional techniques. Suitable host cells are known in the art. The host cell can be a mammalian cell, such as HEK293 cell or CHO cell.

The transfected cells can be cultured by conventional techniques to produce the fusion polypeptides of the invention.

Once the fusion polypeptide of the present invention has been produced, for example, by recombinant expression, it can be purified by any method known in the art. Exemplary protein purification techniques include chromatography (e.g., ion exchange chromatography, affinity chromatography and/or size column chromatography), centrifugation, and differential solubility. The present disclosure provides an isolated fusion polypeptide, which is optionally separated from a cell culture by at least one purification step.

treatment method

The fusion polypeptides of the present invention may be provided in pharmaceutical compositions.

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

The present invention relates to a method for treating a subject with heart failure combined with pulmonary hypertension by administering a heterodimer fusion (or a pharmaceutical composition) as described herein, as well as the use of the heterodimer fusion (or the pharmaceutical composition), and the heterodimer fusion (or the pharmaceutical composition) for use in the method. In particular, the subject can be an animal, preferably a mammal, and more preferably a human.

The use or method may comprise administering a therapeutically effective regimen having less frequent doses of the heterodimer fusion/fusion polypeptide of the invention than a therapeutically effective dosing regimen of a wild-type relaxin molecule.

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" may also include more specific diagnoses, such as heart failure with preserved ejection fraction (HFpEF), heart failure with intermediate ejection fraction, or heart failure with reduced ejection fraction (HFrEF). This may also include heart failure caused by hypertrophic cardiomyopathy or dilated cardiomyopathy.

As used herein, the term "pulmonary hypertension" may be defined as a subject having a mean pulmonary artery pressure of about 20 mmHg or more, preferably 25 mmHg or more, typically when the subject is at rest. It may also be defined as a mean pulmonary artery pressure of about 30 mmHg or more, typically when the subject is or has recently been exercising. Thus, the subject's mean pulmonary artery pressure may be in the range of about 20 mmHg to about 30 mmHg, preferably about 25 mmHg to about 30 mmHg or more. Alternatively or in addition, the subject may have:

a. Right ventricular systolic pressure of about 40 mmHg or higher;

b. Pulmonary artery wedge pressure (PAWP) greater than 15 mmHg; and/or

c. Pulmonary vascular resistance as follows:

i. less than 3.0 Wood units; or

ii.3.0 or more Wood units.

Therefore, in some cases, pulmonary hypertension can be classified as group 2 pulmonary hypertension as defined by the World Health Organization. This can also be called "heart failure due to left heart disease combined with pulmonary hypertension." In other cases, pulmonary hypertension can be classified as group 1 pulmonary hypertension as defined by the World Health Organization (see Ryan et al., 2012, Pulm. Circ. [Pulmonary Circulation] 2(1): 107-121).

Parameters of pulmonary hypertension and heart failure can be measured or estimated using techniques known in the art. For example, these techniques include echocardiography, pulmonary artery catheters, and implantable monitoring devices. In certain embodiments, the subject may have been equipped with a blood pressure monitoring device as known in the art, preferably a pulmonary artery pressure monitoring device. In a specific embodiment, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. Typically, the device is equipped before treatment with the heterodimer fusions of the present invention as described herein. Alternatively, the subject is equipped with the device during or after treatment.

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

"Treatment" refers to improving and/or eliminating one or more symptoms or causes of the target disease. In some embodiments, this may involve adjusting the level of one or more biomarkers or functions to a non-disease range (compared to a healthy cohort). For example, the heterodimer fusion of the present invention can reduce the pulmonary vascular resistance (PVR) of the subject. For example, compared to the baseline PVR (before the heterodimer fusion of the present invention is administered to the subject), the PVR after treatment can be reduced by at least 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, or 1% to 50% or more. Therefore, compared to the baseline PVR (before the heterodimer fusion of the present invention is administered to the subject), the heterodimer fusion of the present invention can reduce the PVR of the subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more. In addition or alternatively, the heterodimer fusion of the present invention can reduce the mean pulmonary artery pressure (mPAP) of the subject. For example, mPAP can be reduced by at least 1 mmHg to 15 mmHg or more. Thus, the heterodimer fusions of the present invention can reduce the mean pulmonary artery pressure of 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 or more. Similarly, the heterodimer fusions of the present invention can reduce the estimated pulmonary artery diastolic pressure (ePAD) of a subject. For example, the ePAD can be reduced by at least 1 mmHg to 15 mmHg or more. Thus, the heterodimer fusions of the invention can reduce the estimated pulmonary artery diastolic pressure of 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 or more. Additionally or alternatively, the heterodimer fusions of the invention can increase the ejection fraction percentage (EF%) of a subject as a measure of cardiac output. For example, EF% can be increased by at least 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, or 1% to 50% or more. Thus, the heterodimer fusion of the present invention can increase the ejection fraction percentage (EF%) of a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more. Additionally or alternatively, in a subject, the heterodimer fusion of the present invention can:

a) Increase the heart's stroke volume (SV);

(b) reduce systemic vascular resistance (SVR) and/or increase estimated glomerular filtration rate (eGFR);

(c) increase ejection fraction; and/or

(d) increase cardiac output;

Compared with pre-administration baseline levels, the decrease in SVR and the increase in eGFR combined indicate improved organ perfusion.

Thus, in a subject, the heterodimeric fusions of the invention may:

a) Reduce PVR;

(b) decrease in mPAP;

(c) reduce ePAD;

(d) Increase the cardiac output (SV);

(e) reduce systemic vascular resistance (SVR) and/or increase estimated glomerular filtration rate (eGFR);

(f) increase ejection fraction; and/or

(g) Increase cardiac output;

Compared to the baseline level before administration. The reduction in SVR and the increase in eGFR combined indicate that organ perfusion is improved. Changes in one or more or all of these parameters can each occur after 1-24 weeks of treatment. In some embodiments, changes in one or more or all of these parameters occur after 24 weeks of treatment.

In certain embodiments, a reduction in mPAP as described herein may improve dyspnea as described in Solomonica A et al. (2013) Circ Heart Fail. 6:53-60.

The fusion polypeptides (thus including heterodimeric fusions) and/or pharmaceutical compositions of the invention are suitable for parenteral administration to a subject or patient. In some embodiments, the subject or patient is a mammal, particularly a human.

Wild-type human relaxin-2 has an in vivo half-life of the order of minutes. Therefore, it must be administered by continuous intravenous infusion in hospitalized patients and exhibits severe side effects, including decreased blood pressure. In contrast, it will be appreciated that embodiments of the fusion polypeptides (thus including heterodimeric fusions) and/or pharmaceutical compositions of the present invention can be administered to a subject or patient by injection (e.g., by intravenous, subcutaneous, or intramuscular injection). In some embodiments, the fusion polypeptides (thus including heterodimeric fusions) and/or pharmaceutical compositions are administered by subcutaneous injection. Administration by injection (e.g., by subcutaneous injection) provides the benefit of greater comfort to the subject or patient and provides the opportunity to administer to a subject or patient outside of a hospital setting. In some embodiments, the fusion polypeptides (thus including heterodimeric fusions) or pharmaceutical compositions are administered by self-administration.

In some embodiments, the fusion polypeptides of the invention (thus including heterodimeric fusions) have an increased half-life compared to wild-type relaxin, which allows for lower total exposure based on molar concentration. For example, the fusion polypeptides of the invention (thus including heterodimeric fusions) can be administered less frequently than wild-type relaxin, thereby providing a more convenient dosing regimen.

A kit comprising the pharmaceutical composition of the present invention can be provided. The kit may include packaging containing the pharmaceutical composition of the present invention and instructions. In certain embodiments, the pharmaceutical composition of the present invention is formulated in a single dose vial or a container closure system (e.g., a prefilled syringe). Optionally, associated with one or more of these containers may be an announcement in the form of regulations of a government agency that manages the manufacture, use or sale of a drug or biological product, which reflects that the agency is administered for human use, to permission for manufacture, use or sale.

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

"About" can generally mean an acceptable degree of error for a measured amount given the nature or precision of the measured value. Exemplary degrees of error are within a percentage (%) of a given value or range of values, typically within 10%, and more typically within 5%.

Embodiments described herein as “comprising” one or more features may also be considered as disclosure of corresponding embodiments “consisting of such features”.

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

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

The above embodiments are to be understood as illustrative examples. Additional embodiments are contemplated. It should be understood that any feature described with respect to any one embodiment may be used alone or in combination with other features described, and may also be used in combination with one or more features of any other embodiment, or any combination of any other embodiment. In addition, equivalents and modifications not described above may also be employed without departing from the scope of the invention as defined by the appended claims.

Other examples and variations of the fusion polypeptides and methods described herein will be apparent to those skilled in the art in light of the present disclosure.

Other examples and variations are within the scope of the present disclosure as indicated in the appended claims.All documents cited herein are incorporated by reference in their entirety, including all data, tables, figures, and text presented in the cited documents.

Examples

Example 1: Production of recombinant heterodimeric Fc relaxin-2 fusion protein

The Fc relaxin-2 fusion proteins described herein have been designed using the heterodimerization properties of the knob-and-hole Fc domain (Fc knob and Fc hole) to induce the correct folding and heterodimerization of the A and B chains of relaxin-2.

More precisely, the relaxin-2 A and B chains have been genetically fused to two complementary Fcs (at the N- and/or C-termini of the Fc) via a linker, as shown in Figure 1. CHO cells were then co-transfected with two expression vectors, each containing a single Fc-relaxin chain (A and/or B). The two complementary Fc parts assembled within the CHO cells and thus facilitated the assembly and correct folding of relaxin-2. As demonstrated in Example 2 below, disulfide bonds were then formed between the complementary Fc chains and between the A and B chains, thereby reproducing the native relaxin-2 structure.

Heterodimeric Fc-Relaxin-2 fusion protein was secreted in the supernatant and then purified by affinity chromatography using an automated system, where the Fc region of the protein was bound to a column matrix.

Example 2: LC-MS analysis of Fc relaxin-2 knob-hole heterodimer

LC-MS analysis was performed on both non-reduced and reduced deglycosylated Fc-Relaxin-2 heterodimers. For deglycosylation, samples were diluted to 1 mg/ml and buffered with 10 mM Tris-Cl at pH 7.80. Peptide-N-glycosidase F (PNGase F) (Roche) was added to the samples at a concentration of 1 unit of enzyme/50 μg of Fc-Relaxin-2 and incubated overnight at 37°C. For non-reduced analysis, samples were diluted to 0.05 mg/ml in water and 20 μL was loaded into a LC-MS certified total recovery vial with a pre-slit cap (Waters part number: 186005663CV). For reduced analysis, 10 mM TCEP was added and the samples were incubated at 37°C for an additional 30 minutes before analysis.

Experiments were performed using an ACQUITY I-Class UPLC (Waters Corporation, Milford, MA) coupled to a Xevo G2-XS Q-TOF instrument, both operated using the UNIFI Scientific Information System. For the LC system, solvent A was water containing 0.1% formic acid, and solvent B was acetonitrile containing 0.1% formic acid (both UPLC-MS grade, BioSolve). The UV detector was set to measure at wavelengths of 220 nm and 280 nm, and the vial was placed in a sample compartment maintained at 4°C. A volume of 1 μL was injected onto a reverse phase ACQUITY UPLC protein BEH C4 column, pore column (Waters p/n: 186004495) and the protein was eluted using an increasing gradient from 5% to 75% solvent B over 6 minutes.

The mass spectrometer was calibrated from 500-5000 m/z by infusion of 2 μg/μL sodium iodide in 50% 2-propanol, and the lockspray was 200 pg/μL Leucine Enkephalin. The instrument was operated in positive ionization mode and sensitivity analyzer mode, with the following key settings: capillary voltage = 3.0 V; sample cone voltage = 40 V; source temperature = 120°C; desolvation temperature = 450°C; cone gas flow = 120 L/h; desolvation gas flow = 1000 L/h; mass range = 500-5000 m/z, scan time = 1.0 sec.

Data were processed in UNIFI software. Spectra were merged from the retention times in the chromatograms where the target protein was eluted. Raw data were background subtracted and deconvoluted using the MaxEnt1 algorithm for macromolecules. The experimental data were compared with the quality of the theoretical sequence, in which disulfide bonds were considered for non-reduced analysis and free cysteine was considered for reduced analysis. After peptide-N-glycosidase F deglycosylation, deamidation of asparagine (+1Da) was also considered.

LC-MS analysis confirmed the formation of disulfide bonds between complementary Fc chains and between the A and B chains, reproducing the native relaxin-2 structure. As an example, FIG2A shows LC-MS data for RELAX0019 and RELAX0023. Non-reducing analysis confirmed the formation of heterodimers with the expected masses of 58932 Da and 59361 Da for RELAX0019 and RELAX0023, respectively: no homodimers were detected. Reducing analysis ( FIG2B ) confirmed the sequence identity of the two chains and showed that they had no modifications.

Non-reducing peptide mapping for identification of disulfide bonds

Heterodimer Fc-Relaxin (50 μg) was placed into a clean sample tube and diluted in 17 μL of 100 mM sodium phosphate (pH 7.0). Alkylation of free cysteines was achieved by adding 0.5 μL of 5 mg/ml iodoacetamide followed by incubation at room temperature for 20 minutes. After alkylation, an additional 2.5 μL of 100 mM sodium phosphate buffer (pH 7.0) and 2.5 μL of sodium chloride were added. The protein was denatured by adding 40 μL of 8.0 M guanidine hydrochloride and incubating at 37°C for 30 minutes. Dilution was achieved by adding 125 μL of 100 mM sodium phosphate buffer (pH 7.0) followed by 0.5 μL of 40 mM EDTA. Endoproteinase Lys-C (Wako Chemicals) was reconstituted in water at a concentration of 1 mg/ml and 5 μL was added to Fc-Relaxin-2. Digestion was performed at 37°C for 2 hours, after which time an additional 5 μL of Lys-C was added and incubation continued for another 2 hours. For peptide analysis, 42.5 μL of sample was transferred to a UPLC vial and 2.5 μL of water was added. For reduction of disulfide bonds, 2.5 μL of 500 mM DTT was added to another 42.5 μL aliquot of the sample and left at room temperature for 15 minutes before LC-MS analysis.

Peptide analysis was performed using an ACQUITY I-Class UPLC (Waters Corporation, Milford, MA) coupled to a Xevo G2-XS Q-TOF instrument, both operated using the UNIFI Scientific Information System. For the LC system, solvent A was water containing 0.1% formic acid, and solvent B was acetonitrile containing 0.1% formic acid (both UPLC-MS grade, Quanqing Company). The UV detector was set to measure at a wavelength of 214 nm, and the vial was placed in a sample chamber maintained at 4°C. A volume of 10 μl was injected into a reverse phase ACQUITY BEH C18 pore column (Waters p/n: 186003687) and the protein was eluted using an increasing gradient of solvent B from 5% to 37% B over 73.5 min and then increasing to 60% B over an additional 2.5 min. After 77.5 min, the column was held at 95% B for 5 min.

The mass spectrometer was calibrated from 100-2600 m/z by infusion of 2 μg/μL sodium iodide in 50% 2-propanol, and the lockspray was 200 pg/μL leucine enkephalin. The instrument was operated in positive ionization mode and sensitivity analyzer mode, with the following key settings: capillary voltage = 3.0 V; sample cone voltage = 25 V; source temperature = 100°C; desolvation temperature = 250°C; cone gas flow = 0 L/h; desolvation gas flow = 500 L/h; mass range = 100-2600 m/z, scan time = 0.5 sec.

Data were processed in UNIFI software by inputting sequences with expected disulfide bonds and searching for matching Lys-C generated peptides. Chromatograms obtained in the absence and presence of reducing agent were overlaid to verify that the identified disulfide bonded peptides were no longer observed once reduced.

A peptide matching the expected mass for a disulfide bonded relaxin-2 peptide incorporating both the A and B chains was identified, as shown at the top of Figure 3 (SLSLSPGGGGGSGGGGSGGGGSGGGGGSQLYSALANKCCHVGCTK=LCGRELVRAQIAICGMSTWS=RSLARFC (SEQ ID NOs 75-77, respectively), expected mass 6836.23 Da including 3 disulfide bonds). Figure 3 (A-D) shows the identification of this peptide for RELAX0019, and confirms that the peptide is no longer observed when a reducing agent is added: panels A and B show extracted ion chromatograms in the absence and presence of DTT, and panels C and D show the corresponding mass spectra of the peptide. Figure 3 (E-H) shows the identification of the same peptide for RELAX0023 and confirms that the peptide is no longer observed when a reducing agent is added: panels E and F show extracted ion chromatograms in the absence and presence of DTT, and panels G and H show the corresponding mass spectra of the peptide. These data confirm that the relaxin A and B chains interact through disulfide bonds within the heterodimers RELAX0019 and RELAX0023.

Example 3: In vitro activity of Fc-Relaxin-2 fusion protein (cell-based cAMP activity assay)

Relaxin-2 fusion polypeptides produced as described above are tested for biological activity, such as stimulation of one or more cellular receptor responses, by the following methods.

Stable cell lines expressing human or mouse receptors produced in CHO cells were purchased from DiscoverX.

-cAMP Hunter ^TM CHO-K1 RXFP1 Gs, cell line (DiscoVocans catalog number 95-0127C2)

-cAMP Hunter ^TM CHO-K1 RXFP2 Gs cell line (DiscoVocas catalog number 95-0140C2)

-cAMP Hunter ^TM CHO-K1 mRXFP1 Gs cell line (DiscoVocas catalog number 95-0180C2)

Activation of these receptors results in the downstream production of the cAMP second messenger which can be measured in functional activity assays.

Conventional cAMP assays were performed using the following bovine serum albumin (BSA) based assay buffer: Hank's Balanced Salt Solution (Sigma #H8264) supplemented with 0.1% BSA (Sigma #A9418) and 0.5 mM IBMX (Sigma #I7018), adjusted to pH 7.4 with 1 M NaOH.

The frozen vials of cells expressing the target receptor are thawed rapidly in a water bath, transferred to a pre-heated cell culture medium and spun at 240xg for 5 minutes. The cells are resuspended in a cell culture medium with an optimized concentration (e.g., 3.33x ¹⁰⁴ cells/mL of hRXFP1), and 30 μL of cell suspensions are added to a 384-well plate (Greiner #781946) coated with poly-D-lysine, and allowed to adhere overnight. The next day, the culture medium is removed from the plate and replaced with 5uL of assay buffer. Using a contactless liquid dispenser (ECHO ^TM , Labcyte), 11-point serial dilutions of the test recombinant peptide or Fc fusion sample are added to the cells. All sample dilutions are made in duplicate. In each well, an additional 5 μL of assay buffer is added, and the plate is incubated at room temperature for 30 minutes.

cAMP levels were measured using a commercially available cAMP dynamics _GS HTRF kit (Cisbio, catalog number 62AM4PEJ) following a two-step protocol as recommended by the manufacturer. Briefly, anti-cAMP cryptate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were prepared separately by diluting each 1/20 in the conjugation and lysis buffer provided in the kit. 5 μL of anti-cAMP cryptate was added to all wells of the assay plate, and 5 μL of cAMP-d2 was added to all wells except the non-specific binding (NSB) wells, to which conjugation and lysis buffer was added. The plate was incubated at room temperature for one hour and then read on an Envision (PerkinElmer) using an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. Data were converted to % ΔF and then to % activation of the maximum native agonist response as described in the manufacturer's instructions and analyzed by a 4-parameter logistic fit to determine EC50 values. In the case of hRXFP1 cells, these results were compared with those of recombinant h-Relaxin-2 (R&D Systems, Catalog No. 6586RN); in the case of mRXFP1 cells, with those of m-Relaxin-1 (R&D Systems, Catalog No. 6637RN); and in the case of hRXFP2 cells, with those of INSL3 (R&D Systems, Catalog No. 4544NS).

Data analysis was performed using statistical analysis software (GraphPad Prism, version 6).

The biological activities of the tested constructs are provided in Table 4 and Figure 4. Table 4 has summarized the average EC50 measurements for both recombinant human relaxin-2 and the fusion polypeptide from several assays.

RELAX0013, RELAX0014 and RELAX0010 are reference proteins, wherein RELAX0013 is recombinant human relaxin-2, RELAX0014 is recombinant mouse relaxin-1, and RELAX0010 is a single-chain fusion protein comprising an A chain, a 15 amino acid linker, a B chain, a 15 amino acid linker, and an Fc comprising the amino acid sequence of SEQ ID NO. 8, described in WO2018/138170.

Table 4: Biological activity of heterodimeric Fc relaxin fusion polypeptides (n: number of repeat sequences).

From the results presented in Table 4, it can be concluded that the heterodimeric Fc relaxin fusion proteins tested were less potent than single chain fusion RELAX0010 or recombinant human relaxin-2 peptide, but they still retained high levels of bioactivity (ranging from about 10 pM to about 80 pM in the human RXFP1 cell line).

These results show that relaxin A and B chains can be fused to either/both termini (linkers can be attached to the N or C termini of the relaxin chains) and either chain of the heterodimeric Fc (X or Y) and retain biological activity. Thus, the format of the heterodimeric Fc relaxin fusion protein described herein constitutes a robust format for the production of long half-life active relaxin.

The presence of disulfide bonds used to stabilize the heterodimeric Fc did not affect the potency of the fusion protein (compare RELAX0023 to RELAX0021 and RELAX0024 to RELAX0022).

The two upper hinge regions used (GGAGGA (SEQ ID NO: 78) and native DKTHT (SEQ ID NO: 79)) did not affect potency (compare RELAX0023 to RELAX0019 and RELAX0024 to RELAX0020). The exact amino acid sequence of the upper hinge is not critical for the activity of the fusion protein.

Example 4: Effect of linker composition and length in heterodimeric relaxin-2 Fc fusion proteins

The linker can be composed of glycine and serine residues (GS), or can be composed of proline and alanine repeats (PA). The length of the linker used herein is 6 to 21 residues. An example of a long GS linker is: GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5) (21 amino acids). An example of a long PA linker is: PAPAPAPAPAPAPAPAPAG (SEQ ID NO: 6) (21 amino acids).

Linkers of varying lengths and compositions can be placed on each Fc chain of the heterodimeric relaxin-2 Fc fusion polypeptide.

Examples of heterodimeric relaxin-2 Fc fusion proteins with various linkers are shown in Table 5. This table also shows information on developability/manufacturability (expression yield and percentage of monomeric/non-aggregated relaxin-2 Fc fusion protein after protein A capture from cell culture supernatant) and biological activity.

Table 5: Effect of linkers on biological activity and developability properties of heterodimeric Fc-Relaxin-2 fusion proteins during small-scale expression.

The length and composition of the linker does have an impact on the developability aspects of the molecule. As shown in Table 5, heterodimeric relaxin-2 Fc fusion polypeptides with a PA linker of less than or equal to 16 amino acids were not well expressed. In contrast, a 21 residue long PA linker significantly increased expression yields. Constructs with a GS linker had more consistent expression yields.

Heterodimeric relaxin-2 Fc fusion proteins with short and asymmetric (different) linkers retained potency. Reduced biological activity was only observed in fusion proteins with low monomer content (RELAX0109, RELAX0110 and RELAX0111).

Example 5: Point mutations in the relaxin-2 sequence

Relaxin single point mutation analogs were prepared as heterodimeric Fc relaxin-2 fusion proteins. Table 6 shows examples of such molecules that retain potency and favorable developability properties.

The targeted native residues are positively charged and likely susceptible to proteolysis but are not involved in the binding of relaxin to its receptor.

For example, the R22X analog of the heterodimeric Fc relaxin-2 fusion protein appears to consistently have improved developability/manufacturability properties.

Table 6: Examples of relaxin-2 analogs that retained potency and favorable developability properties during small-scale expression.

The results presented in Table 6 demonstrate that some variability in the amino acid sequence of the relaxin-2A chain is tolerated without loss of potency while retaining favorable developability properties.

Example 6: PK curve of Fc-Relaxin-2 fusion protein

The pharmacokinetic (PK) profile of relaxin-2 fusion polypeptides was determined using relaxin ELISA assays and/or cAMP assays. Relaxin-2 fusion polypeptides were administered at 6 mg/kg to 6-10 week old male C57BL/6J (Jax) mice (Jackson Laboratories) by subcutaneous (SC) and/or intravenous (IV) routes. For IV route administration, serum samples were collected at 5 minutes, 30 minutes and 60 minutes after drug administration, followed by 3 hours and/or 6 hours and/or 8 hours and 24 hours, followed by a series of minimum 1 day intervals to a maximum of 21 days. For SC route administration, a similar schedule was followed with less frequent collections within the first 8 hours; for example, the first sample was collected at 30 minutes, followed by collections at 3 hours, 8 hours, 24 hours, 30 hours and 48 hours, followed by a series of minimum 1 day intervals to a maximum of 21 days. Samples were collected by cardiac puncture into serum tubes and kept at room temperature for 15 to 30 minutes and then centrifuged at 10000 rpm for 10 minutes within 30 minutes of collection. Aliquots of samples were stored at <-80°C and tested later by ELISA or cAMP activity assay.

For most molecules, PK samples were tested in ELISA using anti-h-Relaxin-2 capture (pre-coated human relaxin-2 Quantikine ELISA kit, R&D Systems, catalog number DRL200) and anti-human Fc detection antibody (AU003 labeled with HRP), except for RELAX0010 (described in WO2018/138170), which was tested in ELISA using anti-human Fc capture and anti-h-Relaxin-2 detection (using polyclonal HRP-labeled antibody from human relaxin-2 ELISA kit (R&D Systems, catalog number DRL200)). In both assays, the plate coated with the capture antibody was blocked with 100 μL of RD1-19 assay diluent at room temperature for one hour. 50 μL of standard or sample was added to each well and incubated for two hours at room temperature. The samples were aspirated and the wells were washed three times with assay wash buffer. 50 μL of HRP-labeled detection antibody was added per well, diluted 1:1000 in PBS/1% BSA in case of anti-human Fc specific detection, or used undiluted in case of anti-h-Relaxin-2 detection. After incubation for 1 hour at room temperature and three washes, 50 μL/well of TMB (SureBlue Reserve KPL 53-00-03) was added, and once a color change occurred, the reaction was stopped by adding 50 μL/well of TMB stop solution (KPL 50-85-06).

Bioactivity of PK samples in cell-based cAMP activity assay.

Serum samples collected from animals as described above were tested for biological activity to measure functional relaxin-2 to assess the integrity of the Fc-Relaxin-2 fusion polypeptide. A stable cell line expressing the human RXFP1 receptor produced in CHO cells was purchased from DiscoVocos. Activation of this receptor results in the production of the downstream cAMP second messenger, which can be measured in a functional activity assay.

The cAMP assay was performed using the following bovine serum albumin (BSA) based assay buffer: Hank's Balanced Salt Solution (Sigma #H8264) supplemented with 0.1% BSA (Sigma #A9418) and 0.5 mM IBMX (Sigma #I7018), adjusted to pH 7.4 with 1 M NaOH.

Dosing solutions of relaxin-2 fusion polypeptide or recombinant relaxin-2 peptide (R&D Systems, Catalog No. 6586-RN) were diluted in assay buffer and a non-contact liquid dispenser (ECHO, Labcyte) was used to generate an 11-point standard curve of four matrix concentrations. The matrix used was blank serum from mock dosed animals and was manually added to the wells at twice the desired concentration and cells were allowed to add. Test samples were transferred from serum tubes to a 384-well source plate, which was used to create four dilutions in assay buffer using a non-contact liquid dispenser (ECHO, Labcyte). All sample dilutions were made in duplicate.

The frozen vials of cells expressing hRXFP1 were thawed rapidly in a water bath, transferred to a pre-heated cell culture medium and spun at 240 x g for 5 minutes. The cells were resuspended in 8 mL of cell culture medium, seeded in a T75 flask containing 10 mL of culture medium, and allowed to adhere overnight. The next day, accutase was used to separate the cells, and spun at 240 x g for 5 minutes. The resulting cell pellet was resuspended at an optimized concentration, and 2.5 μL of cell suspension was added to each well of the assay plate using a Combi-drop dispenser.

cAMP levels were measured using a commercially available cAMP Dynamic 2HTRF kit (Cisbio, Cat. No. 62AM4PEJ) following a two-step protocol as recommended by the manufacturer. Briefly, anti-cAMP cryptate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were prepared separately by diluting each 1/20 in the conjugation and lysis buffer provided in the kit. 2.5 μL of anti-cAMP cryptate was added to all wells of the assay plate, and 2.5 μL of cAMP-d2 was added to all wells except the non-specific binding (NSB) wells, to which conjugation and lysis buffer was added. The plate was incubated at room temperature for one hour and then read on Envision (PerkinElmer) using an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. Data were converted to % ΔF as described in the manufacturer's instructions, and sample values were calculated from the linear portion of the standard curve.

Results and Conclusions

Figure 5 shows a summary of data from a series of in vivo PK experiments in which Fc-Relaxin-2 polypeptides were administered IV to mice. Data were normalized to the 5 minute time point.

After IV administration, the half-life of human relaxin-2 in humans is about 0.09 +/- 0.04 hours, i.e., 5.4 +/- 2.4 minutes (Chen et al. 1993). Recombinant relaxin Fc fusion polypeptides all show improved half-life compared to native relaxin-2. Fc-relaxin polypeptides in which the relaxin A and B chains are linked to different heterodimeric Fc chains (exemplified by RELAX0019, RELAX0023, RELAX0034, RELAX0046, and RELAX0117) have improved PK properties compared to those Fc-relaxin polypeptides in which the relaxin chains are linked to a linker (exemplified by RELAX0010 and RELAX0009). However, since both linker-containing molecules RELAX0088 and RELAX0122 show good in vivo stability, the presence of a linker between the relaxin A and B chains itself is not directly related to the rapid elimination of Fc-relaxin polypeptides in vivo.

Unexpectedly in this study, the heterodimeric Fc-relaxin fusion polypeptides (RELAX0019, RELAX0023, RELAX0034, RELAX0046, RELAX0117, RELAX0088, and RELAX0122) all had significantly improved pharmacokinetic properties compared to the Fc-relaxin fusion polypeptides RELAX0010 and RELAX0009.

Example 7: Reversal of established hypertrophy and fibrosis by RELAX0019 and RELAX0023

Isoproterenol (15 mg/kg/day) was infused into C57B6 mice via a mini pump for 10 days to induce cardiac hypertrophy and fibrosis. Mice infused with vehicle for the same duration were used as baseline controls. After 10 days, the mini pump was removed and mice were given a new mini pump containing r-relaxin-2 (500 ug/kg/day), or mice received the first of two weekly (QW) subcutaneous injections of RELAX0019 (20 mg/kg) or RELAX0023 (20 mg/kg). After the 14-day treatment period, mice were sacrificed and their hearts were collected for analysis of hypertrophy and fibrosis. After removing the vehicle mini pump, hearts from baseline control mice were collected. Hypertrophy was determined as a measure of heart weight relative to tibia length, and fibrosis was established by quantifying collagen content relative to heart weight. In this model, infusion of isoproterenol significantly induced both hypertrophy and fibrosis. QW dosing of RELAX0019 and RELAX0023 returned isoproterenol-induced hypertrophy to baseline levels, as did constant infusion of r-Relaxin-2. All relaxin treatments also reduced cardiac fibrosis by more than 50%. N=8 for each group. **p<0.01, ***p<0.001, ****p<0.0001

Recombinant relaxin Fc fusion proteins RELAX0019 and RELAX0023 were able to reverse hypertrophy and fibrosis in a manner similar to native h-relaxin-2 ( FIG. 6 ).

Example 8: Evaluation of non-specific binding of Fc-Relaxin-2 protein using baculovirus ELISA.

RELAX protein was expressed in CHO cells and purified as described above. The baculovirus ELISA developed to assess non-specific binding of monoclonal antibodies (reference: Hotzel et al., 2012 mAbs 4:6, 753-760) was adapted to determine non-specific binding with modified Fc-Relaxin polypeptides, whereby instead of calculating a "BV score" (baculovirus plate absorbance/blank plate absorbance), non-specific binding was calculated as signal relative to background for baculovirus plates and blank plates separately (where background is the value obtained in the absence of Fc-Relaxin polypeptide). This measure was introduced to reflect the increased non-specific binding of some Fc peptides to coated and uncoated (blank) plates (compared to monoclonal antibodies). Preparations of each protein were prepared at 100 nM or 10 nM in PBS (Gibco 14190-086) + 0.5% BSA (Sigma A9576) and used in duplicate for ELISA assays on 96-well Nunc Maxisorp F plates, which were coated overnight at 4°C with 50 μL/well of 1% baculovirus extract in 50 mM sodium carbonate (BV plates) or with 50 mM sodium carbonate (blank plates). After washing with PBS, the plates were blocked with 300 μL/well of PBS + 0.5% BSA for 1 hour at room temperature and washed three times with PBS. 50 μL/well of PBS + 0.5% BSA (background) or RELAX protein dilutions were added and incubated for 1 h at room temperature. After washing three times in PBS, detection antibody (anti-human Fc-specific-HRP Sigma A0170) diluted 1:5000 in PBS + 0.5% BSA was added at 50 μL/well. The samples were incubated for 1 hour at room temperature and the plates were washed three times in PBS. HRP substrate-TMB (SureBlue Reserve KPL 53-00-03) was then added at 50 μL/well and after the color change, the reaction was stopped by adding 50 μL/well of 0.5M sulfuric acid. The absorbance was measured at 450 nm and the nonspecific binding of each sample was determined. Nonspecific binding (fold binding relative to background) was defined as the ratio of nonspecific binding in the presence of Fc relaxin-2 protein and in the absence of Fc relaxin-2 protein (background). The data of Fc-Relaxin-2 protein tested at 2 different concentrations of 100 nM or 10 nM are shown in Table 7.

Table 7: Binding of Fc-Relaxin fusion proteins at 100 nM and 10 nM in baculovirus ELISA (-001, 002, 003 refer to different batches of the same protein)

As shown in Table 7 and Figure 7, the heterodimeric relaxin-2 Fc fusion polypeptides exhibited lower non-specific binding when the relaxin chain was attached to the C-terminus using a GS linker. Some asymmetric PA linkers, certain point mutations, and positioning of the relaxin chain at the N-terminus, particularly in the case of the bivalent molecule (RELAX0117), increased non-specific binding to both blank and BV-coated plates. Some Fc-relaxin proteins with particularly high non-specific binding exhibited greater non-specific binding to blank plates compared to BV-coated plates at both high (100 nM) and low (10 nM) concentrations. Although the control molecules - linker-containing bivalent RELAX0009, RELAX0010, RELAX0126, RELAX0127, and RELAX0128 all demonstrated high non-specific binding, neither the presence of a linker between the A and B chains of relaxin nor the bivalency itself drove high non-specific binding, as can be demonstrated by the low non-specific binding of RELAX0122.

Example 9: Stability in Solution

The stability of RELAX0023 was evaluated using high performance size exclusion chromatography (HP-SEC) and liquid chromatography-mass spectrometry (LC-MS), and compared with RELAX0127 and RELAX0128. HP-SEC, which detects the absorbance at 280 nm, can be used to measure purity, aggregation and fragmentation. The molecule buffer is exchanged into the optimized formulation composition and then concentrated to up to 10 mg/mL. All samples are placed under stress temperature conditions (40 ° C) for up to 4 weeks. At 1, 2 and 4 weeks, samples are collected and injected into a size exclusion column and eluted isocratically with an aqueous mobile phase at a fixed flow rate. Compared with smaller molecules, larger molecules are excluded from the holes of the size exclusion column to a greater extent, and therefore elute earlier. The peak eluted earlier than the monomer peak is recorded as an aggregate. The peak eluted after the monomer peak (excluding the buffer-related peak) is recorded as a fragment. The results are recorded as purity percentage, aggregate percentage and fragment percentage, and are shown in Figure 8. RELAX0023 is the most stable molecule, with a purity loss rate of only 0.1%/month, while RELAX0128 and RELAX0127 are 7.7% and 9.3%, respectively. Both RELAX0127 and RELAX0128 show signs of aggregation, however, the aggregation level of RELAX0023 does not increase, indicating better physical solution stability. Fragmentation seems to be the main factor for purity loss, with RELAX0127 having 6.6% fragmentation/month and RELAX0128 being 6.8%. RELAX0023 has only a fragmentation rate of 0.7%/month. At the same time, after storage at 40°C for 4 weeks, the total peak area of RELAX0128 decreased from 22403 to 18216 (a decrease of 19%), while RELAX0127 decreased from 22225 to 18823 (a decrease of 15%). This significant loss of total peak area and the high fragmentation rate indicate that these two molecules may be highly chemically degraded. It should be noted that this loss in total area had a large impact on the chromatogram profiles of both molecules. This explains why RELAX0128 and RELAX0127 showed lower percentages of aggregates at 4 weeks compared to earlier time points, despite a significant increase in aggregate peak area after storage. In contrast, RELAX0023 only decreased in total peak area by 0.03%, from 21828 to 21761, indicating a better stability profile compared to RELAX0128 and RELAX0127.

The fragmentation of the molecules was further verified by LC-MS using reduced mass analysis, indicating that the fragment peaks of RELAX0127 and RELAX0128 increased in intensity after storage at 40°C (Figure 9A). In contrast, the fragment peaks of RELAX0023 remained unchanged after stress. The mass spectra under reducing conditions also showed modification of RELAX0127 and RELAX0128 over time, as evidenced by the shift of the peaks to larger masses and the broadening of the peaks, indicating greater heterogeneity (Figure 9B). In contrast, the intact mass spectrum of RELAX0023 remained unchanged, indicating that no modification occurred. This study demonstrates that RELAX0023 has superior physical and chemical stability compared to RELAX0127 and RELAX0128.

Example 10: PK profile of RELAX0023 in cynomolgus monkeys

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

Assay plates were coated with goat anti-human IgG antibody and incubated with cynomolgus monkey sera from animals from Groups 1-4. RELAX0023 bound to the plate was detected by anti-Relaxin antibody conjugated to HRP. Cynomolgus monkey sera were diluted 1:10 before addition to the plate. In 100% serum, the lower limit of quantitation was 0.010 μg/mL and the upper limit of quantitation was 0.300 μg/mL.

Results and Conclusions

Figure 10 shows the mean serum concentration-time curve of RELAX0023 in cynomolgus monkeys after a single dose. After a single dose of SC administration, RELAX0023 exhibited linear PK in a dose range of 0.01 to 10 mg/kg. It was observed that the dose of _Cmax increased proportionally. For 0.1, 1 and 10 mg/kg SC dose groups, the average _Cmax values were 0.400, 4.69, 34.8 μg/mL, respectively. From 0.1 mg/kg to 10 mg/kg SC groups, it was also observed that the dose of AUC _0-last value increased proportionally. For 0.1, 1 and 10 mg/kg SC dose groups, the average AUC _0-last values were 2.01, 25.5, 193 μg days/mL, respectively. Overall, RELAX0023 PK was linear in the range of 0.1 mg/kg to 10 mg/kg, with an average CL/F of 51.0 mL/day/kg and an average t _1/2 of 3.07 days. The SC bioavailability of RELAX0023 was estimated to be 88.2%.

Example 11: Evaluation of the long-term efficacy of RELAX0023 in cynomolgus monkeys (Macaca fascicularis) with heart failure and reduced left ventricular ejection fraction (LVEF)

The long-term efficacy of RELAX0023 on cardiac function was evaluated in obese and aged cynomolgus monkeys (Macaca fascicularis). Cynomolgus monkeys were selected as the selected test species, rather than other lower mammalian species, because they are closely related to humans both phylogenetically and physiologically. Aged cynomolgus monkeys fed with a high-fat diet for at least 2 years have the same risk factors as human patients susceptible to cardiovascular disease, and a metabolic syndrome that can characteristically progress to heart failure and reduced LVEF occurs. The effect of RELAX0023 on LVEF was evaluated when administered at different dose levels by subcutaneous (SC) injection for 20 weeks, with the first dose administered in the first week of the study, followed by an 18-week observation period. In a collection of approximately 100 obese and aged cynomolgus monkeys aged 12-20 years and weighing 6-15 kg that have been fed a high-fat diet for at least 2 years, 38 monkeys were identified as having LVEF of 30%-60% by 2D echocardiographic screening. Healthy monkeys of this age weigh 5-8 kg and have an LVEF of 70%-75%, so an LVEF of 60% or less represents a model of HFrEF. The identified animals were selected and randomly assigned to 3 treatment groups of 8 monkeys each and a vehicle group of 14 monkeys. The dosing period consisted of subcutaneous administration of RELAX0023 once weekly at 3 ascending dose levels (all less than 10 mg/kg; referred to as "low", "mid" and "high" doses, respectively).

Cardiac function measurements were determined 9 times by 2D echocardiography at baseline Week -2 and during the dosing and post-dosing observation periods at Weeks 5, 9, 13, 17, 21, 25, 29, 33. A further 2D echocardiogram was scheduled for Week 39 (end of study). Parameters (including LVEF) were based on apical two-chamber and four-chamber views and a biplane approach. HDO (high definition oscillometric) was used to measure parameters including mean arterial pressure (MAP) and heart rate (HR).

Results and Conclusions

Compared to vehicle control, RELAX0023 was able to significantly improve LVEF at all RELAX0023 dose levels at weeks 5, 9, 13, 17, and 21 without affecting heart rate or blood pressure (Figure 12). After the end of treatment, it was observed throughout the washout period to the 33rd week of the study that LVEF was significantly improved after treatment with RELAX0023 compared to week 0 (baseline). These striking results show that the hemodynamics of the treated animals were significantly improved and clearly demonstrate the efficacy of RELAX0023 in the treatment of heart failure in this model. In addition, to the best of the inventors' knowledge, the extent of the sustained response after treatment is not achieved by any other known compound targeting this mechanism of action pathway. The monkeys will continue to be monitored until the 39th week of the study.

Example 12: Phase 1 (Ph1) study in healthy volunteers and patients with heart failure

Study D8330C00001 is a Phase Ia/b, randomized, single-blind, placebo-controlled, first-in-human (FTIH) study (ClinicalTrials.gov identifier NCT04630067). The primary objective of this study is to evaluate the safety and tolerability of single-ascending and multiple-ascending doses of RELAX0023 (also known as "AZD3427"), and the secondary objectives are to evaluate the (i) pharmacokinetics (PK) and (ii) immunogenicity of single-ascending and multiple-ascending doses of AZD3427.

The study was conducted in 2 parts, Part A and Part B. Part A was a single ascending dose (SAD) study in healthy participants (males and females of infertile potential), and Part B was a multiple ascending dose (MAD) study in HF participants (males and females of infertile potential).

Part B included 48 patients in 6 cohorts (8 participants per cohort). Of these, 3 cohorts consisted of HFrEF participants (cohorts 1b, 3b, and 5b), and 3 cohorts consisted of HF participants with EF ≥ 41% (cohorts 2b, 4b, and 6b). The dose levels for the HFrEF and HF cohorts with EF ≥ 41% were 5 mg (cohorts 1b, 2b), 15 mg (cohorts 3b, 4b), and 45 mg (cohorts 5b, 6b), administered once a week (QW) for 5 weeks (i.e., a total of 5 doses).

For Part B, which included 48 patients in 6 cohorts, inclusion criteria included: (i) all cohorts: patients with a known clinical diagnosis of stage C HF (NYHA class I to III) and receiving stable medical therapy for at least 12 weeks before screening, without significant dose changes or addition of new medications during this period, (ii) cohorts 1b, 3b, 5b: patients diagnosed with HFrEF (defined as EF ≤ 40%), (iii) cohorts 2b, 4b, 6b: patients diagnosed with HF with EF ≥ 41% (including patients diagnosed with HFpEF (defined as EF ≥ 50%)), (iv) all cohorts: BMI between 18 and 40 kg/ ^m2 (inclusive) and body weight at least 55 kg and not more than 120 kg (inclusive), and (v) all cohorts: previously documented NT-proBNP > 125 pg/mL or BNP > 35 pg/mL.

Example 13: Results of the Ph1 AZD3427 MAD study in patients with HF

In the Part B MAD cohort, data from HFpEF and HFrEF patients were combined. Trends indicated that AZD3427 improved cardiac function, including improved cardiac output and stroke volume (SV), reduced systemic vascular resistance (SVR), and improved organ perfusion (SVR and eGFR) (Figures 13A-6F).

Although the hypertension status of subjects in the MAD cohort was not determined, the observed improvements in cardiac output and stroke volume (SV), reduction in systemic vascular resistance (SVR), increase in estimated glomerular filtration rate (eGFR), and thus improvement in organ perfusion (SVR and eGFR) are expected to benefit patients with HF+PH.

Example 14: Ph2b study - a randomized, placebo-controlled, multicenter, dose-ranging study of AZD3427 in participants with heart failure due to left heart disease and pulmonary hypertension (World Health Organization [WHO], Group 2).

This study (Study ID: D8330C00003) was designed to evaluate the ability of AZD3427 to reduce pulmonary vascular resistance (PVR) after 24 weeks of treatment in participants with heart failure (HF) and 2 groups of pulmonary hypertension (PH).

Approximately 220 participants will be randomized to 4 treatment groups (in a 1:1:1:1 ratio) to receive subcutaneous (SC) injections of AZD3427 or placebo every 2 weeks for 24 weeks. This study will evaluate 3 dose levels of AZD3427: Dose A, Dose B, and Dose C. Dose adjustment is not applicable for this study. The study will be conducted in approximately 60 research centers in an estimated 15 countries. The study will include approximately 16 study visits: 2 visits during the screening period, 13 visits during the treatment period, and one visit during the follow-up period. The expected total duration of the study is 32 to 37 weeks, depending on the length of the screening period.

Participants will receive a single subcutaneous dose of AZD3427 (doses A, B, or C) or placebo every 2 weeks from day 1 to day 155 for 24 weeks.

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

Secondary outcome measures include:

Change from baseline in mean pulmonary artery pressure (mPAP)

Change from baseline in pulmonary artery wedge pressure (PAWP)

Change in cardiac output from baseline

Change in stroke volume (SV) from baseline

Change from baseline in ejection fraction (EF)

Change from baseline in left ventricular global longitudinal strain (LVGLS)

Change from baseline in pulmonary artery systolic pressure (PASP)

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

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

Change in tricuspid regurgitation velocity (TRV) from baseline

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

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

Change in inferior vena cava (IVC) diameter with inspiratory collapse relative to baseline

Change from baseline in systemic vascular resistance

Change from baseline in 6-minute walk 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 in serum creatinine from baseline

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. Participants must be ≥18 years old (inclusive). 2. Participants must be pre-diagnosed with HF, NYHA functional class (FC) II to IV, and pre-diagnosed with PH-LHD or probable or intermediate probability of PH due to left heart disease according to the 2022 European Society of Cardiology/European Society of Respiratory Diseases (ESC/ESR) guidelines for pulmonary hypertension due to left heart disease (PH-LHD). Participants must receive stable HF standard care medications, including diuretics. 3. Participants must have a combination of echocardiographic parameters showing an intermediate or high probability of PH according to the 2022 ESC/ERS guidelines. 4. At the second screening visit, participants must have an elevated pulmonary artery pressure on study in the RHC performed according to the RHC manual provided by the sponsor: (a) PAWP ≥ 15 mmHg (b) mPAP ≥ 20 mmHg 5. Minimum weight is 50 kg (inclusive). 6. Able to sign informed consent.

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

Claims (33)

1. A method for treating a subject with heart failure and pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimer fusion, the heterodimer fusion comprising: (i) a first heterodimerization domain linked to at least one Relaxin A chain polypeptide or variant thereof; and (ii) a second heterodimerization domain linked to at least one relaxin B chain polypeptide or variant thereof, wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and wherein the heterodimer fusion has relaxin activity. 2. The method of claim 1, wherein the relaxin A chain polypeptide and the relaxin B chain polypeptide of the heterodimeric fusion are covalently bound by at least one interchain disulfide bond. 3. The method of claim 1 or 2, wherein the relaxin A chain and the relaxin B chain of the heterodimer fusion are not covalently linked to each other by an amino acid linker. 4. The method of any one of the preceding claims, wherein the relaxin A chain of the heterodimeric fusion is relaxin-2A chain and the relaxin B chain of the heterodimeric fusion is relaxin-2B chain. 5. The method according to any of the preceding claims, wherein the relaxin A chain of the heterodimer fusion is linked to the first heterodimerization domain via a linker, and the relaxin B chain of the heterodimer fusion is linked to the second heterodimerization domain via a linker, optionally one or preferably both linkers are polypeptides. 6. The method according to claim 5, wherein one or preferably both linkers of the heterodimer fusion are 6 to 40 amino acids in length, such as one or preferably both linkers are 21 amino acids in length. 7. The method according to any of the preceding claims, wherein the first and second heterodimerization domains of the heterodimeric fusion are derived from immunoglobulin Fc regions ("first Fc region" and "second Fc region", respectively), optionally wherein the first and second Fc regions comprise constant domains CH2 and CH3. 8. The method of claim 7, wherein the C-terminus of the first Fc region is linked to the N-terminus of the relaxin A chain and the C-terminus of the second Fc region is linked to the N-terminus of the relaxin B chain. 9. The method according to claim 7 or 8, wherein the first and second Fc regions comprise amino acid mutations and/or modifications that promote heterodimerization, optionally wherein the amino acid mutations that promote heterodimerization are "Fc knob" and "Fc hole" mutations, such as "Fc knob" and "Fc hole" mutations present in the CH3 domain. 10. The method according to any one of claims 7 to 9, wherein the first and second Fc regions are derived from human IgG1 immunoglobulins. 11. The method of claim 10, wherein the amino acid mutations that promote heterodimerization comprise: a. "Fc hole" mutations Y349C, T366S, L368A and Y407V in one CH3 domain; and b. "Fc knob" mutations S354C and T366W in another CH3 domain, Therein the amino acid numbering is according to the EU index as in Kabat. 12. The method according to claim 11, wherein: a. the first Fc region comprises an "Fc knob" mutation and the second Fc region comprises an "Fc hole" mutation; or b. The second Fc region comprises an "Fc knob" mutation and the first Fc region comprises an "Fc hole" mutation. 13. The method according to any one of claims 10 to 12, wherein the first and/or second Fc region comprises the amino acid mutations L234F, L235E and P331S, wherein the amino acid numbering is according to the EU index as in Kabat. 14. The method of any one of claims 4 to 13, wherein the relaxin-2A chain polypeptide of the heterodimeric fusion comprises the sequence as shown in SEQ ID NO: 1 or a variant thereof, and the relaxin-2B chain polypeptide of the heterodimeric fusion comprises the sequence as shown in SEQ ID NO: 2 or a variant thereof. 15. The method of claim 14, wherein the relaxin-2A chain polypeptide of the heterodimer fusion comprises the amino acid mutations K9H, K17M or K17I. 16. The method according to any one of claims 5 to 15, wherein both linkers of the heterodimer fusion have the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5]. 17. A method of treating a subject with heart failure and pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising: (i) FcX-con-A fusion polypeptide; and (ii) Fcγ-con-B fusion polypeptide, in: A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof; B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof; Fcγ is an Fc region comprising the constant domains CH2 and CH3 of a human IgG1 immunoglobulin, and comprises an "Fc hole" amino acid mutation and/or modification, preferably an amino acid mutation Y349C:T366S:L368A:Y407V; FcX is an Fc region with an "Fc knob" amino acid mutation and/or modification, preferably comprising the constant domains CH2 and CH3 of a human IgG1 immunoglobulin, and comprises an "Fc knob" amino acid mutation and/or modification, preferably the amino acid mutation S354C:T366W; and con is a linker polypeptide, preferably having the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5], wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX and FcY heterodimerize, and wherein the heterodimeric fusion has relaxin activity. 18. The method according to any one of the preceding claims, wherein the heterodimeric fusion comprises a fusion polypeptide having the amino acid sequence of SEQ ID NO: 11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO: 20. 19. The method according to any one of claims 8 to 18, wherein the heterodimeric fusion further comprises one or more Fabs, optionally wherein the heterodimeric fusion comprises one Fab linked to the N-terminus of the first Fc region and a second Fab linked to the N-terminus of the second Fc region. 20. The method of any one of claims 8 to 19, wherein the heterodimeric fusion further comprises a second relaxin A chain polypeptide or variant thereof linked to the N-terminus of the first Fc region and a second relaxin B chain polypeptide or variant thereof linked to the N-terminus of the second Fc region, optionally wherein the second relaxin A chain is linked to the first Fc region via a linker polypeptide, and the second relaxin B chain is linked to the second Fc region via a linker polypeptide. 21. A method of treating a subject with heart failure and pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising: (i) FcX-B-L-A and FcY, optionally FcY-B-L-A; or (ii) FcY-B-L-A and FcX, optionally FcX-B-L-A; in: Fcγ is an immunoglobulin Fc region having an "Fc hole" amino acid mutation and/or modification, preferably comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V; FcX is an immunoglobulin Fc region with an "Fc knob" amino acid mutation and/or modification, preferably comprising a CH3 domain with an amino acid mutation S354C:T366W; B is relaxin B chain or a variant thereof, such as relaxin 2 B chain or a variant thereof; A is relaxin A chain or a variant thereof, such as relaxin 2A chain or a variant thereof; and L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60], wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX and FcY heterodimerize, and wherein the heterodimeric fusion has relaxin activity. 22. The method of claim 21, wherein the relaxin B chain of the heterodimer fusion is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, such as 21 amino acids in length. 23. The method of any one of the preceding claims, wherein the ratio of relaxin activity of the heterodimeric fusion to the relaxin activity of a reference relaxin protein is from about 0.001 to about 10. 24. The method according to any of the preceding claims, wherein the heart failure is heart failure with reduced ejection fraction, heart failure with intermediate ejection fraction, or heart failure with preserved ejection fraction. 25. The method according to any of the preceding claims, wherein the subject has a mean pulmonary artery pressure of about 25 mmHg or more, a pulmonary artery wedge pressure (PAWP) greater than 15 mmHg and/or a right ventricular systolic pressure of about 40 mmHg or more. 26. The method of any of the preceding claims, wherein the subject has a pulmonary vascular resistance of less than 3.0 Wood units. 27. The method of any one of claims 1-25, wherein the subject has a pulmonary vascular resistance of 3.0 or more Wood units. 28. The method according to any of the preceding claims, wherein the subject is equipped with a blood pressure monitoring device, preferably a pulmonary artery pressure monitoring device. 29. The method of claim 28, wherein the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. 30. The method according to any one of the preceding claims, wherein the heterodimeric fusion is administered as a pharmaceutical composition comprising the heterodimeric fusion and a pharmaceutically acceptable excipient. 31. The method according to any of the preceding claims, wherein the heterodimeric fusion or pharmaceutical composition is administered to the subject by subcutaneous injection. 32. The method according to any one of the preceding claims, wherein the heterodimeric fusion or pharmaceutical composition is administered by self-administration. 33. The method according to any of the preceding claims, wherein administration of the heterodimeric fusion or pharmaceutical composition achieves one or more of the following: a) decreased PVR; (b) decreased mPAP; (c) ePAD reduction; (d) Increased cardiac output (SV); (e) decreased systemic vascular resistance (SVR) and/or increased estimated glomerular filtration rate (eGFR); (f) increased ejection fraction; and/or (g) Increased cardiac output; Compared with baseline levels before administration.