Treatment with heterodimeric relaxin fusions

Abstract

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

Description

[Technical field]

[0001] The present invention relates to therapeutic methods using heterodimeric relaxin fusions, in particular to therapeutic methods using relaxin2 fusions. [Background technology]

[0002] Relaxin is a peptide hormone that belongs to the insulin superfamily. In humans, the relaxin peptide family includes seven peptides with high structural but low sequence similarity: relaxins 1, 2, and 3, and insulin-like peptides INSL3, INSL4, INSL5, and INSL6. Natural relaxin is composed of A and B polypeptide chains covalently linked by two interchain disulfide bonds. The A chain has an additional intrachain disulfide bond. The relaxin gene encodes a prohormone with the structure BCA (B and A polypeptide chains linked by a C peptide). The prohormone undergoes intracellular proteolytic cleavage by PC1 and PC2 enzymes to remove the C peptide and then secrete mature relaxin.

[0003] Relaxin is a pleiotropic hormone known to mediate adaptive changes in systemic hemodynamics and in the kidney during pregnancy. Relaxin has also been shown to have antifibrotic properties and beneficial effects in heart failure, such as acute decompensated heart failure (ADHF). Heart failure is associated with significant morbidity and mortality. Heart failure is characterized by complex tissue remodeling with increased myocardial cell death and interstitial fibrosis. Relaxin activates many signaling cascades that have been shown to be beneficial in situations such as ischemia-reperfusion and heart failure. These signaling pathways include activation of the phosphoinositide 3-kinase pathway and activation of the nitric oxide signaling pathway (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4).

[0004] A significant subpopulation of heart failure patients also suffers from pulmonary hypertension (HF+PH patients). It has been estimated that approximately 50% of heart failure patients with preserved ejection fraction also suffer from pulmonary hypertension, and this increases to 60% in heart failure patients with reduced ejection fraction (Non-Patent Document 5, Non-Patent Document 6). Patients with heart failure complicated with pulmonary hypertension have been shown to have reduced survival rates compared to patients with heart failure without pulmonary hypertension (Non-Patent Document 7). In heart failure patients, a 3 mmHg increase or decrease in estimated pulmonary artery diastolic pressure (ePAD) (corresponding to an increase or decrease of approximately 4 mmHg in mean pulmonary artery pressure (mPAP)) was associated with a 24% increase or 19% decrease in cardiovascular mortality, respectively (Non-Patent Document 8). A 4 mmHg decrease in mPAP is also associated with improved dyspnea in patients with heart failure and pulmonary hypertension (Non-Patent Document 9).

[0005] Clinical trials have been conducted using serelaxin, an unmodified recombinant human relaxin 2. Continuous intravenous administration of serelaxin to hospitalized patients improved cardiac, renal, and liver damage and congestion (Non-Patent Document 10, Non-Patent Document 11, Non-Patent Document 12). Serelaxin has also demonstrated improvements in pulmonary artery pressure, cardiac output, and systemic and pulmonary vascular resistance in patients who received continuous infusions for approximately 20 hours (Non-Patent Document 13). However, because serelaxin is rapidly cleared from the patient's circulation, its therapeutic effect is limited, and the positive effects disappear rapidly once intravenous injection is stopped. Furthermore, approximately one-third of patients experienced a significant drop in blood pressure (>40 mm Hg) after intravenous administration of serelaxin, leading to the conclusion that the dose must be reduced by half or even more.

[0006] US Patent No. 5,999,233 and US Patent No. 5,999,233 describe recombinant relaxin polypeptides in which relaxin A and relaxin B are fused in a single chain by a linker peptide. US Patent No. 5,999,233 describes recombinant relaxin comprising a linker peptide of at least 5 amino acids and less than 15 amino acids. US Patent No. 5,999,233 describes recombinant relaxin comprising a linker peptide of at least 15 amino acids.

[0007] Given the promising clinical studies conducted to date with unmodified recombinant relaxin, there remains a need for additional recombinant relaxins that retain the biological activity of relaxin and offer advantages such as extended half-life, a convenient route of administration, and convenient dosing. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2013 / 004607 Brochure [Patent Document 2] International Publication No. 2018 / 138170 Brochure [Non-patent literature]

[0009] [Non-Patent Document 1] Bathgate RA et al.(2013)Physiol.Rev.93(1):405-480 [Non-Patent Document 2] Mentz RJ et al. (2013) Am.Heart J.165(2):193-199 [Non-Patent Document 3] Tietjens J et al. (2016) Heart 102:95-99 [Non-Patent Document 4] Wilson SS et al.(2015)Pharmacology 35:315-327 [Non-Patent Document 5] Guazzi,(2014)Circ Heart Fail.,7:367-377 [Non-Patent Document 6] Miller et al.,(2013)JACC Heart Fail.,1(4):290-299 [Non-Patent Document 7] Barnett and De Marco,(2012)Heart Fail.Clin.8:447-459 [Non-Patent Document 8] Zile MR,et al.(2017)Circ Heart Fail.,10:e003594 [Non-Patent Document 9] Solomonica A,et al.(2013)Circ Heart Fail.,6:53-60 [Non-Patent Document 10] Felker GM et al.(2014)J.Am.Coll.Cardiol.64(15):1591-1598 [Non-Patent Document 11] Metra M et al.(2013)J.Am.Coll.Cardiol.61(2):196-206 [Non-Patent Document 12] Teerlink JR et al. (2013) Lancet 381(9860):29-39 [Non-Patent Document 13] Ponikowski et al.,(2014)European Heart Journal 35:431-441 Summary of the Invention [Means for solving the problem]

[0010] The present invention relates to the use of heterodimeric fusions with relaxin activity in the treatment of subjects with heart failure combined with pulmonary hypertension (HF+PH). There remains a significant unmet need in the treatment of HF+PH subjects.

[0011] Thus, in one aspect, the invention provides a method of treating a subject having heart failure complicated by pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion, the heterodimeric fusion comprising: (i) a first heterodimerization domain linked to at least one relaxin A chain polypeptide or variant thereof; (ii) a second heterodimerization domain linked to at least one relaxin B chain polypeptide or variant thereof; The first heterodimerization domain heterodimerizes with the second heterodimerization domain, and the heterodimeric fusion has relaxin activity.

[0012] Similarly, the present invention provides a heterodimeric fusion for use in treating a subject having heart failure complicated by pulmonary hypertension, the heterodimeric fusion comprising: (i) a first heterodimerization domain linked to at least one relaxin A chain polypeptide or variant thereof; (ii) a second heterodimerization domain linked to at least one relaxin B chain polypeptide or variant thereof; The first heterodimerization domain heterodimerizes with the second heterodimerization domain, and the heterodimeric fusion has relaxin activity.

[0013] Similarly, the invention provides the use of a heterodimeric fusion in the manufacture of a medicament for treating a subject having heart failure complicated by pulmonary hypertension, the heterodimeric fusion comprising: (i) a first heterodimerization domain linked to at least one relaxin A chain polypeptide or variant thereof; (ii) a second heterodimerization domain linked to at least one relaxin B chain polypeptide or variant thereof; The first heterodimerization domain heterodimerizes with the second heterodimerization domain, and the heterodimeric fusion has relaxin activity.

[0014] In some embodiments, the relaxin A chain and relaxin B chain of the heterodimeric fusion are covalently linked 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.

[0015] In some embodiments, the relaxin A chain is a relaxin 2A chain and the relaxin B chain is a relaxin 2B chain.

[0016] In a preferred embodiment, the first and second heterodimerization domains are derived from an immunoglobulin Fc region, such as an immunoglobulin G (IgG) Fc region ("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.

[0017] In another embodiment, the first and second heterodimerization domains are derived from an immunoglobulin Fab region.

[0018] In yet another embodiment, the first and second heterodimerization domains heterodimerize to form a parallel coiled-coil.

[0019] In some embodiments, the relaxin A chain is linked to a first heterodimerization domain (e.g., a first Fc region) via a connector and the relaxin B chain is linked to a second heterodimerization domain (e.g., a second Fc region) via a connector. In preferred embodiments, one or preferably both connectors are polypeptides.

[0020] In some embodiments, at least one connector is a polypeptide having a length of 6 to 40 amino acids. Preferably, both connectors are polypeptides having a length of 6 to 40 amino acids. In a preferred embodiment, at least one connector is a polypeptide having a length of 21 amino acids. In a particularly preferred embodiment, both connectors are polypeptides having a length of 21 amino acids. In a particular embodiment, both connectors have the sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5).

[0021] 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 another 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.

[0022] In some embodiments, the first and second heterodimerization domains (e.g., the first and second Fc regions) comprise heterodimerization-promoting amino acid mutations and / or modifications, preferably asymmetric heterodimerization-promoting amino acid mutations and / or modifications. In preferred embodiments, the heterodimerization-promoting amino acid mutations are "Fc knob" and "Fc hole" mutations. In particularly preferred embodiments, the "Fc knob" and "Fc hole" mutations are present in the CH3 domain. In preferred embodiments, 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 heterodimerization-promoting amino acid mutations include, in one CH3 domain, "Fc hole" mutations Y349C, T366S, L368A and Y407V, or conservative substitutions thereof; and, in the other CH3 domain, "Fc knob" mutations S354C and T366W, or conservative substitutions thereof, where amino acid numbering is according to EU index of Kabat.

[0023] In embodiments of any aspect of the invention, the relaxin 2A chain polypeptide comprises the sequence set forth in SEQ ID NO: 1 or a variant thereof, and the relaxin 2B chain polypeptide comprises the sequence set forth in SEQ ID NO: 2 or a variant thereof. In some embodiments, the relaxin 2A chain polypeptide comprises the amino acid mutation K9H, K17M, or K17I, preferably K9H.

[0024] The present invention further provides a method of treating a subject having heart failure complicated by pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion, the heterodimeric fusion comprising: (i) an FcX-con-A fusion polypeptide, (ii) an FcY-con-B fusion polypeptide, During the ceremony, A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; FcY is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof; con is a connector, e.g., a connector polypeptide preferably having the sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:5); where amino acid numbering is according to EU index of Kabat, FcX heterodimerizes with FcY and the heterodimeric fusion has relaxin activity.

[0025] Similarly, the present invention provides a heterodimeric fusion for use in a method of treating a subject having heart failure complicated by pulmonary hypertension, the heterodimeric fusion comprising: (i) an FcX-con-A fusion polypeptide, (ii) an FcY-con-B fusion polypeptide, During the ceremony, A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; FcY is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof; con is a connector, e.g., a connector polypeptide preferably having the sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:5); where amino acid numbering is according to EU index of Kabat, FcX heterodimerizes with FcY and the heterodimeric fusion has relaxin activity.

[0026] Similarly, the invention provides the use of a heterodimeric fusion in the manufacture of a medicament for treating a subject having heart failure complicated by pulmonary hypertension, the heterodimeric fusion comprising: (i) an FcX-con-A fusion polypeptide, (ii) an FcY-con-B fusion polypeptide, During the ceremony, A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; FcY is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof; con is a connector, e.g., a connector polypeptide preferably having the sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:5); where amino acid numbering is according to EU index of Kabat, FcX heterodimerizes with FcY and the heterodimeric fusion has relaxin activity.

[0027] In a particularly preferred embodiment, the heterodimeric fusion comprises a fusion polypeptide with the amino acid sequence of SEQ ID NO:11 and a fusion polypeptide with the amino acid sequence of SEQ ID NO:20.

[0028] 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).

[0029] In some embodiments of any aspect 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 connector polypeptide and the second relaxin B chain is linked to the second heterodimerization domain (e.g., the second Fc region) via a connector polypeptide.

[0030] In another aspect, the invention provides a method of treating a subject having heart failure complicated by pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion, the heterodimeric fusion comprising: (i) FcX-BLA and FcY, optionally FcY-BLA, or (ii) FcY-BLA and FcX, optionally comprising FcX-BLA; During the ceremony, FcY is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; L is a linker polypeptide preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60); wherein amino acid numbering is according to EU index of Kabat, FcX heterodimerizes with FcY, and 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 connector, optionally a connector polypeptide having a length of 6-40 amino acids, e.g., 21 amino acids.

[0031] Similarly, the present invention provides a heterodimeric fusion for use in a method of treating a subject having heart failure complicated by pulmonary hypertension, the heterodimeric fusion comprising: (i) FcX-BLA and FcY, optionally FcY-BLA, or (ii) FcY-BLA and FcX, optionally comprising FcX-BLA; During the ceremony, FcY is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; L is a linker polypeptide preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60); wherein amino acid numbering is according to EU index of Kabat, FcX heterodimerizes with FcY, and 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 connector, optionally a connector polypeptide having a length of 6-40 amino acids, e.g., 21 amino acids.

[0032] Similarly, the invention provides the use of a heterodimeric fusion in the manufacture of a medicament for treating a subject having heart failure complicated by pulmonary hypertension, the heterodimeric fusion comprising: (i) FcX-BLA and FcY, optionally FcY-BLA, or (ii) FcY-BLA and FcX, optionally comprising FcX-BLA; During the ceremony, FcY is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; L is a linker polypeptide preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60); wherein amino acid numbering is according to EU index of Kabat, FcX heterodimerizes with FcY, and 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 connector, optionally a connector polypeptide having a length of 6-40 amino acids, e.g., 21 amino acids.

[0033] In yet another aspect, the invention provides a method of treating a subject having heart failure complicated by pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion, the heterodimeric fusion comprising: (i) FcX-ALB and FcY, optionally FcY-ALB, or (ii) FcY-ALB and FcX, optionally FcX-ALB; During the ceremony, FcY is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof; A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; L is a linker polypeptide preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60); wherein amino acid numbering is according to EU index of Kabat, FcX heterodimerizes with FcY, and 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 connector, optionally a connector polypeptide having a length of 6-40 amino acids, e.g., 21 amino acids.

[0034] Similarly, the present invention provides a heterodimeric fusion for use in a method of treating a subject having heart failure complicated by pulmonary hypertension, the heterodimeric fusion comprising: (i) FcX-ALB and FcY, optionally FcY-ALB, or (ii) FcY-ALB and FcX, optionally FcX-ALB; During the ceremony, FcY is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof; A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, such as a relaxin 2 B chain or variant thereof; L is a linker polypeptide preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60); wherein amino acid numbering is according to EU index of Kabat, FcX heterodimerizes with FcY, and 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 connector, optionally a connector polypeptide having a length of 6-40 amino acids, e.g., 21 amino acids.

[0035] Similarly, the invention provides the use of a heterodimeric fusion in the manufacture of a medicament for treating a subject having heart failure complicated by pulmonary hypertension, the heterodimeric fusion comprising: (i) FcX-ALB and FcY, optionally FcY-ALB, or (ii) FcY-ALB and FcX, optionally FcX-ALB; During the ceremony, FcY is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an immunoglobulin (e.g., IgG1) Fc region having "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof; A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; L is a linker polypeptide preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60); wherein amino acid numbering is according to EU index of Kabat, FcX heterodimerizes with FcY, and 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 connector, optionally a connector polypeptide having a length of 6-40 amino acids, e.g., 21 amino acids.

[0036] According to all aspects of the invention, the heart failure may be heart failure with reduced ejection fraction, heart failure with mildly reduced ejection fraction, or heart failure with preserved ejection fraction.

[0037] According to all aspects of the present invention, the subject may have a mean pulmonary artery pressure of about 25 mmHg or greater, and / or a right ventricular systolic pressure of about 40 mmHg or greater, typically prior to treatment with a heterodimeric fusion of the present invention.

[0038] According to all aspects of the present 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 a heterodimeric fusion of the present invention.

[0039] According to all aspects of the invention, the subject may be fitted with a blood pressure monitor, preferably a pulmonary artery pressure monitor. Preferably, the pulmonary artery pressure monitor is a CardioMEMS pressure monitor.

[0040] In some embodiments of any aspect of the invention, the ratio of the relaxin activity of the heterodimeric fusion to the relaxin activity of the reference relaxin protein is about 0.001 to about 10.

[0041] According to all aspects of the present invention, the heterodimeric fusion may be administered as a pharmaceutical composition comprising the heterodimeric fusion of the present invention.

[0042] Also described herein are nucleic acid molecules (e.g., DNA molecules) encoding the heterodimeric fusions of the invention, vectors containing the nucleic acid molecules, host cells containing the vectors or nucleic acids, and methods of producing the heterodimeric fusions of the invention by culturing the host cells and harvesting the fusion proteins.

[0043] 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]

[0044] [Figure 1] 1 shows exemplary formats of heterodimeric fusions according to some embodiments of the invention. The format of each fusion polypeptide of the heterodimeric fusion is given in terms of FcX, FcY, A, B, con, and L, where FcX ("Fc knob") and FcY ("Fc hole") are two Fc regions containing heterodimerization-promoting amino acid mutations and / or modifications, A ("Rlx A") and B ("Rlx B") are relaxin A chain and relaxin B chain polypeptides, "con" is a connector polypeptide, L is a linker polypeptide, HC X and HC Y are antibody heavy chains, LC is antibody light chain, hinge is the antibody hinge region, and Fab is an antibody Fab fragment. [Diagram 2] FIG. 2 shows LC-MS analysis of RELAX0019 and RELAX0023. A) RELAX0019 and RELAX0023 deglycosylated and non-reduced analysis shows the mass of the intact molecules, and B) RELAX0019 and RELAX0023 deglycosylated and reduced analysis shows the mass of the individual Fc fusion chains (nobrilaxin chain A and whole relaxin chain B). [Diagram 3]FIG. 3 shows the analysis of the C-terminal peptides of RELAX0019 and RELAX0023 by non-reduced peptide mapping using LC-MS. The amino acid sequences of the C-terminal peptides with the predicted disulfide bonds represented by lines are shown in the top panels. Panels A and E - extracted ion chromatograms of the C-terminal peptides in the absence of reducing agent (-DTT). Panels C and G - deconvoluted mass spectrum of the C-terminal peptides in the absence of reducing agent. Panels B and F - extracted ion chromatograms in the presence of reducing agent (+DTT) and Panels D and H - deconvoluted mass spectrum in the presence of reducing agent. FIG. 3 discloses SEQ ID NOs: 75, 77, and 76, respectively, in order of appearance. [Figure 4] FIG. 1 shows the in vitro biological activity of several heterodimeric fusions of the invention as measured by cAMP induction in cells expressing recombinant human RXFP1. [Diagram 5] FIG. 1 shows in vivo pharmacokinetic (PK) profiles from a series of ELISA experiments in which heterodimeric fusions of the invention were administered intravenously to mice. Data are normalized as %cMax at 5 minutes (T1). [Figure 6] FIG. 1 shows the reversal of isoproterenol-induced myocardial fibrosis and hypertrophy in mice treated with RELAX0019 and RELAX0023. Fibrosis and hypertrophy levels are shown for (1) vehicle (baseline), (2) isoproterenol, (3) isoproterenol + relaxin2, (4) isoproterenol + RELAX0019, and (5) isoproterenol + RELAX0023. [Figure 7] FIG. 1 shows in vitro non-specific binding of heterodimeric fusions of the invention in a Baculovirus (BV) ELISA assay. [Figure 8] The percentage of purity loss, aggregation and fragmentation of RELAX0023, RELAX0127 and RELAX0128 in solution upon storage is shown. [Figure 9]Figure 1 shows the stability of RELAX0023, RELAX0127, and RELAX0128 over time in solution as assessed by reduced LC-MS analysis: A) total ion chromatogram, B) mass spectrum of the reduced molecules. [Figure 10] 1 shows the PK profile of RELAX0023 in cynomolgus monkeys after intravenous and subcutaneous injections. [Figure 11] The nucleotide sequences encoding parts of the polypeptides of the present invention are shown (SEQ ID NOs: 80 to 140, respectively, in order of appearance). [Figure 12] Results of a long-term efficacy study of RELAX0023 in cynomolgus monkeys (Macaca fascicularis) with heart failure and reduced left ventricular ejection fraction (LVEF) are shown in (A), (B), and (C), respectively. The effects of RELAX0023 on LVEF, heart rate (HR), and mean arterial pressure (MAP) in these monkeys treated with relatively low, medium, or high doses of RELAX0023 or vehicle control for 20 weeks, followed by an observation period, are shown. Each data point represents the group mean (n=8 for treatment groups, n=14 for vehicle group). The x-axis in each of (A), (B), and (C) represents the number of weeks since the start of the treatment period. [Figure 13] [Figures 13A-13F] Cardiac function, systemic vascular resistance, and organ perfusion in the MAD cohort after RELAX0023 administration. Figure 6A shows the ejection fraction (EF) of HFpEF subjects. Figure 6B shows the EF of HFrEF subjects. Figure 6C shows the cardiac output of pooled subjects. Figure 6D shows the systemic vascular resistance (SVR) of pooled subjects. Figure 6E shows the stroke volume (SV) of pooled subjects. Figure 6F shows the estimated glomerular filtration rate (eGFR) of pooled subjects. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Significant (p<0.1) compared to placebo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Brief Description of the Sequence Listing

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[0061] [Table 16]

[0062] Detailed Description Relaxin The present invention is based, at least in part, on the discovery that the heterodimeric fusions described herein can exhibit relaxin activity when the relaxin A chain and the relaxin B chain are not covalently linked to each other via an amino acid linker. This is surprising according to the disclosures in WO 2013 / 004607 and WO 2018 / 138170, which describe recombinant relaxins in which relaxin A and relaxin B are fused in a single chain. The inventors further found that heterodimerization of the heterodimerization domain induces correct folding and heterodimerization of the relaxin A chain and the relaxin B chain (see Example 2). In addition, unlike wild-type relaxin protein, the fusion polypeptides of the present invention do not require endoproteolytic processing for biological activity.

[0063] As used herein, the term "heterodimeric fusion" refers to a heterodimer of fusion polypeptides, where one fusion polypeptide contains a first heterodimerization domain linked to a first subunit of the heterodimeric protein (e.g., relaxin A chain) and the other fusion polypeptide contains a second heterodimerization domain linked to a second subunit of the heterodimeric protein (e.g., relaxin B chain).

[0064] The heterodimeric fusions of the invention may comprise relaxin A chain and B chain polypeptides from the group of relaxins selected from relaxin 1, relaxin 2 and relaxin 3. In a preferred embodiment, the relaxin A chain polypeptide of the invention is a relaxin 2 A chain polypeptide or a variant thereof and the relaxin B chain polypeptide of the invention is a relaxin 2 B chain polypeptide or a variant thereof. In a particular embodiment, the relaxin A chain polypeptide comprises a human relaxin 2 A chain polypeptide or a variant thereof and a human relaxin 2 B chain polypeptide or a variant thereof.

[0065] The terms "chain," "polypeptide," and "peptide" may be used interchangeably herein to refer to a chain of two or more amino acids linked through peptide bonds.

[0066] In some embodiments, the relaxin 2A chain polypeptide has a sequence set forth in SEQ ID NO: 1 or a variant thereof, and the relaxin 2B chain polypeptide has a sequence set forth in SEQ ID NO: 2 or a variant thereof. The variants may include one or more amino acid substitutions, deletions and / or insertions. In some embodiments, the relaxin 2A chain polypeptide includes one or more amino acid mutations selected from K9E, K9H, K9L, K9M, R18E, R18H, R22A, R22I, R22M, R22Q, R22S, R22Y, F23E, F23A and F23I. In a preferred embodiment, the relaxin 2A chain includes the amino acid mutation K9H.

[0067] Mutants of relaxin A and B chains are known in the art. Moreover, guidance for the design of mutants of relaxin A and B chains is available to the skilled artisan. For example, it will be understood that the mutants may retain amino acids required for relaxin function. For example, mutants of relaxin2 B chain may contain the conserved motifs Arg-XXX-Arg-XX-lle (Claasz AA et al. (2002) Eur. J. Biochem. 269(24):6287-6293) or Arg-XXX-Arg-XX-Val (Bathgate RA et al. (2013) Physiol Rev. 93(1):405-480). Mutants may contain one or more amino acid substitutions and / or insertions. For example, the relaxin2 B chain mutant may have one or more additional amino acids, such as K30 and R31, N-terminal V-2, A-1 and M-1, compared to SEQ ID NO:62. Alternatively, or in addition, the variant may contain one or more amino acid derivatives, for example the first amino acid of a relaxin2 B chain variant may be pyroglutamic acid.

[0068] In a preferred embodiment, the relaxin A chain and the relaxin B chain are covalently linked by two interchain disulfide bonds (see Example 2).

[0069] The relaxin family of peptides mediate their biological effects, at least in part, through activation of G protein-coupled receptors (GPCRs) and subsequent stimulation or inhibition of the cAMP signaling pathway by the Gs or Gi protein subunits, respectively. Relaxin 2 is known to activate the GPCR RXFP1 (also known as LGR7) and, to a lesser extent, the GPCR RXFP2 (also known as LGR8), thus stimulating the Gs-cAMP-dependent signaling pathway, leading to an increase in the second messenger molecule cAMP.

[0070] As used herein, the term "relaxin activity" refers to the ability of a relaxin molecule to bind to and / or activate a relaxin receptor and / or initiate a signaling cascade inside a cell. In embodiments where the relaxin activity is relaxin2 activity, the relaxin activity may refer to the ability to bind to and / or activate the receptors RXFP1 and / or RXFP2. The term "relaxin activity" may be used interchangeably with "biological activity."

[0071] Relaxin activity may be determined by measuring binding of a relaxin molecule to a relaxin receptor and / or by measuring events downstream of binding to the relaxin receptor.

[0072] Relaxin activity may be determined in vitro and / or in vivo, hi some embodiments, relaxin activity is determined in vitro.

[0073] Relaxin activity may be determined by measuring the amount and / or presence of molecules downstream of relaxin activation of receptor. For example, relaxin activity may be determined by measuring cAMP production after relaxin activation of receptor. Methods for detection of relaxin-induced cAMP production are known in the art. Such methods include cAMP ELISA, HTRF cAMP assay and HitHunter® cAMP assay. In some embodiments, relaxin activity is determined by measuring relaxin-induced cAMP production by HTRF cAMP assay, for example as performed in Example 3. Relaxin activity may also be determined by measuring nitric oxide (NO) production after relaxin activation of receptor. Relaxin activity may also be determined by measuring activation of molecules downstream of relaxin activation of receptor. For example, relaxin activity may be determined by measuring activation of p42 / 44 MAPK.

[0074] Alternatively or additionally, relaxin activity may be determined by measuring the activation of known relaxin target genes. For example, relaxin activity may be determined by measuring the transcriptional activation of a known relaxin target gene, VEGF, in THP-1 cells. Methods for determining transcriptional activation of genes are known in the art and include quantitative PCR analysis of mRNA. The relative expression of VEGF mRNA can be measured by quantitative real-time PCR induction of VEGF transcripts after incubation of THP-1 cells with relaxin, as described in Xiao et al. (2013) Nat Commun. 4:1953.

[0075] Alternatively or additionally, relaxin activity may be determined by measuring one or more downstream effects of relaxin. For example, reduction in cardiac hypertrophy may be measured by echocardiography, left ventricular weight compared to body weight and / or calf length by standard methods. In another example, relaxin activity may be determined by measuring reduction in fibrosis by Masson's trichrome staining. In another example, relaxin activity may be determined by measuring changes in connective tissue metabolism, such as inhibition of profibrotic factors (such as TGF-beta), inhibition of fibroblast proliferation and differentiation, and / or activation of MMP-mediated extracellular matrix degradation (Bathgate RA et al. (2013) Physiol Rev. 93(1):405-480).

[0076] In some embodiments, relaxin activity is determined by measuring the reversal of isoproterenol-induced cardiac hypertrophy (measured as heart weight relative to tibia length) and fibrosis (measured as collagen content relative to heart weight), for example as performed in Example 7.

[0077] The activity of the heterodimeric fusion of the present invention can be determined by comparing it with 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 an array of relaxin A chain and relaxin B chain of mature relaxin protein. Recombinant relaxins having an array of relaxin A chain and relaxin B chain of mature relaxin protein are commercially available. For example, recombinant human relaxin2, mouse relaxin1 and INSL3 are available from R&D systems (catalogue numbers 6586-RN, 6637-RN and 4544-NS, respectively).

[0078] In some embodiments, the reference relaxin protein has the same relaxin A chain and relaxin B chain as the heterodimeric fusion of the invention or differs from the relaxin A chain and relaxin B chain of the heterodimeric fusion of the invention by 10 or fewer amino acids, e.g., 1 or 2 amino acids. In one embodiment, the first amino acid of the B chain of the reference relaxin2 is D, and this amino acid is deleted in the relaxin B chain of the heterodimeric fusion of the invention.

[0079] The reference relaxin protein is (i) recombinant human relaxin 2 (referred to herein as RELAX0013), and (ii) recombinant mouse relaxin1 (referred to herein as RELAX0014), and (iii) a recombinant Fc-fused relaxin2 (referred to herein as RELAX0010 and described in WO 2018 / 138170), in which relaxin A and relaxin B are fused into a single chain and Fc is a half-life extended Fc region; and (iv) recombinant Fc-fused relaxin2, in which relaxin A and relaxin B are fused into a single chain and Fc is a half-life extended Fc region (referred to herein as RELAX0009 and described in WO 2018 / 138170); and (v) recombinant Fc-fused relaxin2 in which relaxin A and relaxin B are fused into a single chain (referred to herein as RELAX0126 and described in WO 2013 / 004607); and (vi) recombinant Fc-fused relaxin2 in which relaxin A and relaxin B are fused into a single chain (referred to herein as RELAX0127 and described in WO 2013 / 004607); and (vii) A recombinant Fc-fusion relaxin in which relaxin A and relaxin B are fused into a single chain (referred to herein as RELAX0128 and described in WO 2013 / 004607).

[0080] In a particularly preferred embodiment, the reference relaxin protein is a relaxin 2 protein having the relaxin 2 chain A and relaxin 2 B chain arrays of the mature relaxin 2 protein disclosed in UniProtKB / Swiss-Prot Accession No. P04090.1.

[0081] Heterodimeric fusions of the invention may be considered to have relaxin activity if they exhibit at least a portion of the activity of a reference relaxin protein. For example, a fusion polypeptide may be considered to have relaxin activity if it has at least about half the activity of a reference relaxin protein. Heterodimeric fusions of the invention may have relaxin activity if the ratio of the activity of the fusion polypeptide to the activity of the reference relaxin protein is greater than or equal to about 10. -5 Between about 1 and about 10 -4 Between about 1 and about 10 -3 Between about 1 and about 10 -2 and about 1, between about 1 / 50 and about 1, between about 1 / 20 and about 1, between about 1 / 15 and about 1, between about 1 / 10 and about 1, between about 1 / 5 and about 1, between about 1 / 2 and about 1. Alternatively, a heterodimeric fusion of the invention may 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 and about 10. 5 , about 1 to about 104 , 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 can be considered to have relaxin activity.

[0082] In some embodiments, the relaxin activity of the heterodimeric fusion relative to the relaxin activity of the reference relaxin protein is about 0.001 to about 10.

[0083] Relaxin activity may be determined as an EC50 value. As used herein, the term "EC50" (median effective concentration) refers to the effective concentration of a therapeutic compound that induces a response halfway between the baseline and maximum after a particular exposure time.

[0084] Heterodimerization domain The heterodimeric fusions of the present invention comprise a first heterodimerization domain and a second heterodimerization domain, hi a preferred embodiment, the first and second heterodimerization domains are derived from an immunoglobulin Fc region.

[0085] The term "Fc region" defines the C-terminal region of an immunoglobulin heavy chain, which can be generated by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally contains two constant domains, a CH2 domain and a CH3 domain, and optionally contains a CH4 domain.

[0086] 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.

[0087] The Fc region may be derived from an immunoglobulin (e.g., IgG) from any species, preferably human (e.g., human IgG). In embodiments where the Fc region is derived from an IgG, the Fc region may be derived from an IgG of any subclass (e.g., IgG1, IgG2, IgG3, IgG4), preferably IgG1. Preferably, the first and second Fc regions are derived from a human IgG1 immunoglobulin. In other embodiments, the first and second Fc regions are derived from a human IgG4 immunoglobulin.

[0088] In a preferred embodiment, the first and second Fc regions contain heterodimerization-promoting amino acid mutations and / or modifications. Such modifications may include the introduction of asymmetric complementary modifications into each of the first and second Fc regions such that both chains are compatible with each other and therefore capable of forming heterodimers, but each chain is unable to dimerize with itself. Such modifications may include insertions, deletions, conservative and non-conservative substitutions, and rearrangements. The incorporation of such modifications provides a method for increasing the yield of heterodimers produced by recombinant cell culture over other unwanted end-products such as homodimers.

[0089] The first and second Fc regions can contain any heterodimerization-promoting amino acid mutations and / or modifications known in the art. A combination of modifications can be used to maximize the efficiency of assembly while minimizing the impact on antibody stability.

[0090] In the "knob-in-hole" method, heterodimerization can be promoted by introducing steric hindrance between contacting residues. A "protrusion" is generated 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 compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the other Fc region ("Fc hole") by replacing the amino acid with the 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, for example, in Ridgway JB et al. (1996) Protein Eng. 9(7) 617-621 and Merchant AM et al. (1998) Nat. Biotechnol. 16(7):677-681.

[0091] Other modifications that can be used to generate heterodimers include, but are not limited to, modifications that generate favorable electrostatic interactions between 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 of the other Fc region. Alternatively or additionally, the Fc region can be modified to include a mutation that introduces a cysteine ​​residue capable of forming a disulfide bond. Alternatively or additionally, the Fc region can include one or more modifications to hydrophilic and hydrophobic residues at the interface between the chains to make heterodimer formation more entropically and enthalpically favorable than homodimer formation.

[0092] Thus, in some embodiments, heterodimerization-promoting amino acid mutations and / or modifications create steric hindrance between contact residues (e.g., by "knobs-in-holes"), create favorable electrostatic interactions between the two Fc regions, introduce cysteine ​​residues capable of forming disulfide bonds, and / or modify hydrophilic and hydrophobic residues at the interface between the two Fc regions.

[0093] In a preferred embodiment, the heterodimerization-promoting amino acid mutations are "Fc knob" and "Fc hole" mutations. In a preferred embodiment, the "Fc knob" and "Fc hole" mutations are in the CH3 domain.

[0094] In some embodiments, the first and second Fc regions are derived from a human IgG1 immunoglobulin and comprise "Fc X" and "Fc Y" with mutations in the CH3 domain, where the "Fc X" and "Fc Y" mutations are selected from the combinations (or conservative substitutions thereof) set forth in Table 2.

[0095] [Table 17]

[0096] In a preferred embodiment, "Fc Y" is an "Fc hole" with the mutations Y349C, T366S, L368A and Y407V, or conservative substitutions thereof, and "Fc X" is an "Fc knob" with the mutations S354C and T366W, or conservative substitutions thereof, where amino acid numbering is according to the EU index of Kabat.

[0097] The term "Kabat EU index" refers to the numbering system for the human lgG1 EU antibody as described in Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service. National Institutes of Health. Bethesda, MD. All amino acid positions referenced in this application refer to positions in the EU index.

[0098] In some embodiments, the first Fc region has an "Fc hole" mutation and the second Fc region has an "Fc knob" mutation. In another preferred embodiment, the first Fc region has an "Fc knob" mutation and the second Fc region has an "Fc hole" mutation.

[0099] It will be understood that the Fc region may further comprise other amino acid modifications relative to the wild-type Fc region. The Fc region can be modified, for example, to increase the affinity of the IgG molecule for FcRn. WO 02 / 060919 discloses modified immunoglobulins comprising an Fc region with one or more amino acid modifications, and is incorporated herein by reference in its entirety. Methods for making Fc regions with one or more amino acid modifications are known in the art.

[0100] In some embodiments, the first and / or second Fc region may comprise one or more amino acid modifications to reduce or eliminate an effector function of the Fc region, hi some embodiments, the amino acid modifications reduce or avoid cytotoxicity, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0101] 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.

[0102] In some embodiments, the first and / or second Fc region comprises a combination of the following 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) at least one of M252Y, S254T, T256E, L234F, L235E, and P331S, or a conservative substitution thereof; Here, the amino acid numbering is according to the EU index of Kabat.

[0103] In some embodiments, the first and / or second Fc region may comprise the amino acid mutations L234F, L235E and P331S, or conservative substitutions thereof, where amino acid numbering is according to the EU index of Kabat.

[0104] In some embodiments, the Fc region comprising an "Fc hole" mutation has the sequence set forth in SEQ ID NO:3 or a variant thereof, and the Fc region comprising an "Fc knob" mutation has the sequence set forth in SEQ ID NO:4 or a variant thereof.

[0105] In some embodiments, the Fc region comprises a SEQ ID NO:3 variant having an amino acid mutation Y349C reverting Y349 and a SEQ ID NO:4 variant having an amino acid mutation S354C reverting S354, such that the Fc region is unable to form a stabilizing disulfide bond.

[0106] 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 substituted with the sequence DKTHTACPPC (SEQ ID NO:70). In another embodiment, this region is substituted with the sequence GGAGGACPPC (SEQ ID NO:71). In another embodiment, this region is substituted with the sequence ACPPC (SEQ ID NO:72).

[0107] In another embodiment, the first and second heterodimerization domains are derived from an immunoglobulin Fab region. In some embodiments, the heterodimerization domain comprises a CH1 and a CL region. The Fab region, which comprises an L chain and an Fd chain, has been shown to mediate efficient heterodimerization (Schoonjans R et al. (2000) J. Immunol. 165(12):7050-7057). Thus, in another embodiment, the heterodimerization domain comprises an L chain and an Fd chain. In some embodiments, the L chain and the Fd chain heterodimerize to form a disulfide bridge stabilized heterodimer.

[0108] In yet another embodiment, the first and second heterodimerization domains heterodimerize to form a parallel coiled-coil. Heterodimeric coiled-coils are described, for example, in Aronsson et al. (2015) Sci. Rep. 5:14063. In some embodiments, the heterodimerization domain comprises amino acid mutations and / or modifications to prevent the formation of undesired folded assemblies and / or to promote the formation of parallel coiled-coils.

[0109] The first and second heterodimerization domains (e.g., the 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.

[0110] As used herein, the term "half-life" is used to refer to the time it takes for the concentration of a fusion protein in plasma to fall to 50% of its original level. The "half-life" of a protein in plasma can vary depending on various factors, such as the size of the protein, its stability, its rate of elimination, turnover rate, in vivo proteolysis, and the rate of absorption by the body or specific tissues. Methods for measuring the half-life of a protein are known in the art and are described in the Examples below.

[0111] The present inventors have demonstrated that the heterodimeric fusions of the present 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 comparison, the half-life of human relaxin 2 after IV administration is approximately 0.09 + / - 0.04 hours, or 5.4 + / - 2.4 minutes in humans (Chen SA et al. (1993) Pharm. Res. 10(6):834-838).

[0112] It will be appreciated that an increased half-life is advantageous as it allows a therapeutic protein to be administered according to a safe and convenient dosing schedule, e.g., lower doses that can be administered less frequently. Furthermore, achieving lower doses may provide additional benefits, such as providing an improved safety profile and / or activating multiple mechanisms of action in vivo.

[0113] connector One or both of the relaxin A and B chains may be linked to their respective heterodimerization domains by a connector polypeptide. In some embodiments, the relaxin A chain is linked to a first heterodimerization domain (e.g., a first Fc region) via a connector polypeptide and the relaxin B chain is linked to a second heterodimerization domain (e.g., a second Fc region) via a connector polypeptide.

[0114] The connector polypeptide may be of any suitable length, for example, about 6-40 amino acids in length, preferably about 6-21 amino acids in length. In some embodiments, the connector 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 connector polypeptide is less than 40 amino acids in length. Connector polypeptides of different or the same length can be used in each arm of the heterodimeric fusion of the invention. In some embodiments, at least one connector polypeptide has a length of 21 amino acids. In a preferred embodiment, both connector polypeptides have a length of 21 amino acids. Connector polypeptides may have any amino acid sequence. Connector polypeptides of different or the same amino acid composition can be used in each arm of the heterodimeric fusion of the invention.

[0115] In some embodiments, one or preferably both connector polypeptides comprise a proline and alanine repeat (PA)x (SEQ ID NO:73), preferably where x is 3 to 15, preferably the connector polypeptide has a length of greater than 16 amino acids, preferably the connector polypeptide is composed of the 21 amino acid sequence PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO:6).

[0116] In some embodiments, one or preferably both connector polypeptides comprise glycine and serine repeats, such as those described in Chen X et al. (2013) Adv. Drug. Deliv. Rev. 65(10):1357-1369. In some embodiments, one or both connector polypeptides comprise the motif (GGGGS)n (SEQ ID NO:74), where n can be 1-8, e.g., n is 4. In some embodiments, one or more connector polypeptides are comprised of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:5). In certain embodiments, both connector polypeptides are comprised of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:5).

[0117] In some embodiments, one connector polypeptide contains proline and alanine repeats as described herein and the other connector polypeptide contains glycine and serine repeats as described herein.

[0118] Alternatively, one or both of the relaxin A and B chains may be linked to their respective heterodimerization domains by a synthetic connector polypeptide, such as a polyethylene glycol (PEG) polymer chain. Thus, the relaxin A chain may be linked to a first heterodimerization domain (e.g., a first Fc region) via a synthetic connector, such as a polyethylene glycol (PEG) polymer chain, and the relaxin B chain may be linked to a second heterodimerization domain (e.g., a second Fc region) via a synthetic connector, such as a polyethylene glycol (PEG) polymer chain, where the synthetic connector may be covalently or non-covalently attached to the heterodimerization domain (e.g., the Fc region). PEGylation, i.e., the process of attaching a PEG polymer chain to a molecule, may be carried out according to methods known in the art.

[0119] stability The inventors 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.

[0120] 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.

[0121] Surprisingly, the inventors have found that the heterodimeric fusions of the invention have superior physical and chemical stability compared to recombinant Fc-fused relaxins in which relaxin A and relaxin B are fused to a single chain (rather than relaxin A and B in separate fusion polypeptides). WO 2013 / 004607 describes recombinant single-chain relaxin fusion polypeptides fused to an immunoglobulin Fc region, such as the fusion polypeptides referred to herein as RELAX0127 and RELAX0128. Thus, in some embodiments, the heterodimeric fusions of the invention have superior physical and / or chemical stability compared to RELAX0127 and RELAX0128.

[0122] The heterodimeric fusion may comprise a half-life extending moiety in addition to the first and second heterodimerization domains. In some embodiments, the half-life extending moiety is a proteinaceous half-life extending moiety. The proteinaceous 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 that is not a protein or peptide, such as a polyethylene glycol (PEG) polymer chain.

[0123] The half-life extending moiety can be attached to the N-terminus or C-terminus of the first or second heterodimerization domain. In some embodiments, the half-life extending moiety is attached to the N-terminus of the first or second heterodimerization domain. In other embodiments, the half-life extending moiety is attached to the C-terminus of the first or second heterodimerization domain. Methods for attaching half-life extending moieties to heterodimeric fusions are known in the art. For example, the half-life extending moiety can be attached by chemical conjugation or recombinant techniques. The half-life extending moiety can be attached to the heterodimeric fusion directly or via a connector (e.g., a connector polypeptide). The use of a connector polypeptide may be particularly suitable when the fusion polypeptide comprises a proteinaceous half-life extending moiety, such as an Fc region.

[0124] Exemplary embodiments The heterodimeric fusions of the present invention may have a variety of formats and / or sequences.

[0125] The terms "fusion polypeptide of the invention" and "fusion polypeptide of the invention" may be used to refer to a first heterodimerization domain fused to a relaxin A chain and / or a second heterodimerization domain fused to a relaxin B chain. The fusion polypeptide of the invention may be a recombinant fusion polypeptide, i.e., one produced by recombinant DNA techniques.

[0126] In preferred embodiments, 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.

[0127] In another 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 relaxin A chain polypeptide and / or the relaxin B chain polypeptide have a free N-terminus.

[0128] The heterodimeric fusions of the invention may further comprise one or more Fabs, hi some embodiments, the heterodimeric fusions comprise 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).

[0129] Heterodimeric fusions of the 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 a first heterodimerization domain (e.g., a first Fc region) and the second relaxin B chain polypeptide or variant thereof is linked to the N-terminus of a second heterodimerization domain (e.g., a second Fc region), optionally in which case the second relaxin A chain is linked to the first heterodimerization domain (e.g., the first Fc region) via a connector (e.g., a connector polypeptide) and the second relaxin B chain is linked to the second heterodimerization domain (e.g., the second Fc region) via a connector (e.g., a connector polypeptide).

[0130] Thus, in some embodiments, the format of the heterodimeric fusion is: (i) FcX-con-A / FcY-con-B (see, for example, Figure 1 ); (ii) FcX-con-B / FcY-con-A (see, e.g., Figure 1 ); (iii) A-con-FcX / B-con-FcY (see, e.g., Figure 1 ); (iv) B-con-FcX / A-con-FcY (see, e.g., Figure 1 ); (v) Fab-FcX-con-A / Fab-FcY-con-B (see, for example, Figure 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-BLA, and FcY, optionally FcY-con-BLA (see, e.g., FIG. 1 ); (x) FcY-con-BLA, and FcX, optionally FcX-con-BLA; (xi) FcX-con-ALB, and FcY, optionally FcY-con-ALB, and (xii) selected from FcY-con-ALB, and FcX, optionally FcX-con-ALB; During the ceremony, FcY is preferably an immunoglobulin Fc region having "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is preferably an Fc region with "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain with the amino acid mutations S354C:T366W or conservative substitutions thereof; "con" is a connector polypeptide, B is a relaxin B chain or a variant thereof; A is a relaxin A chain or a variant thereof; L is preferably a linker polypeptide having the amino acid sequence GGGSGGGSGG (SEQ ID NO:60).

[0131] In another aspect, the present invention provides a method for producing a composition comprising: (i) XBLA and Y, optionally YBLA; or (ii) providing a heterodimeric fusion comprising YBLA and X, optionally XBLA; During the ceremony, X and Y are heterodimerization domains as described herein; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; L is a linker polypeptide preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60); wherein X heterodimerizes with Y, and the heterodimeric fusion has relaxin activity.

[0132] In yet another aspect, the present invention provides a method for producing a composition comprising: (i) XALB and Y, optionally YALB; or (ii) providing a heterodimeric fusion comprising YALB and X, and optionally XALB; During the ceremony, X and Y are heterodimerization domains as described herein; A is a relaxin A chain or variant thereof, e.g., relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; L is preferably a linker polypeptide having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60); wherein X heterodimerizes with Y, and the heterodimeric fusion has relaxin activity.

[0133] In a particularly preferred embodiment according to all aspects of the invention, the heterodimeric fusion comprises the fusion polypeptides Rlx011DD as set forth in SEQ ID NO: 11 and Rlx014DD as set forth in SEQ ID NO: 20. The heterodimeric fusion may also be referred to as "RELAX0023" or "AZD3427".

[0134] In another preferred embodiment, the heterodimeric fusion comprises the fusion polypeptides Rlx013DD set forth in SEQ ID NO:17 and Rlx012DD set forth in SEQ ID NO:14.

[0135] In an embodiment of the invention, a heterodimeric fusion is provided comprising a combination of fusion polypeptides selected from the FcX and FcY combinations listed in Table 3.

[0136] [Table 18]

[0137] [Table 19]

[0138] In accordance with all aspects of the present invention there is provided a heterodimeric fusion comprising the fusion polypeptides set forth in SEQ ID NO:11 and SEQ ID NO:20 for use in a method of treating a subject with heart failure complicated by pulmonary hypertension as described herein.

[0139] Alternatively, in accordance with all aspects of the present invention there is provided a heterodimeric fusion comprising the fusion polypeptides set forth in SEQ ID NO: 17 and SEQ ID NO: 14 for use in a method of treating a subject with heart failure complicated by pulmonary hypertension as described herein.

[0140] The fusion polypeptides of the invention may be produced by any method known in the art, in some embodiments, the fusion polypeptides of the invention are produced by recombinant expression of a nucleic acid molecule encoding the fusion polypeptide in a host cell.

[0141] Methods known to those skilled in the art can be used to construct expression vectors containing nucleic acid molecules encoding the fusion polypeptides of the invention. Suitable vectors include, for example, plasmid, phagemid, phage, or viral vectors.

[0142] The vector containing the nucleic acid molecule encoding the fusion polypeptide of the present invention can be transferred into a host cell by conventional techniques. Suitable host cells are known in the art. The host cell can be a mammalian cell, such as a HEK293 cell or a CHO cell.

[0143] The transfected cells may be cultured by conventional techniques to produce the fusion polypeptide of the invention.

[0144] Once a fusion polypeptide of the invention is produced, e.g., by recombinant expression, it may be purified by any method known in the art. Exemplary protein purification techniques include chromatography (e.g., ion exchange, affinity, and / or sizing column chromatography), centrifugation, and differential solubility. The present disclosure provides isolated fusion polypeptides separated from cell culture, optionally by at least one purification step.

[0145] Treatment method The fusion polypeptides of the invention may be provided in pharmaceutical compositions.

[0146] The pharmaceutical compositions of the present invention may include one or more excipients. Pharmaceutically acceptable excipients are known in the art, see, for example, Remington's Pharmaceutical Sciences (Joseph P. Remington, 18th ed., Mack Publishing Co., Easton, PA), which is incorporated herein in its entirety.

[0147] The present invention relates to a method of treating a subject with heart failure complicated by pulmonary hypertension by administering a heterodimeric fusion (or a pharmaceutical composition) as described herein, as well as to the use of said heterodimeric fusion (or said pharmaceutical composition), and said heterodimeric fusion (or said pharmaceutical composition) for use in said method. In particular, the subject may be an animal, preferably a mammal, more preferably a human.

[0148] The use or method may include administering a therapeutically effective dosing schedule in which the heterodimeric fusion / fusion polypeptide of the present invention is administered less frequently than a therapeutically effective dosing schedule for a wild-type relaxin molecule.

[0149] 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 mildly reduced ejection fraction, or heart failure with reduced ejection fraction (HFrEF). It may also include heart failure caused by hypertrophic or dilated cardiomyopathy.

[0150] As used herein, the term "pulmonary hypertension" may be defined as a subject having a mean pulmonary artery pressure of about 20 mmHg or greater, preferably 25 mmHg or greater, typically at rest. It may also be defined as a mean pulmonary artery pressure of about 30 mmHg or greater, typically when the subject is exercising or has recently exercised. Thus, a subject may have a mean pulmonary artery pressure in the range of about 20 mmHg to about 30 mmHg, preferably about 25 mmHg to about 30 mmHg, or greater. Alternatively or additionally, a subject may have: Right ventricular systolic pressure of approximately 40 mmHg or more; b. Pulmonary artery occlusion pressure (PAWP) greater than approximately 15 mmHg; and / or C. Pulmonary vascular resistance of: i. Less than 3.0 wood units, or ii.3.0 or more Wood Units may have:

[0151] Thus, in some cases, pulmonary hypertension may be classified as Group 2 pulmonary hypertension as defined by the World Health Organisation, which may also be referred to as "pulmonary hypertension complicated by heart failure due to left heart disease". In other cases, pulmonary hypertension may be classified as Group 1 pulmonary arterial hypertension as defined by the World Health Organisation (see Ryan et al., 2012, Pulm. Circ. 2(1):107-121).

[0152] Pulmonary hypertension and heart failure parameters can be measured or estimated using techniques known in the art. For example, these include echocardiography, pulmonary artery catheterization, and implantable monitoring devices. In certain embodiments, the subject may wear a blood pressure monitor, preferably a pulmonary artery pressure monitor, as known in the art. In certain embodiments, the pulmonary artery pressure monitor is a CardioMEMS pressure monitor. Typically, the device is worn before treatment with the heterodimeric fusions of the present invention described herein. Alternatively, the subject wears the device during or after the treatment period.

[0153] As used herein, the term "heart failure complicated by pulmonary hypertension" refers to the subset of heart failure subjects who also suffer from pulmonary hypertension (HF+PH subjects).

[0154] "Treatment" refers to the amelioration and / or elimination of one or more symptoms or causes of a target disease. In some embodiments, this may involve altering the levels of one or more biological markers or functions to be within a healthy range (compared to a healthy cohort). For example, the heterodimeric fusions of the invention may decrease the pulmonary vascular resistance (PVR) of a subject. For example, the PVR after treatment may be decreased by at least 1%-10%, 1%-20%, 1%-30%, 1%-40%, or 1%-50%, or more, compared to the baseline PVR (before administering the heterodimeric fusions of the invention to the subject). Thus, the heterodimeric fusions of the invention may decrease the PVR of a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, or more, compared to the baseline PVR (before administering the heterodimeric fusions of the invention to the subject). Additionally or additionally, the heterodimeric fusions of the invention may reduce the mean pulmonary artery pressure (mPAP) of a subject. For example, the mPAP may be reduced by at least 1 mmHg to 15 mmHg or more. Thus, the heterodimeric fusions of the invention may 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 heterodimeric fusions of the invention may reduce the estimated pulmonary artery diastolic pressure (ePAD) of a subject. For example, the ePAD may be reduced by at least 1 mmHg to 15 mmHg or more. Thus, the heterodimeric fusions of the invention may reduce estimated pulmonary artery diastolic pressure in a subject by at least 1 mmHg, at least 2 mmHg, at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 11 mmHg, at least 12 mmHg, at least 13 mmHg, at least 14 mmHg, or at least 15 mmHg, or more.Additionally or alternatively, the heterodimeric fusions of the invention may increase a subject's percent ejection fraction (EF%) as an index of cardiac output. For example, EF% may increase by at least 1%-10%, 1%-20%, 1%-30%, 1%-40%, or 1%-50%, or more. Thus, the heterodimeric fusions of the invention may increase a subject's percent ejection fraction (EF%) by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, or more. Additionally or alternatively, the heterodimeric fusions of the invention may increase a subject's percent ejection fraction (EF%) as compared to pre-administration baseline levels. a) It can increase the cardiac stroke volume (SV); (b) reducing systemic vascular resistance (SVR) and / or increasing estimated glomerular filtration rate (eGFR); (c) may increase the ejection fraction; and / or (d) It may increase cardiac output. The combination of a decrease in SVR and an increase in eGFR indicates improved organ perfusion.

[0155] Thus, the heterodimeric fusions of the present invention reduce the level of inflammatory bowel disease in subjects compared to pre-administration baseline levels. a) may reduce PVR; (b) reducing mPAP; (c) may reduce ePAD; (d) may increase cardiac stroke volume (SV); (e) reducing systemic vascular resistance (SVR) and / or increasing estimated glomerular filtration rate (eGFR); (f) may increase the ejection fraction; and / or (g) may increase cardiac output. A combination of a decrease in SVR and an increase in eGFR indicates improved organ perfusion. A change in one or more or all of these parameters may occur after 1-24 weeks of treatment, respectively. In some embodiments, a change in one or more or all of these parameters occurs after 24 weeks of treatment.

[0156] In certain embodiments, a reduction in mPAP as described herein may cause improvement in dyspnea, as described in Solomonica A, et al. (2013) Circ Heart Fail. 6:53-60.

[0157] The fusion polypeptides (and thus heterodimeric fusions) and / or pharmaceutical compositions of the present invention are suitable for parenteral administration to a subject or patient. In some embodiments, the subject or patient is a mammal, particularly a human.

[0158] Wild-type human relaxin2 has a half-life of several minutes in vivo. As a result, it must be administered by continuous intravenous infusion in hospitalized patients, causing severe side effects including hypotension. In contrast, it will be understood that the fusion polypeptide (and thus the heterodimeric fusion) and / or pharmaceutical composition embodiments of the present invention can be administered to a subject or patient by injection, for example, by intravenous, subcutaneous, or intramuscular injection. In some embodiments, the fusion polypeptide (and thus the heterodimeric fusion) and / or pharmaceutical composition is administered by subcutaneous injection. Administration by injection, such as by subcutaneous injection, offers the advantage of being more comfortable for the subject or patient and the opportunity to administer to the subject or patient outside of a hospital. In some embodiments, the fusion polypeptide (and thus the heterodimeric fusion) or pharmaceutical composition is administered by self-administration.

[0159] In some embodiments, the fusion polypeptides of the invention (and thus heterodimeric fusions) have increased half-life compared to wild-type relaxin, which can result in lower overall exposure on a molar basis. For example, the fusion polypeptides of the invention (and thus heterodimeric fusions) can be administered less frequently than wild-type relaxin, providing a more convenient dosing schedule.

[0160] A kit may be provided that includes the pharmaceutical composition of the present invention. The kit may include a package containing the pharmaceutical composition of the present invention and instructions. In some embodiments, the pharmaceutical composition of the present invention is formulated in a single-dose vial or container closure system (e.g., a pre-filled syringe). Such containers may optionally be affixed with a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological substances, indicating approval by that agency for manufacture, use, or sale for human administration.

[0161] As used herein, the articles "a" and "an" may refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0162] "About" may generally mean an acceptable degree of error for the quantity measured, given the nature or precision of the measurement method. Exemplary degrees of error are within a percent (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0163] An embodiment described herein as "comprising" one or more features may also be considered to disclose a corresponding embodiment "consisting of" such features.

[0164] The term "pharmaceutical acceptable," as used herein, means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans.

[0165] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format, with it being understood that such range formats are used merely for convenience and brevity and should be interpreted flexibly not only to include the numerical values ​​expressly recited as the limits of the range, but also to include all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were expressly recited.

[0166] The above-described embodiments should be understood as illustrative examples. Further embodiments are envisioned. 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. Moreover, equivalents and modifications not described above may also be used without departing from the scope of the invention as defined in the appended claims.

[0167] Other examples and variations of the fusion polypeptides and methods described herein will be apparent to those of skill in the art in light of the present disclosure.

[0168] Other examples and variations are within the scope of this disclosure as set forth in the following claims. All documents cited herein are hereby fully incorporated by reference, including all data, tables, figures, and text presented in the cited documents. EXAMPLES

[0169] Example 1: Generation of recombinant heterodimeric Fc-relaxin2 fusion protein The Fc-relaxin2 fusion proteins described herein are designed using the heterodimerization properties of the knob-in-hole Fc domain (Fc knob and Fc hole) to induce correct folding and heterodimerization of chains A and B of relaxin2.

[0170] More precisely, as shown in Figure 1, relaxin2 chains A and B are genetically fused to two complementary Fc (N- and / or C-termini of Fc) via connectors. 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 moieties facilitate the assembly and correct folding of relaxin2 as it assembles in the CHO cells. As shown in Example 2 below, disulfide bonds are subsequently formed between the complementary Fc chains and between chain A and chain B, reconstituting the native relaxin2 structure.

[0171] The heterodimeric Fc-relaxin2 fusion protein is secreted into the supernatant and purified using an automated system by affinity chromatography, where the Fc region of the protein binds to a column matrix.

[0172] Example 2: LC-MS analysis of Fc relaxin2 knob-in-hole heterodimer LC-MS analysis was performed on both non-reduced and reduced deglycosylated Fc-relaxin2 heterodimer. For deglycosylation, samples were diluted to 1 mg / ml and buffered at pH 7.80 with 10 mM Tris-Cl. PNGase F (Roche) was added to the samples at a concentration of 1 enzyme unit per 50 μg Fc-relaxin2 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 LC-MS certified total recovery vials with pre-slit caps (Waters part number: 186005663CV). For reduced analysis, 10 mM TCEP was added and samples were further incubated at 37°C for 30 min before analysis.

[0173] Experiments were performed using an ACQUITY I-Class UPLC interfaced to a Xevo G2-XS Q-TOF instrument (Waters, Milford, MA), both operated using the UNIFI Scientific Information System. For the LC system, solvent A was water with 0.1% formic acid, and solvent B was acetonitrile with 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 in the sample chamber was kept at a temperature of 4°C. A volume of 1 μL was injected onto a reversed-phase ACQUITY UPLC Protein BEH C4 Column (300 Å pore size column) (Waters part number: 186004495), and the protein was eluted using an increasing gradient of solvent B from 5% to 75% over 6 minutes.

[0174] The mass spectrometer was calibrated from 500-5000 m / z by injecting 2 μg / μL sodium iodide in 50% 2-propanol and lockspray 200 pg / μL leucine enkephalin. The instrument was operated in positive ionization mode and sensitivity analysis 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 rate = 120 L / hr; desolvation gas flow rate = 1000 L / hr; mass range = 500-5000 m / z, scan time = 1.0 s.

[0175] Data were processed with UNIFI software. Spectra were aligned from the retention time in the chromatogram where the protein of interest eluted. Raw data were background subtracted and deconvoluted using the MaxEnt1 algorithm for macromolecules. Experimental data were compared to theoretical sequence masses taking into account disulfide bonds for non-reduced analysis and free cysteines for reduced analysis. After PNGasE F deglycosylation, asparagine deamidation (+1 Da) was also taken into account.

[0176] LC-MS analysis confirmed the formation of disulfide bonds between the complementary Fc chains and between chain A and chain B, reconstituting the native relaxin2 structure. Figure 2A shows the LC-MS data of RELAX0019 and RELAX0023 as an example. Non-reduced 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. Reduced analysis (Figure 2B) confirmed the sequence identity of both chains and revealed their lack of modifications.

[0177] Non-reduced peptide mapping to identify disulfide bonds Heterodimeric Fc-relaxin (50 μg) was placed in a clean sample tube and diluted with 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 min. After alkylation, an additional 2.5 μL of 100 mM sodium phosphate buffer pH 7.0 was added followed by 2.5 μL of sodium chloride. The protein was denatured by the addition of 40 μL of 8.0 M guanidine HCl and incubated at 37° C. for 30 min. Dilution was achieved by the addition of 125 μL of 100 mM sodium phosphate buffer pH 7.0 followed by the addition of 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-relaxin2. Digestion was carried out for 2 h at 37°C, after which an additional 5 μL of Lys-C was added and incubation continued for another 2 h. For peptide analysis, 42.5 μL of the 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 min before LC-MS analysis.

[0178] Analysis of peptides was performed using an ACQUITY I-Class UPLC interfaced to a Xevo G2-XS Q-TOF instrument (Waters, Milford, MA), both operated using the UNIFI Scientific Information System. For the LC system, solvent A was water with 0.1% formic acid, and solvent B was acetonitrile with 0.1% formic acid (both UPLC-MS grade, BioSolve). The UV detector was set to measure at a wavelength of 214 nm, and the vial in the sample chamber was kept at a temperature of 4°C. A volume of 10 μL was injected onto a reversed-phase ACQUITY BEH C18 300 Å pore size column (Waters part number: 186003687), and proteins were eluted using an increasing gradient of solvent B, increasing from 5% to 37% over 73.5 min, and then to 60% B in 2.5 min. After 77.5 min, the column was held at 95% B for 5 min.

[0179] The mass spectrometer was calibrated from 100-2600 m / z by injecting 2 μg / μL sodium iodide in 50% 2-propanol and lockspray 200 pg / μL leucine enkephalin. The instrument was operated in positive ionization and sensitivity analysis modes 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 rate = 0 L / hr; desolvation gas flow rate = 500 L / hr; mass range = 100-2600 m / z, scan time = 0.5 s.

[0180] The data were processed in UNIFI software by importing sequences with predicted disulfide bonds and performing a search for matching Lys-C generated peptides. Chromatograms obtained in the absence and presence of reducing agent were overlaid to confirm that identified disulfide bonded peptides were no longer observed once reduced.

[0181] As depicted at the top of FIG. 3, a peptide was identified that matched the predicted mass of a disulfide-linked relaxin 2 peptide incorporating both chains A and B (SLSLSPGGGGGSGGGGSGGGGSGGGGGSQLYSALANKCCHVGCTK=LCGRELVRAQIAICGMSTWS=RSLARFC (SEQ ID NOs: 75-77, respectively), predicted mass with three disulfide bonds 6836.23 Da). FIG. 3(A-D) shows the identification of this peptide for RELAX0019 and its disappearance upon addition of a reducing agent. Panels A and B show extracted ion chromatograms in the absence and presence of DTT, and panels C and D show the mass spectrum of the corresponding peptide. FIG. 3(E-H) shows the identification of the same peptide for RELAX0023 and its disappearance upon addition of a reducing agent. Panels E and F show extracted ion chromatograms in the absence and presence of DTT, and panels G and H show mass spectra of the corresponding peptides. These data support that relaxin chains A and B interact through a disulfide bond within the heterodimers RELAX0019 and RELAX0023.

[0182] Example 3: In vitro activity of Fc-Relaxin2 fusion proteins (cell-based cAMP activity assay) Relaxin2 fusion polypeptides produced as described above were tested for biological activity, eg, stimulation of one or more cellular receptor responses, by the following methods.

[0183] Stable cell lines expressing the human or mouse receptors generated in CHO cells were purchased from DiscoverX. -cAMP Hunter™ CHO-K1 RXFP1 Gs cell line (DiscoverX Catalog No. 95-0127C2) -cAMP Hunter™ CHO-K1 RXFP2 Gs cell line (DiscoverX Catalog No. 95-0140C2) -cAMP Hunter™ CHO-K1 mRXFP1 Gs cell line (DiscoverX Catalog No. 95-0180C2)

[0184] Activation of these receptors results in the downstream production of the cAMP second messenger, which can be measured in a functional activity assay.

[0185] Routine cAMP assays were performed using a bovine serum albumin (BSA)-based assay buffer: Hanks' Balanced Salt Solution (Sigma# H8264) supplemented with 0.1% BSA (Sigma# A9418) and 0.5 mM IBMX (Sigma# I7018) and adjusted to pH 7.4 with 1 M NaOH.

[0186] Frozen cryovials of cells expressing the receptor of interest were quickly thawed in a water bath, transferred to pre-warmed cell culture medium, and spun at 240 x g for 5 min. Cells were cultured at an optimized concentration (e.g., 3.33 x 10 4 Cells were resuspended in cell culture medium at 1000 x g (cells / ml of hRXFP1) and 30 μL cell suspension was added to poly-D-lysine coated 384-well plates (Greiner #781946) and allowed to adhere overnight. The following day, medium was gently flicked off the plates and replaced with 5 μL assay buffer. Eleven-step serial dilutions of test recombinant peptide or Fc fusion samples were added to the cells using a non-contact liquid dispenser (ECHO™, Labcyte). All sample dilutions were made in duplicate. An additional 5 μL assay buffer was added to each well and the plate was incubated at room temperature for 30 min.

[0187] cAMP levels were measured using a commercially available cAMP dynamic G assay following a two-step protocol as recommended by the manufacturer. SHTRF kit (Cisbio, Cat #62AM4PEJ) was used to measure. Briefly, anti-cAMP cryptate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were prepared separately by diluting each 1 / 20 in the conjugate & lysis buffer provided in the kit. 5 μL anti-cAMP cryptate was added to all wells of the assay plate, and 5 μL cAMP-d2 was added to all wells except for the non-specific binding (NSB) wells, which were supplemented with conjugate and lysis buffer. Plates were incubated at room temperature for 1 hour and then read on an Envision (Perkin Elmer) using an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. Data were converted to %Delta F as described in the manufacturer's guidelines, then converted to percent activation relative to maximum natural agonist response, and analyzed by a 4-parameter logistic fit to determine EC50 values. These results are compared to corresponding results for recombinant hRRelaxin2 (R&D Systems Cat #6586 RN) in hRXFP1 cells, mRelaxin-1 (R&D Systems Cat# 6637 RN) in mRXFP1 cells, and INSL3 (R&D Systems Cat# 4544 NS) in hRXFP2 cells.

[0188] Data analysis was performed using statistical analysis software (GraphPad Prism, V6).

[0189] The biological activities of the tested constructs are listed in Table 4 and Figure 4. The average EC50 measurements for both recombinant human relaxin2 and the fusion polypeptide from several assays are summarized in Table 4.

[0190] RELAX0013, RELAX0014, and RELAX0010 are reference proteins, where RELAX0013 is recombinant human relaxin 2, RELAX0014 is recombinant mouse relaxin 1, and RELAX0010 is a single chain fusion protein comprising chain A, a 15 amino acid linker, chain B, a 15 amino acid connector, and Fc, and comprises the amino acid sequence of SEQ ID NO: 8 as described in WO 2018 / 138170.

[0191] [Table 20]

[0192] From the results shown in Table 4, it can be concluded that the heterodimeric Fc-relaxin fusion proteins tested were less potent than the single chain fusion RELAX0010 or recombinant human relaxin2 peptide, but still retained high levels of biological activity (approximately 10 pM to approximately 80 pM in the human RXFP1 cell line).

[0193] These results demonstrate that relaxin A and B chains can be fused to one or both termini (connectors can be attached to the N- or C-terminus of the relaxin chains) of a heterodimeric Fc (X or Y) and either chain retains biological activity. Thus, the heterodimeric Fc-relaxin fusion protein format described herein constitutes a robust format for generating active relaxin with a long half-life.

[0194] The presence of a disulfide bond to stabilize the heterodimeric Fc did not affect the potency of the fusion proteins (compare RELAX0023 with RELAX0021, and RELAX0024 with RELAX0022).

[0195] The two upper hinge regions used (GGAGGA (SEQ ID NO:78) and native DKTHT (SEQ ID NO:79)) did not affect potency (compare RELAX0023 with RELAX0019, and RELAX0024 with RELAX0020). The exact amino acid sequence of the upper hinge is not critical for the activity of the fusion protein.

[0196] Example 4: Effect of connector composition and length in heterodimeric relaxin2Fc fusion proteins The connector can be composed of glycine and serine residues (GS) or proline and alanine repeats (PA). The connectors used here had lengths between 6 and 21 residues. An example of a long GS connector is GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5) (21 amino acids). An example of a long PA connector is PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO: 6) (21 amino acids).

[0197] Connectors of different length and composition can be positioned on each Fc chain of a heterodimeric relaxin2Fc fusion polypeptide.

[0198] Examples of heterodimeric relaxin2Fc fusion proteins with various connectors are shown in Table 5. The table also provides information on the generability / manufacturability (expression yield and percentage of monomeric / non-aggregated relaxin2Fc fusion proteins after Protein A capture from cell culture supernatant) and biological activity.

[0199] [Table 21]

[0200] [Table 22]

[0201] The length and composition of the connector affect generative aspects of the molecule. As shown in Table 5, heterodimeric relaxin2Fc fusion polypeptides with PA connectors of 16 amino acids or less were not well expressed. In contrast, a PA connector of 21 residues in length significantly increased the expression yield. The expression yield of constructs with GS connectors is more consistent.

[0202] Heterodimeric relaxin2Fc fusion proteins with short and asymmetric (different) connectors retained potency. Reduction in biological activity was only observed for fusion proteins with low monomer content (RELAX0109, RELAX0110 and RELAX0111).

[0203] Example 5: Point mutations in the relaxin 2 sequence Relaxin single point mutant analogs were generated as heterodimeric Fc-relaxin2 fusion proteins. Table 6 shows examples of such molecules that retain potency and favorable gene expression properties.

[0204] The targeted native residues are positively charged and may be prone to proteolysis, but were not involved in binding of relaxin to its receptor.

[0205] For example, the R22X analog of the heterodimeric Fc-relaxin2 fusion protein appears to have consistently improved generability / manufacturability.

[0206] [Table 23]

[0207] The results presented in Table 6 demonstrate that some variability in the amino acid sequence of relaxin 2 chain A can be tolerated without loss of efficacy while retaining favorable gener- alities.

[0208] Example 6: PK profile of Fc-Relaxin2 fusion protein The pharmacokinetic (PK) profile of the relaxin2 fusion polypeptide was determined using a relaxin ELISA assay and / or a cAMP assay. The relaxin2 fusion polypeptide was administered at 6 mg / kg to 6-10 week old male C57BL / 6J (Jax) mice (Jackson Laboratories) by either the subcutaneous (SC) and / or intravenous (IV) route. For the IV route of administration, serum samples were collected at 5 min, 30 min, and 60 min, followed by 3 h and / or 6 h and / or 8 h, and 24 h, and then at minimum daily intervals up to a maximum of 21 days. For the SC route of administration, a similar schedule was followed, but with less frequent collections within the first 8 hours; e.g., the first sample was collected at 30 min, followed by 3 h, 8 h, 24 h, 30 h, 48 h, and then at minimum daily intervals up to a maximum of 21 days. Samples were collected by cardiac puncture into serum tubes, kept at room temperature for 15-30 min, and then centrifuged at 10,000 rpm for 10 min within 30 min of collection. Aliquots of samples were stored at <-80°C and later tested by ELISA or cAMP activity assay.

[0209] For most molecules, PK samples were tested by ELISA using anti-hRelaxin2 capture (pre-coated Human Relaxin-2 Quantikine ELISA Kit, R&D Systems Cat# DRL200) and anti-human Fc detection antibody (AU003 labeled with HRP), except for RELAX0010 (described in WO 2018 / 138170), which was tested by ELISA using anti-human Fc capture and anti-hRelaxin2 detection (using polyclonal HRP-labeled antibody from Human Relaxin-2 ELISA kit, R&D Systems Cat# DRL200). In both assays, plates coated with capture antibody were blocked with 100 μL RD1-19 assay diluent for 1 hour at room temperature. 50 μL of standard or sample was added to each well and incubated for 2 hours at room temperature. Samples were aspirated and wells were washed 3 times with assay wash buffer. HRP-labeled detection antibodies were added at 50 μL per well, diluted 1:1000 in PBS / 1% BSA for anti-human Fc specific detection, or used undiluted for anti-hRelaxin2 detection. After 1 hour incubation at room temperature followed by 3 washes, 50 μL TMB (SureBlue Reserve KPL 53-00-03) was added per well and once a color change had occurred the reaction was stopped by adding 50 μL TMB stop solution (KPL 50-85-06) per well.

[0210] Biological activity of PK samples in a cell-based cAMP activity assay. Serum samples collected from animals as outlined above were tested for biological activity to measure functional relaxin2 to assess the integrity of the Fc-relaxin2 fusion polypeptide. A stable cell line expressing the human RXFP1 receptor produced in CHO cells was purchased from DiscoverX. Activation of this receptor results in downstream production of the cAMP second messenger, which can be measured in a functional activity assay.

[0211] cAMP assays were performed using a bovine serum albumin (BSA)-based assay buffer: Hanks' Balanced Salt Solution (Sigma# H8264) supplemented with 0.1% BSA (Sigma# A9418) and 0.5 mM IBMX (Sigma# I7018) and adjusted to pH 7.4 with 1 M NaOH.

[0212] Relaxin2 fusion polypeptide or recombinant relaxin2 peptide (R&D Systems Cat#6586-RN) dosing solutions were diluted in assay buffer and then an 11-point standard curve at four matrix concentrations was generated using a non-contact liquid dispenser (ECHO, Labcyte). The matrix used was blank serum from mock-dosed animals, which was added manually to the wells at twice the concentration required to allow for the addition of cells. Test samples were transferred from serum tubes to a 384-well source plate, which was used by a non-contact liquid dispenser (ECHO, Labcyte) to set up four dilutions in assay buffer. All sample dilutions were made in duplicate.

[0213] Frozen cryovials of cells expressing hRXFP1 were rapidly thawed in a water bath, transferred to pre-warmed cell culture medium, and spun at 240 x g for 5 min. Cells were resuspended in 8 mL cell culture medium, seeded into T75 flasks containing 10 mL culture medium, and allowed to attach overnight. The next day, cells were detached using Accutase and spun at 240 x g for 5 min. The resulting cell pellet was resuspended at an optimal concentration, and 2.5 μL of cell suspension was added to each well of the assay plate using a combi-drop dispenser.

[0214] cAMP levels were measured using a commercially available cAMP dynamic 2 HTRF kit (Cisbio, Cat#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 conjugate & 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, which were added with conjugate and lysis buffer. Plates were incubated at room temperature for 1 hour and then read on an Envision (Perkin Elmer) using an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. Data were converted to % delta F as described in the manufacturer's guidelines, and sample values ​​were calculated from the linear portion of the standard curve.

[0215] Results and Conclusions 5 summarizes data from a series of in vivo PK experiments in which Fc-relaxin2 polypeptide was administered IV to mice. Data is normalized to the 5 minute time point.

[0216] The half-life of human relaxin2 after IV administration is approximately 0.09 + / - 0.04 hours, or 5.4 + / - 2.4 minutes in humans (Chen et al. 1993). All recombinant relaxin Fc fusion polypeptides show improved half-life compared to native relaxin2. Fc-relaxin polypeptides in which 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 Fc-relaxin polypeptides in which the relaxin chains are linked by a linker (exemplified by RELAX0010 and RELAX0009). However, since both linker-containing molecules, RELAX0088 and RELAX0122, exhibit good in vivo stability, the presence of the connecting linker between relaxin chain A and relaxin chain B is not solely related to the rapid in vivo clearance of the Fc-relaxin polypeptide.

[0217] Unexpectedly, in this study, the heterodimeric Fc-relaxin fusion polypeptides (RELAX0019, RELAX0023, RELAX0034, RELAX0046, RELAX0117, RELAX0088 and RELAX0122) all have significantly improved pharmacokinetic properties compared to the Fc-relaxin fusion polypeptides RELAX0010 and RELAX0009.

[0218] Example 7: Reversal of established hypertrophy and fibrosis by RELAX0019 and RELAX0023 Isoproterenol was infused into C57B6 mice via minipumps (15 mg / kg / day) for 10 days to induce cardiac hypertrophy and fibrosis. Mice infused with vehicle for the same period served as baseline controls. After 10 days, the minipumps were removed and mice were administered new minipumps containing rRelaxin2 (500 ug / kg / day) or received the first of two QW subcutaneous injections of RELAX0019 (20 mg / kg) or RELAX0023 (20 mg / kg). After a 14-day treatment period, mice were sacrificed and their hearts harvested for analysis of hypertrophy and fibrosis. Hearts were harvested from baseline control mice after removal of the vehicle minipumps. Hypertrophy was determined as a measure of heart weight relative to tibia length, and fibrosis was confirmed by quantification of collagen content relative to heart weight. Infusion of isoproterenol significantly induced both hypertrophy and fibrosis in this model. QW administration of RELAX0019 and RELAX0023 reversed isoproterenol-induced hypertrophy to baseline levels, as did continuous infusion of rRelaxin2. All relaxin treatments also reduced myocardial fibrosis by more than 50%. N=8 for each group. **p<0.01, ***p<0.001, ****p<0.0001

[0219] The recombinant relaxin Fc fusion proteins RELAX0019 and RELAX0023 were able to restore hypertrophy and fibrosis, similar to native hRelaxin2 (Figure 6).

[0220] Example 8: Assessment of non-specific binding of Fc-Relaxin2 protein using Baculovirus ELISA. The RELAX protein was expressed in CHO cells and purified as previously described. The Baculovirus ELISA (Ref: Hotzel et al 2012 mAbs 4:6,753-760), developed to assess nonspecific binding of monoclonal antibodies, was modified to determine nonspecific binding of Fc-Relaxin polypeptides; in this case, instead of calculating the "BV score" (baculovirus plate absorbance / blank plate absorbance), nonspecific binding was calculated separately for the baculovirus plate and the blank plate as a signal over background (in this case the background is the Fc-Relaxin polypeptide). This measurement was introduced to reflect the increased nonspecific binding of some Fc peptides to both coated and uncoated (blank) plates compared to monoclonal antibodies. Preparations of each protein were made at 100 nM or 10 nM in PBS (Gibco 14190-086) + 0.5% BSA (Sigma A9576) and used in duplicate in ELISA assays on 96-well Nunc Maxisorp F plates coated overnight at 4°C with 50 μL / well of either 1% Baculovirus extract in 50 mM sodium carbonate (BV plates) or 50 mM sodium carbonate (blank plates). After washing with PBS, 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 either PBS + 0.5% BSA (background) or RELAX protein dilutions were added and incubated at room temperature for 1 hour. After washing three times with PBS, 50 μL / well of detection antibody (anti-human Fc specific-HRP Sigma A0170) diluted 1:5000 in PBS+0.5% BSA was added. Samples were incubated for 1 hour at room temperature and the plate was washed three times with PBS. Next, 50 μL / well of HRP substrate TMB (SureBlue Reserve KPL 53-00-03) was added and after color change, the reaction was stopped by adding 50 μL / well of 0.5 M sulfuric acid.Absorbance was measured at 450 nm to determine non-specific binding for each sample. Non-specific binding (fold binding over background) was defined as the ratio of non-specific binding in the presence of Fc relaxin2 protein and in the absence of Fc relaxin2 protein (background). Data for Fc-relaxin2 protein tested at two different concentrations, either 100 nM or 10 nM, are shown in Table 7.

[0221] [Table 24]

[0222] [Table 25]

[0223] [Table 26]

[0224] [Table 27]

[0225] As shown in Table 7 and Figure 7, heterodimeric relaxin2Fc fusion polypeptides show lower non-specific binding when the relaxin chain is attached to the C-terminus using a GS connector. Some asymmetric and PA connectors, certain point mutations and placement of the relaxin chain at the N-terminus increase non-specific binding to both blank and BV-coated plates, especially for the bivalent molecule (RELAX0117). Some Fc-relaxin proteins with particularly high non-specific binding show higher non-specific binding to blank plates than to BV-coated plates at both high (100 nM) and low (10 nM) concentrations. The control molecules (bivalent linker-containing RELAX0009, RELAX0010, RELAX0126, RELAX0127 and RELAX0128) all show high non-specific binding, but as can be demonstrated by the low non-specific binding of RELAX0122, neither the presence of the linker between chains A and B of relaxin nor the bivalency by itself drives high non-specific binding.

[0226] Example 9: Stability in solution The stability of RELAX0023 was evaluated and compared to RELAX0127 and RELAX0128 using high performance size exclusion chromatography (HP-SEC) and liquid chromatography-mass spectrometry (LC-MS). HP-SEC with detection by absorbance at 280 nm can be used to measure purity, aggregation and fragmentation. Molecules were concentrated to 10 mg / mL after buffer exchange into the optimized formulation composition. All samples were placed in stress temperature conditions (40° C.) for up to 4 weeks. At 1, 2 and 4 weeks, samples were collected and injected into a size exclusion column and eluted isocratically with a constant flow rate of aqueous mobile phase. Larger molecules elute earlier because they are excluded from the pores of the size exclusion column in greater amounts than smaller molecules. Peaks eluting earlier than the monomer peak are recorded as aggregates. Peaks eluting after the monomer peak (excluding buffer-related peaks) are recorded as fragments. Results are recorded as percent purity; percent aggregates; and percent fragments, and are shown in FIG. 8. RELAX0023 is the most stable molecule with a purity loss (%) of only 0.1% per month compared to 7.7% and 9.3% for RELAX0128 and RELAX0127, respectively. Both RELAX0127 and RELAX0128 showed signs of aggregation, but the aggregate levels of RELAX0023 did not increase, indicating good physical solution stability. Fragmentation appeared to be the main factor in purity loss with RELAX0127 having 6.6% fragmentation per month and RELAX0128 having 6.8%. RELAX0023 has a fragmentation rate of only 0.7% per month. Furthermore, after 4 weeks of storage at 40°C, the total peak area of ​​RELAX0128 decreased from 22403 to 18216 (a 19% decrease) and RELAX0127 decreased from 22225 to 18823 (a 15% decrease). This significant loss of total peak area, together with the high fragmentation rate, indicated the high probability of chemical degradation associated with these two molecules. It should be noted that this loss of total area had a strong impact on the chromatographic profiles of these two molecules.This explains why RELAX0128 and RELAX0127 showed lower aggregate percentages at 4 weeks compared to earlier time points, despite a clear increase in aggregate peak area after storage. In contrast, the total peak area of ​​RELAX0023 only decreased by 0.03%, from 21828 to 21761, indicating a better stability profile compared to RELAX0128 and RELAX0127.

[0227] The fragmentation of the molecules was further verified by LC-MS with reducing mass spectrometry, which showed 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 did not change after stress. Mass spectra under reducing conditions also showed changes over time for RELAX0127 and RELAX0128, as evidenced by the shift of the peaks to higher 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 changes had occurred. This study indicates that RELAX0023 has superior physical and chemical stability compared to RELAX0127 and RELAX0128.

[0228] Example 10: PK profile of RELAX0023 in cynomolgus monkeys The pharmacokinetic (PK) profile of RELAX0023 in cynomolgus monkeys was determined using a sandwich ELISA immunoassay. RELAX0023 was administered to a total of 12 female cynomolgus monkeys randomly assigned to four groups, three per group. Animals in groups 1, 2, and 3 received RELAX0023 SC at 0.1, 1, and 10 mg / kg, respectively. Animals in group 4 received an IV bolus of RELAX0023 at 10 mg / kg. 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.

[0229] Assay plates were coated with goat anti-human IgG antibody and incubated with cynomolgus monkey serum from animals in groups 1-4. Plate-bound RELAX0023 was detected with an anti-relaxin antibody conjugated with HRP. Cynomolgus monkey serum was diluted 1:10 before addition to the plate. The lower limit of quantification in 100% serum is 0.010 μg / mL, and the upper limit of quantification is 0.300 μg / mL.

[0230] Results and Conclusions Figure 10 shows the mean serum concentration-time profile of RELAX0023 in cynomolgus monkeys after a single dose. After a single dose administered SC, RELAX0023 demonstrated linear PK over the dose range of 0.01 to 10 mg / kg. max A dose-proportional increase in mean C max The values ​​were 0.400, 4.69, and 34.8 μg / mL in the 0.1, 1, and 10 mg / kg SC dose groups, respectively. AUC 0-last A dose-proportional increase in values ​​was observed from the 0.1 mg / kg to 10 mg / kg SC groups. Mean AUC 0-last Values ​​were 2.01, 25.5, and 193 μg·day / mL in the 0.1, 1, and 10 mg / kg SC dose groups, respectively. Overall, RELAX0023 PK was linear over the 0.1 mg / kg to 10 mg / kg range, with a mean CL / F of 51.0 mL / day / kg and a mean t 1 / 2 The SC bioavailability of RELAX0023 was estimated to be 88.2%.

[0231] 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 elderly cynomolgus monkeys (Macaca fascicularis). Cynomolgus monkeys were chosen as the test species over other lower mammalian species because they are closely related to humans both phylogenetically and physiologically. Aged cynomolgus monkeys fed a high-fat diet for at least 2 years have the same risk factors as human patients predisposed to cardiovascular disease and characteristically develop metabolic syndrome that may progress to heart failure and reduced LVEF. The effect of RELAX0023 on LVEF was evaluated when administered by subcutaneous (SC) injection at various dose levels for 20 weeks, where the first dose was given in the first week of the study, followed by an 18-week observation period. From a pool of approximately 100 obese and elderly cynomolgus monkeys, aged 12-20 years, weighing 6-15 kg, fed a high-fat diet for at least 2 years, 38 monkeys were identified by cross-sectional echocardiographic screening as having an LVEF of 30-60%. Healthy monkeys of this age weigh 5-8 kg and have an LVEF of 70-75%, so an LVEF of 60% or less would be a HFrEF model. Identified animals were selected and randomly assigned to three treatment groups of eight animals each, and a vehicle group of 14 animals. The treatment period consisted of weekly subcutaneous administration of RELAX0023 at three escalating dose levels (all less than 10 mg / kg, the so-called "low", "medium", and "high" doses, respectively).

[0232] Cardiac function measurements by two-dimensional echocardiography were performed nine times: at baseline on week -2, and at weeks 5, 9, 13, 17, 21, 25, 29, and 33 during the treatment and post-treatment observation periods. A further two-dimensional echocardiogram is scheduled for week 39 (end of study). Parameters including LVEF were based on apical two- and four-chamber views, and biplane views. HDO (High Definition Oscillometry) was used to measure parameters including mean arterial pressure (MAP) and heart rate (HR).

[0233] Results and Conclusions RELAX0023 was able to significantly improve LVEF at weeks 5, 9, 13, 17, and 21 without affecting heart rate or blood pressure at all RELAX0023 dose levels compared to vehicle controls (Figure 12). Notably, the improvement in LVEF after treatment with RELAX0023 compared to week 0 (baseline) was observed throughout the washout period from the end of treatment to week 33 of the study. These striking results indicate a significant improvement in hemodynamics in treated animals, clearly demonstrating the efficacy of RELAX0023 in treating heart failure in this model. Moreover, the magnitude of sustained response after treatment is, to the best of our knowledge, not previously achieved with other known compounds targeting this mechanism of action pathway. Monkey monitoring will continue through week 39 of the study.

[0234] Example 12: Phase 1 (Ph1) study in healthy volunteers and patients with heart failure Study D8330C00001 was a Phase Ia / b, randomized, single-blind, placebo-controlled, first-time-in-human (FTIH) study (ClinicalTrials.gov Identifier NCT04630067). The primary objective of the study was to evaluate the safety and tolerability of single and multiple ascending doses of RELAX0023 (also referred to as "AZD3427"), and secondary objectives were to evaluate (i) the pharmacokinetics (PK) and (ii) the immunogenicity of single and multiple ascending doses of AZD3427.

[0235] The study was conducted in two parts, Part A and Part B. Part A was a single ascending dose (SAD) study in healthy participants (men and women of non-childbearing potential) and Part B was a multiple ascending dose (MAD) study in participants (men and women of non-childbearing potential) with HF.

[0236] Part B included 48 patients across six cohorts (8 participants in each cohort), of which three cohorts consisted of participants with HFrEF (Cohorts 1b, 3b, and 5b) and three cohorts consisted of participants with HF with EF ≥ 41% (Cohorts 2b, 4b, and 6b). Dose levels in the HFrEF and HF with EF ≥ 41% cohorts were 5 mg (Cohorts 1b, 2b), 15 mg (Cohorts 3b, 4b), and 45 mg (Cohorts 5b, 6b), administered once weekly (QW) for 5 weeks (i.e., 5 times in total).

[0237] In Part B, which included 48 patients across six cohorts, inclusion criteria included: (i) all cohorts: patients with a known clinical diagnosis of stage C HF (NYHA class I-III) and stable medical therapy for at least 12 weeks prior to screening, with no significant dose changes or addition of new medications during this period; (ii) cohorts 1b, 3b, and 5b: patients with a diagnosis of HFrEF, defined as EF ≤ 40%; (iii) cohorts 2b, 4b, and 6b: patients with a diagnosis of HF with EF ≥ 41% (including patients with a diagnosis of HFpEF, defined as EF ≥ 50%); and (iv) all cohorts: BMI between 18 and 40 kg / m2. 2 (18 and 40 kg / m 2 (v) all cohorts: had previously documented NT-proBNP >125 pg / mL or BNP >35 pg / mL.

[0238] Example 13: Outcomes of the Ph1 AZD3427 MAD Study in HF Patients Data from HFpEF and HFrEF patients were pooled in the MAD cohort in Part B. Trends suggest 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).

[0239] Although the hypertension status of subjects was not measured in the MAD cohort, the observed improvements in cardiac output and stroke volume (SV), reductions in systemic vascular resistance (SVR), increases in estimated glomerular filtration rate (eGFR), and thus improvements in organ perfusion (SVR and eGFR) are expected to translate to benefit in patients with HF+PH.

[0240] Example 14: Ph2b Study - A randomized, placebo-controlled, multicentre, dose-finding study of AZD3427 in participants with heart failure and pulmonary hypertension due to left heart disease (World Health Organisation (WHO) Group 2). The study (Study ID number: D8330C00003) is intended to evaluate the ability of AZD3427 to reduce pulmonary vascular resistance (PVR) after 24 weeks of treatment in participants with heart failure (HF) and pulmonary hypertension (PH) group 2.

[0241] Approximately 220 participants will be randomized (in a 1:1:1:1 ratio) into four treatment arms to receive subcutaneous (SC) injections of AZD3427 or placebo every 2 weeks for 24 weeks. The study will evaluate three dose levels of AZD3427 (Dose A, Dose B, and Dose C). No dose modifications will be applied in the study. The study will be conducted at approximately 60 study centers across an estimated 15 countries. The study will include approximately 16 study visits: two visits during the screening period, 13 visits during the treatment period, and one visit during the follow-up period. The expected total study duration is 32-37 weeks depending on the length of the screening period.

[0242] Participants will receive a single subcutaneous dose of AZD3427 (Dose A, B, or C) or placebo once every 2 weeks from Day 1 to Day 155 for 24 weeks.

[0243] The primary outcome measure is the change from baseline in pulmonary vascular resistance (PVR) after 24 weeks of treatment. The effect of AZD3427 on PVR parameters, measured by right heart catheterization (RHC), will also be assessed compared to placebo after 24 weeks of treatment in participants in HF and PH groups 2.

[0244] Secondary outcome measures included: Change from baseline in mean pulmonary artery pressure (mPAP) Change from baseline in pulmonary artery occlusion pressure (PAWP) Change from baseline in cardiac output Change from baseline in stroke volume (SV) Change from baseline in ejection fraction (EF) Change from baseline in left ventricular long axis 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) ratio Change from baseline in tricuspid regurgitation velocity (TRV) Change from baseline in TAPSE / PASP (tricuspid annular systolic excursion / pulmonary artery systolic pressure) Change from baseline in right ventricular strain / pulmonary artery systolic pressure (RVS / PASP) Change from baseline in inferior vena cava (IVC) diameter with inspiratory collapse Change from baseline in systemic vascular resistance Change from baseline in 6-minute 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 from baseline in serum creatinine Change from baseline in N-terminal pro-brain natriuretic peptide (NT-proBNP) Change from baseline in cystatin C Change from baseline in eGFR (estimated glomerular filtration rate)

[0245] Inclusion criteria: 1. Participants must be ≥18 years of age (inclusive). 2. Participants must have a pre-existing diagnosis of HF, NYHA functional class (FC) II-IV, and a pre-existing diagnosis of PH-LHD, or a possible or moderate probability of pulmonary hypertension due to left heart disease (PH-LHD) according to the 2022 European Society of Cardiology / European Respiratory Society (ESC / ESR) guidelines. Participants must be receiving standard of care treatment for stable HF, including diuretics. 3. Participants must have a combination of echocardiographic parameters indicating moderate or high probability of PH according to the 2022 ESC / ERS guidelines. 4. Participants must have an elevated pulmonary artery pressure during the study from RHC performed according to the sponsor-provided RHC manual at Screening Visit 2: (a) PAWP ≥15mmHg (b) mPAP ≥20mmHg. 5. Minimum body weight is 50 kg (inclusive). 6. Able to provide signed informed consent.

[0246] Exclusion Criteria 1. Diagnosis of 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 contraindication to RHC. 5. History of hypersensitivity to SC injections or devices. 6. History of hypersensitivity to drugs with a similar chemical structure or class to AZD3427 or any component of the AZD3427 medicinal product, or ongoing clinically significant allergy / hypersensitivity. 7. Known pulmonary disease with forced expiratory volume / vital capacity in the first second (FEV1 / VC) <30%. 8. Congenital long QT syndrome. 9. Ventricular arrhythmias requiring treatment. Participants with atrial fibrillation or flutter and rate-controlled heart will be allowed. 10. History or planned cardiac transplantation or implantation of a ventricular assist device. 11. Known planned (scheduled) highly invasive cardiovascular (CV) procedure (e.g., coronary revascularization, atrial fibrillation / flutter ablation, valve repair / replacement, aortic aneurysm surgery, etc.). 12. Participants who have previously received AZD3427.

Claims

[Claim 1] The invention described herein and in the drawings.

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