Heterodimeric relaxin fusions and uses thereof

Abstract

The present invention relates to heterodimeric relaxin fusion polypeptides, particularly heterodimeric relaxin2 fusion polypeptides, and uses thereof. Accordingly, the present invention provides relaxin fusion polypeptides, nucleic acid molecules, vectors, host cells, pharmaceutical compositions, and kits comprising the pharmaceutical compositions, as well as uses of the pharmaceutical compositions, including methods of treatment. The polypeptides and compositions of the present invention may be particularly useful in the treatment of cardiovascular diseases, such as the treatment of heart failure.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on June 11, 2021, is titled 201011(PCT)_SL.txt, and is 236,203 bytes in size.

[0002] The present invention relates to heterodimeric relaxin fusions and uses thereof. In particular, the present invention relates to relaxin-2 fusions and uses thereof. [Background technology]

[0003] Relaxin is a peptide hormone belonging to the insulin superfamily. In humans, the relaxin peptide family includes seven peptides with high structural similarity but low sequence similarity: relaxins 1, 2, and 3 and insulin-like peptides INSL3, INSL4, INSL5, and INSL6. Natural relaxins are 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, followed by secretion of mature relaxin.

[0004] Relaxin is a pleiotropic hormone known to mediate adaptive changes in systemic hemodynamics and renal function during pregnancy. Relaxin also has antifibrotic properties and has been shown to have 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 accompanied by increased myocardial cell death and interstitial fibrosis. Relaxin activates numerous signaling cascades that have been shown to be beneficial in conditions such as ischemia-reperfusion and heart failure. These signaling pathways include activation of the phosphoinositide 3-kinase pathway and the nitric oxide signaling pathway (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4).

[0005] Clinical trials have been conducted using serelaxin, an unmodified recombinant human relaxin 2. Continuous intravenous administration of serelaxin to hospitalized patients improved markers of cardiac, renal, and liver damage and congestion (Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7). However, due to the rapid elimination of serelaxin from the patient's circulation, the therapeutic effect was limited, and once the intravenous injection was stopped, the positive effects rapidly disappeared. Furthermore, approximately one-third of patients experienced a severe drop in blood pressure (>40 mm Hg) after receiving intravenous serelaxin, leading to the conclusion that the dose had to be reduced by half or even more.

[0006] Patent Documents 1 and 2 describe recombinant relaxin polypeptides in which relaxin A and relaxin B are fused into a single chain via a linker peptide. Patent Document 1 describes a recombinant relaxin comprising a linker peptide of at least 5 amino acids and less than 15 amino acids. Patent Document 2 describes a recombinant relaxin comprising a linker peptide of at least 15 amino acids. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2013 / 004607 Brochure [Patent Document 2] International Publication No. 2018 / 138170 Brochure [Non-patent literature]

[0008] [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] Felker GM et al.(2014)J.Am.Coll.Cardiol.64(15):1591-1598 [Non-patent document 6] Metra M et al.(2013)J.Am.Coll.Cardiol.61(2):196-206 [Non-Patent Document 7] Teerlink JR et al. (2013) Lancet 381(9860):29-39 Summary of the Invention [Means for solving the problem]

[0009] Given the promising clinical studies conducted to date with unmodified recombinant relaxin, there remains a need for additional recombinant relaxins that retain relaxin biological activity and offer advantages such as a prolonged half-life and convenient dosing.

[0010] The present invention relates to heterodimeric fusions with relaxin activity.

[0011] Thus, in one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, 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; and providing a heterodimeric fusion comprising: wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and said heterodimeric fusion has relaxin activity.

[0012] In some embodiments, the relaxin A chain and the relaxin B chain 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 the relaxin B chain are not covalently linked to each other by an amino acid linker.

[0013] In some embodiments, the relaxin A chain is a relaxin-2A chain and the relaxin B chain is a relaxin-2B chain.

[0014] 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.

[0015] In another embodiment, the first and second heterodimerization domains are derived from an immunoglobulin Fab region.

[0016] In yet another embodiment, the first and second heterodimerization domains heterodimerize to form a parallel coiled-coil.

[0017] In some embodiments, the relaxin A chain is tethered to a first heterodimerization domain (e.g., a first Fc region) via a connector, and the relaxin B chain is tethered to a second heterodimerization domain (e.g., a second Fc region) via a connector. In preferred embodiments, one or preferably both connectors are polypeptides.

[0018] 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 specific embodiment, both connectors have the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5].

[0019] 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 tethered 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.

[0020] 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 the EU index of Kabat.

[0021] 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.

[0022] The present invention also provides the following: (i) an FcX-con-A fusion polypeptide; (ii) an FcY-con-B fusion polypeptide; and Heterodimeric fusions comprising: where: A is a relaxin A chain or variant thereof, e.g., a relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; FcY is an immunoglobulin (e.g., IgG1) Fc region having "Fc-hole" amino acid mutations and / or modifications, preferably comprising a CH3 domain with the amino acid mutations Y349C:T366S:L368A:Y407V or conservative substitutions thereof; FcX comprises an immunoglobulin (e.g., IgG1) Fc region with "Fc knob" amino acid mutations and / or modifications, preferably a CH3 domain with 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]; Here, the amino acid numbering is according to the EU index of Kabat, and FcX heterodimerizes with FcY, and the heterodimer fusion product has relaxin activity.

[0023] 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.

[0024] In some embodiments of any aspect of the invention, the heterodimeric fusion further comprises one or more Fabs; optionally, 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).

[0025] 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., a 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., a 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.

[0026] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) FcX-BLA and FcY, optionally FcY-BLA; or (ii) FcY-BLA and FcX, optionally FcX-BLA; and providing a heterodimeric fusion comprising: where: FcY is an immunoglobulin (e.g., IgG1) Fc region having "Fc-hole" amino acid mutations and / or modifications, preferably comprising a CH3 domain with the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is an immunoglobulin (e.g., IgG1) Fc region with "Fc knob" amino acid mutations and / or modifications, preferably comprising a CH3 domain with 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., a relaxin 2 A chain or variant thereof; L is preferably a linker polypeptide having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60], wherein amino acid numbering is according to the EU index of Kabat, and 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 tethered to FcX and / or FcY via a connector, optionally a connector polypeptide having a length of 6 to 40 amino acids, e.g., 21 amino acids.

[0027] In yet another aspect, the present invention provides a method for producing a composition comprising: (i) FcX-ALB and FcY, optionally FcY-ALB; or (ii) FcY-ALB and FcX, optionally FcX-ALB; and providing a heterodimeric fusion comprising: where: FcY is an immunoglobulin (e.g., IgG1) Fc region having "Fc-hole" amino acid mutations and / or modifications, preferably comprising a CH3 domain with the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is an immunoglobulin (e.g., IgG1) Fc region with "Fc knob" amino acid mutations and / or modifications, preferably comprising a CH3 domain with the amino acid mutations S354C:T366W, or conservative substitutions thereof; A is a relaxin A chain or variant thereof, e.g., a relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; L is a linker polypeptide preferably having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60], wherein amino acid numbering is according to the EU index as in Kabat, and wherein 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 tethered to FcX and / or FcY via a connector, optionally a connector polypeptide having a length of 6 to 40 amino acids, e.g., 21 amino acids.

[0028] 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 from about 0.001 to about 10.

[0029] In related aspects, the invention provides 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 collecting the fusion proteins.

[0030] In another aspect, the invention provides pharmaceutical compositions comprising the heterodimeric fusions of the invention, kits comprising the same, and uses of the heterodimeric fusions in therapy, such as methods of treating a subject with heart failure.

[0031] 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. The present disclosure includes, for example: [Section 1] below: (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; a heterodimeric fusion comprising: A heterodimeric fusion, wherein the first heterodimerization domain heterodimerizes with a second heterodimerization domain, and wherein the heterodimeric fusion has relaxin activity. [Section 2] The heterodimeric fusion of paragraph 1, wherein the relaxin A chain polypeptide and the relaxin B chain polypeptide are covalently linked by at least one interchain disulfide bond. [Section 3] 3. The heterodimeric fusion of paragraph 1 or 2, wherein the relaxin A chain and the relaxin B chain are not covalently linked to each other by an amino acid linker. [Section 4] Item 4. The heterodimeric fusion product according to any one of Items 1 to 3, wherein the relaxin A chain is a relaxin-2A chain and the relaxin B chain is a relaxin-2B chain. [Section 5] 5. The heterodimeric fusion of any one of paragraphs 1 to 4, wherein the relaxin A chain is connected to the first heterodimerization domain via a connector and the relaxin B chain is connected to the second heterodimerization domain via a connector, and optionally, one or preferably both connectors are polypeptides. [Section 6] Item 6. The heterodimeric fusion of Item 5, wherein one or preferably both of the connectors have a length of 6 to 40 amino acids, for example, one or preferably both of the connectors have a length of 21 amino acids. [Section 7] 7. The heterodimeric fusion of any one of paragraphs 1 to 6, wherein the first and second heterodimerization domains are derived from immunoglobulin Fc regions (the "first Fc region" and the "second Fc region," respectively), and optionally the first and second Fc regions comprise constant domains CH2 and CH3. [Section 8] The heterodimeric fusion of paragraph 7, wherein the C-terminus of the first Fc region is linked to the N-terminus of the relaxin A chain and the C-terminus of the second Fc region is linked to the N-terminus of the relaxin B chain. [Section 9] 9. The heterodimeric fusion of paragraph 7 or 8, wherein the first and second Fc regions comprise heterodimerization-promoting amino acid mutations and / or modifications, and optionally the heterodimerization-promoting amino acid mutations are "Fc knob" and "Fc hole" mutations, e.g., "Fc knob" and "Fc hole" mutations present in the CH3 domain. [Section 10] Item 10. The heterodimeric fusion product according to any one of Items 7 to 9, wherein the first and second Fc regions are derived from human IgG1 immunoglobulin. [Section 11] the heterodimer-promoting amino acid mutations are selected from the group consisting of: a. "Fc hole" mutations Y349C, T366S, L368A, and Y407V in one CH3 domain; and b. "Fc knob" mutations S354C and T366W in the other CH3 domain Including, Item 11. The heterodimeric fusion of item 10, wherein the amino acid numbering is according to the EU index of Kabat. [Section 12] a. the first Fc region comprises the "Fc knob" mutation and the second Fc region comprises the "Fc hole" mutation; or b. The heterodimeric fusion of paragraph 11, wherein the second Fc region comprises the "Fc knob" mutation and the first Fc region comprises the "Fc hole" mutation. [Section 13] 13. The heterodimeric fusion of any one of items 10 to 12, wherein the first and / or second Fc region comprises the amino acid mutations L234F, L235E, and P331S, wherein the amino acid numbering is according to the EU index of Kabat. [Section 14] Item 14. The heterodimeric fusion product of any one of items 4 to 13, wherein 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. [Section 15] 15. The heterodimeric fusion of paragraph 14, wherein the relaxin-2A chain polypeptide comprises the amino acid mutation K9H, K17M, or K17I. [Section 16] 16. The heterodimeric fusion of any one of paragraphs 5 to 15, wherein both connectors have the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5]. [Section 17] below: (i) an FcX-con-A fusion polypeptide; (ii) an FcY-con-B fusion polypeptide; and a heterodimeric fusion comprising: A is a relaxin A chain or variant thereof, e.g., a relaxin-2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin-2 B chain or variant thereof; FcY is an Fc region comprising the constant domains CH2 and CH3 of human IgG1 immunoglobulin, and containing "Fc hole" amino acid mutations and / or modifications, preferably amino acid mutations Y349C:T366S:L368A:Y407V; FcX is an Fc region, preferably comprising the constant domains CH2 and CH3 of human IgG1 immunoglobulin, with "Fc knob" amino acid mutations and / or modifications, preferably the amino acid mutations S354C:T366W; con is preferably a connector polypeptide having the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5], wherein the amino acid numbering is according to the EU index of Kabat, and FcX heterodimerizes with FcY, and the heterodimeric fusion has relaxin activity. [Section 18] Item 18. The heterodimeric fusion product according to any one of items 1 to 17, comprising a fusion polypeptide having the amino acid sequence of SEQ ID NO: 11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO: 20. [Section 19] 19. The heterodimeric fusion of any one of items 8 to 18, wherein the heterodimeric fusion further comprises one or more Fabs, optionally comprising one Fab linked to the N-terminus of the first Fc region and a second Fab linked to the N-terminus of the second Fc region. [Section 20] 20. The heterodimeric fusion of any one of items 8 to 19, further comprising a second relaxin A chain polypeptide or a variant thereof linked to the N-terminus of the first Fc region and a second relaxin B chain polypeptide or a variant thereof linked to the N-terminus of the second Fc region, optionally wherein the second relaxin A chain is linked to the first Fc region via a connector polypeptide and the second relaxin B chain is linked to the second Fc region via a connector polypeptide. [Section 21] below: (i) FcX-BLA and FcY, optionally FcY-BLA; or (ii) FcY-BLA and FcX, optionally FcX-BLA; a heterodimeric fusion comprising: FcY is preferably an immunoglobulin Fc region with "Fc hole" amino acid mutations and / or modifications comprising a CH3 domain with the amino acid mutations Y349C:T366S:L368A:Y407V; FcX is preferably an immunoglobulin Fc region with "Fc knob" amino acid mutations and / or modifications comprising a CH3 domain with amino acid mutations S354C:T366W; 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., a relaxin 2 A chain or variant thereof; L is preferably a linker polypeptide having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60], wherein the amino acid numbering is according to the EU index as in Kabat, and in this case FcX heterodimerizes with FcY, and the heterodimeric fusion has relaxin activity. [Section 22] 22. The heterodimeric fusion of claim 21, wherein the relaxin B chain is connected to FcX and / or FcY via a connector, optionally a connector polypeptide having a length of 6 to 40 amino acids, e.g., a length of 21 amino acids. [Section 23] Item 23. The heterodimer fusion product according to any one of Items 1 to 22, wherein the ratio of the relaxin activity of the heterodimer fusion product to the relaxin activity of a reference relaxin protein is about 0.001 to about 10. [Section 24] A nucleic acid molecule encoding the heterodimeric fusion product according to any one of items 1 to 23. [Section 25] A vector comprising the nucleic acid molecule of item 24. [Section 26] A host cell comprising the vector of paragraph 25 or the nucleic acid molecule of paragraph 24. [Section 27] 24. A method for producing the heterodimeric fusion protein of any one of claims 1 to 23, comprising culturing the host cell of claim 26 and collecting the fusion protein. [Section 28] 24. A pharmaceutical composition comprising the heterodimer fusion product according to any one of items 1 to 23 and a pharmaceutically acceptable excipient. [Section 29] The heterodimeric fusion product according to any one of Items 1 to 23 or the pharmaceutical composition according to Item 28 for use in therapy. [Section 30] The heterodimeric fusion of any one of Items 1 to 23 or the pharmaceutical composition of Item 28 for use in treating a subject with heart failure, wherein the heterodimeric fusion or pharmaceutical composition is administered to the subject. [Section 31] 31. The heterodimeric fusion for use according to paragraph 29 or 30, or the pharmaceutical composition for use according to paragraph 29 or 30, wherein the heterodimeric fusion or pharmaceutical composition is administered to the subject by subcutaneous injection. [Section 32] 32. The heterodimeric fusion for use according to any one of paragraphs 29 to 31, or the pharmaceutical composition for use according to any one of paragraphs 29 to 31, wherein the fusion polypeptide or pharmaceutical composition is administered by self-administration. [Section 33] Item 29. A kit comprising the pharmaceutical composition of Item 28. [Section 34] A method for treating a subject having a disease or disorder, comprising administering to the subject the heterodimeric fusion product of any one of Items 1 to 23 or the pharmaceutical composition of Item 28. [Section 35] A method for treating a subject with heart failure, comprising administering to the subject the heterodimeric fusion product of any one of Items 1 to 23 or the pharmaceutical composition of Item 28. [Section 36] 36. The method of paragraph 34 or 35, wherein the heterodimeric fusion or pharmaceutical composition is administered to the subject by subcutaneous injection. [Section 37] Item 37. The method of any one of Items 34 to 36, wherein the heterodimeric fusion or pharmaceutical composition is administered by self-administration.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING [Brief explanation of the drawings]

[0033] [Figure 1]1 shows an exemplary format of a heterodimeric fusion 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 the antibody light chain; hinge is the antibody hinge region; and Fab is the antibody Fab fragment. [Figure 2] Figure 2 shows LC-MS analyses of RELAX0019 and RELAX0023. A) Deglycosylated and non-reduced analyses of RELAX0019 and RELAX0023 show the masses of the intact molecules, and B) Deglycosylated and reduced analyses of RELAX0019 and RELAX0023 show the masses of the individual Fc fusion chains (nobrilaxin chain A and whole relaxin chain B). [Figure 3] Figure 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 bond represented by a line, are shown in the top panel. Panels A and E—extracted ion chromatograms of the C-terminal peptides in the absence of a reducing agent (-DTT). Panels C and G—deconvoluted mass spectra of the C-terminal peptides in the absence of a reducing agent. Panels B and F—extracted ion chromatograms in the presence of a reducing agent (+DTT) and panels D and H—deconvoluted mass spectra in the presence of a reducing agent. Figure 3 discloses SEQ ID NOs: 75, 77, and 76, respectively, in order of appearance. [Figure 4] FIG. 4 shows the in vitro biological activity of several heterodimeric fusions of the invention as measured by cAMP induction in cells expressing recombinant human RXFP1. [Figure 5]5 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] Figure 6 shows the reversal of isoproterenol-induced myocardial fibrosis and hypertrophy in mice treated with RELAX0019 and RELAX0023. Levels of fibrosis and hypertrophy are shown for (1) vehicle (baseline), (2) isoproterenol, (3) isoproterenol + relaxin2, (4) isoproterenol + RELAX0019, and (5) isoproterenol + RELAX0023. [Figure 7] FIG. 7 shows the in vitro non-specific binding of heterodimeric fusions of the invention in a Baculovirus (BV) ELISA assay. [Figure 8] FIG. 8 shows the percentage of purity loss, aggregation, and fragmentation of RELAX0023, RELAX0127, and RELAX0128 in solution upon storage. [Figure 9] Figure 9 shows the stability of RELAX0023, RELAX0127, and RELAX0128 in solution over time as assessed by reduced LC-MS analysis. A) Total ion chromatogram, B) Mass spectrum of the reduced molecules. [Figure 10] FIG. 10 shows the PK profile of RELAX0023 in cynomolgus monkeys after intravenous and subcutaneous injections. [Figure 11] FIG. 11 shows the nucleotide sequences encoding parts of the polypeptides of the present invention (SEQ ID NOs: 80 to 140, respectively, in order of appearance).

[0034] [Table 1]

[0035] [Table 2]

[0036] Table 3

[0037] Table 4

[0038] Table 5

[0039] Table 6

[0040] Table 7

[0041] Table 8

[0042] Table 9

[0043] Table 10

[0044] Table 11

[0045] Table 12

[0046] [Table 13]

[0047] [Table 14]

[0048] [Table 15] DETAILED DESCRIPTION OF THE INVENTION

[0049] 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 given the disclosures in WO 2013 / 004607 and WO 2018 / 138170, which describe recombinant relaxins in which relaxin A and relaxin B are fused into a single chain. The inventors further found that heterodimerization of the heterodimerization domains induces correct folding and heterodimerization of the relaxin A chain and the relaxin B chain (see Example 2). In addition, unlike wild-type relaxin proteins, the fusion polypeptides of the present invention do not require endoproteolytic processing for biological activity.

[0050] 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., a relaxin A chain) and the other fusion polypeptide contains a second heterodimerization domain linked to a second subunit of the heterodimeric protein (e.g., a relaxin B chain).

[0051] 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 preferred embodiments, 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 certain embodiments, 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.

[0052] The terms "chain," "polypeptide," and "peptide" may be used interchangeably herein to refer to a chain of two or more amino acids linked via peptide bonds.

[0053] In some embodiments, the relaxin2 A chain polypeptide has the sequence set forth in SEQ ID NO: 1 or a variant thereof, and the relaxin2 B chain polypeptide has the sequence set forth in SEQ ID NO: 2 or a variant thereof. The variants can include one or more amino acid substitutions, deletions, and / or insertions. In some embodiments, the relaxin2 A 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 relaxin2 A chain includes the amino acid mutation K9H.

[0054] Mutants of relaxin A and B chains are known in the art. Moreover, guidance for designing mutants of relaxin A and B chains is available to those skilled in the art. For example, it will be understood that mutants may retain amino acids required for relaxin function. For example, a relaxin 2 B chain mutant may contain the conserved motif Arg-XXX-Arg-XX-Ile (Claasz AA et al., (2002) Eur. J. Biochem. 269(24):6287-6293) or Arg-XXX-Arg-XX-Val (Bathgate RA et al., (2013) Physiol Rev. 93(1):405-480). The mutant may contain one or more amino acid substitutions and / or insertions. For example, a relaxin 2 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 additionally, the variant may comprise one or more amino acid derivatives, for example, the first amino acid of a relaxin-2 B chain variant may be pyroglutamic acid.

[0055] In a preferred embodiment, the relaxin A chain and the relaxin B chain are covalently linked by two interchain disulfide bonds (see Example 2).

[0056] The relaxin family of peptides mediates 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 and leading to an increase in the second messenger molecule cAMP.

[0057] 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."

[0058] 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 a relaxin receptor.

[0059] Relaxin activity may be determined in vitro and / or in vivo, hi some embodiments, relaxin activity is determined in vitro.

[0060] Relaxin activity may be determined by measuring the amount and / or presence of molecules downstream of relaxin-mediated activation of the receptor. For example, relaxin activity may be determined by measuring cAMP production after relaxin-mediated activation of the receptor. Methods for detecting 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 an HTRF cAMP assay, for example, as performed in Example 3. Relaxin activity may also be determined by measuring nitric oxide (NO) production after relaxin-mediated activation of the receptor. Relaxin activity may also be determined by measuring the activation of molecules downstream of relaxin-mediated activation of the receptor. For example, relaxin activity may be determined by measuring the activation of p42 / 44 MAPK.

[0061] 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 gene transcriptional activation 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.

[0062] Alternatively or additionally, relaxin activity may be determined by measuring one or more downstream effects of relaxin. For example, reduction in cardiac hypertrophy can be measured by echocardiography, left ventricular weight relative to body weight and / or 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).

[0063] 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), as performed, for example, in Example 7.

[0064] The activity of the heterodimeric fusion of the present invention can be determined in comparison 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 the relaxin A chain and relaxin B chain sequence of a mature relaxin protein. Recombinant relaxins having the relaxin A chain and relaxin B chain sequence of a mature relaxin protein are commercially available. For example, recombinant human relaxin 2, mouse relaxin 1, and INSL3 are available from R&D systems (catalog numbers 6586-RN, 6637-RN, and 4544-NS, respectively).

[0065] 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 relaxin-2 is D, and this amino acid is deleted in the relaxin B chain of the heterodimeric fusion of the invention.

[0066] The reference relaxin protein is: (i) recombinant human relaxin 2 (referred to herein as RELAX0013); (ii) recombinant mouse relaxin1 (referred to herein as RELAX0014); (iii) a recombinant Fc-fused relaxin 2 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 RELAX0010 and described in WO 2018 / 138170); (iv) a recombinant Fc-fused relaxin 2 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); (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); (vi) a recombinant Fc-fused relaxin 2 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) 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). You can choose from:

[0067] In a particularly preferred embodiment, the reference relaxin protein is a relaxin2 protein having the relaxin2 chain A and relaxin2 chain B arrangement of the mature relaxin2 protein disclosed in UniProtKB / Swiss-Prot accession number P04090.1.

[0068] Heterodimeric fusions of the invention may be considered to have relaxin activity if they exhibit at least a portion of the activity of the 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 the reference relaxin protein. Heterodimeric fusions of the invention may be considered to have relaxin activity if the ratio of the activity of the fusion polypeptide to the activity of the reference relaxin protein is about 10 -5 and about 1, about 10 -4 and about 1, about 10 -3 and about 1, about 10 -2 and about 1, between about 1 / 50 and about 1, between about 1 / 20 and about 1, between about 1 / 15 and about 1, between about 1 / 10 and about 1, between about 1 / 5 and about 1, or between about 1 / 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 105 , about 1 to about 10 4 , about 1 to about 10 3 , about 1 to about 100, about 1 to about 50, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 2, can be considered to have relaxin activity.

[0069] 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.

[0070] 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 elicits a response halfway between baseline and maximum after a particular exposure time.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The Fc region may be derived from an immunoglobulin (e.g., IgG) from any species, preferably human (e.g., human IgG). In embodiments in which the Fc region is derived from IgG, the Fc region may be derived from 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.

[0075] In preferred embodiments, the first and second Fc regions comprise 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 thus 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.

[0076] The first and second Fc regions can comprise any heterodimerization-promoting amino acid mutations and / or modifications known in the art. A combination of modifications can be used to maximize assembly efficiency while minimizing impact on antibody stability.

[0077] In the "knobs-in-holes" approach, heterodimerization can be promoted by introducing steric hindrance between contacting residues. A "protrusion" is created by replacing one or more small amino acid side chains from the interface of one Fc region ("Fc knob") with a larger side chain (e.g., tyrosine or tryptophan). A 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 a larger side chain with an amino acid with a smaller side chain (e.g., alanine or valine). "Knobs-in-hole" modifications are described in detail, for example, in Ridgway JB et al. (1996) Protein Eng. 9(7) 617-621; Merchant AM et al. (1998) Nat. Biotechnol. 16(7):677-681.

[0078] Other modifications that can be used to generate heterodimers include, but are not limited to, modifications that create favorable electrostatic interactions between two Fc regions. For example, one or more positively charged amino acids may be introduced into one Fc region and one or more negatively charged amino acids may be introduced into the corresponding positions of the other Fc region. Alternatively or additionally, the Fc region may be modified to contain mutations that introduce cysteine ​​residues capable of disulfide bond formation. Alternatively or additionally, the Fc region may contain one or more modifications to hydrophilic and hydrophobic residues at the interchain interface to make heterodimer formation more entropically and enthalpically favorable than homodimer formation.

[0079] 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 disulfide bond formation, and / or modify hydrophilic and hydrophobic residues at the interface between the two Fc regions.

[0080] In preferred embodiments, the heterodimerization-promoting amino acid mutations are "Fc knob" and "Fc hole" mutations. In preferred embodiments, the "Fc knob" and "Fc hole" mutations are in the CH3 domain.

[0081] In some embodiments, the first and second Fc regions are derived from human IgG1 immunoglobulin and comprise "FcX" and "FcY" with mutations in the CH3 domain, wherein the "FcX" and "FcY" mutations are selected from the combinations (or conservative substitutions thereof) set forth in Table 2.

[0082] [Table 16]

[0083] In a preferred embodiment, "FcY" is an "Fc hole" having the mutations Y349C, T366S, L368A and Y407V, or conservative substitutions thereof, and "FcX" is an "Fc knob" having the mutations S354C and T366W, or conservative substitutions thereof, where amino acid numbering is according to the EU index of Kabat.

[0084] The term "Kabat EU index" refers to the numbering system for the human IgG1 EU antibody as described in Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service. National Institutes of Health. Bethesda, MD. All amino acid positions referenced in this application refer to EU index positions.

[0085] 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.

[0086] 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 producing Fc regions with one or more amino acid modifications are known in the art.

[0087] 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).

[0088] 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.

[0089] In some embodiments, the first and / or second Fc region comprises the following combination of amino acid mutations: (i) M252Y, S254T and T256E, or conservative substitutions thereof; (ii) L234F, L235Q and K322Q, or conservative substitutions thereof; (iii) L234F, L235E and P331S, or conservative substitutions thereof; (iv) M252Y, S254T, T256E, L234F, L235Q, and K322Q, or conservative substitutions thereof; or (v) M252Y, S254T, T256E, L234F, L235E, and P331S, or conservative substitutions thereof wherein amino acid numbering is according to the EU index of Kabat.

[0090] 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.

[0091] In some embodiments, the Fc region comprising the "Fc hole" mutation has the sequence set forth in SEQ ID NO: 3 or a variant thereof, and the Fc region comprising the "Fc knob" mutation has the sequence set forth in SEQ ID NO: 4 or a variant thereof.

[0092] In some embodiments, the Fc region comprises a SEQ ID NO:3 variant having an amino acid mutation Y349C that reverts Y349 and a SEQ ID NO:4 variant having an amino acid mutation S354C that reverts S354, such that the Fc region is unable to form a stabilizing disulfide bond.

[0093] 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).

[0094] 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. Fab regions comprising an L chain and an Fd chain have been found 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.

[0095] 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.

[0096] 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.

[0097] As used herein, the term "half-life" refers to the time it takes for the concentration of a fusion protein in plasma to decrease 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 clearance rate, metabolic turnover rate, in vivo proteolysis, and absorption rate by the body or specific tissues. Methods for determining the half-life of a protein are known in the art and are described in the Examples below.

[0098] The present inventors have demonstrated that heterodimeric fusions of the invention having first and second heterodimerization domains derived from immunoglobulin Fc have a half-life of at least 5 hours in a mouse model (see Example 6). By 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).

[0099] It will be appreciated that a prolonged half-life is advantageous because it allows a therapeutic protein to be administered according to a safe and convenient dosing schedule, e.g., by allowing smaller doses to be administered less frequently. Furthermore, achieving a lower dose may provide additional benefits, such as providing an improved safety profile and / or activating multiple mechanisms of action in vivo.

[0100] connector One or both of the relaxin A and B chains may be connected 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.

[0101] Connector polypeptides can be of any suitable length, for example, about 6 to 40 amino acids in length, preferably about 6 to 21 amino acids in length. In some embodiments, connector polypeptides are at least 6 amino acid residues in length, preferably at least 11 amino acids in length, and preferably at least 16 amino acids in length. In some embodiments, connector polypeptides are less than 40 amino acids in length. Connector polypeptides of different or the same length can be used in each arm of the heterodimeric fusions of the 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 can have any amino acid sequence. Connector polypeptides of different or the same amino acid composition can be used in each arm of the heterodimeric fusions of the invention.

[0102] 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, and preferably the connector polypeptide consists of the 21 amino acid sequence PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO:6).

[0103] In some embodiments, one or preferably both connector polypeptides comprise glycine and serine repeats as 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 consist of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:5). In particular embodiments, both connector polypeptides consist of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:5).

[0104] In some embodiments, one connector polypeptide comprises proline and alanine repeats as described herein, and the other connector polypeptide comprises glycine and serine repeats as described herein.

[0105] Alternatively, one or both of the relaxin A and B chains may be linked to their respective heterodimerization domains by synthetic connector polypeptides, such as polyethylene glycol (PEG) polymer chains. 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 noncovalently attached to the heterodimerization domain (e.g., the Fc region). PEGylation, the process of attaching PEG polymer chains to a molecule, can be carried out according to methods known in the art.

[0106] 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.

[0107] 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.

[0108] 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 (rather than relaxin A and B in separate fusion polypeptides) are fused into a single chain. 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.

[0109] 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.

[0110] 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 a half-life extending moiety to a heterodimeric fusion 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 appropriate when the fusion polypeptide comprises a proteinaceous half-life extending moiety, such as an Fc region.

[0111] Illustrative Embodiments The heterodimeric fusions of the present invention can have a variety of formats and / or sequences.

[0112] 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. A fusion polypeptide of the invention may be a recombinant fusion polypeptide, i.e., one produced by recombinant DNA techniques.

[0113] 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.

[0114] In another embodiment, the N-terminus of a first heterodimerization domain (e.g., a first Fc region) is linked to the C-terminus of a relaxin A chain and the N-terminus of a second heterodimerization domain (e.g., a second Fc region) is linked to the C-terminus of a relaxin B chain. In some embodiments, the relaxin A chain polypeptide and / or the relaxin B chain polypeptide have a free N-terminus.

[0115] 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).

[0116] The 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 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).

[0117] Thus, in some embodiments, the format of the heterodimeric fusion is: (i) FcX-con-A / FcY-con-B (see, e.g., 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, e.g., Figure 1 ); (vi)Fab-FcX-con-B / Fab-FcY-con-A; (vii) A-con-FcX-con-A / B-con-FcY-con-B (see, e.g., 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., Figure 1 ); (x) FcY-con-BLA, and FcX, optionally FcX-con-BLA; (xi) FcX-con-ALB, and FcY, optionally FcY-con-ALB; and (xii) FcY-con-ALB, and FcX, optionally FcX-con-ALB is selected from wherein FcY is an immunoglobulin Fc region having "Fc-hole" amino acid mutations and / or modifications, preferably comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V, or conservative substitutions thereof; FcX is an Fc region with "Fc knob" amino acid mutations and / or modifications, preferably 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).

[0118] In another aspect, the present invention provides a method for producing a composition comprising: (i) XBLA and Y, optionally YBLA; or (ii) YBLA and X, optionally XBLA and providing a heterodimeric fusion comprising: wherein X and Y are heterodimerization domains 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., a 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.

[0119] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) XALB and Y, optionally YALB or (ii) YALB and X, optionally XALB a heterodimeric fusion comprising: wherein X and Y are heterodimerization domains described herein; A is a relaxin A chain or variant thereof, e.g., a relaxin 2 A chain or variant thereof; B is a relaxin B chain or variant thereof, e.g., a relaxin 2 B chain or variant thereof; 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.

[0120] In a particularly preferred embodiment, the heterodimeric fusion comprises the fusion polypeptides Rlx011DD set forth in SEQ ID NO: 11 and Rlx014DD set forth in SEQ ID NO: 20. 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.

[0121] In an embodiment of the present invention, a heterodimeric fusion is provided comprising a combination of fusion polypeptides selected from the FcX and FcY combinations listed in Table 3.

[0122] [Table 17]

[0123] [Table 18]

[0124] In one aspect, a heterodimeric fusion is provided comprising the fusion polypeptides set forth in SEQ ID NO:11 and SEQ ID NO:20.

[0125] In another aspect, a heterodimeric fusion is provided comprising the fusion polypeptides set forth in SEQ ID NO:17 and SEQ ID NO:14.

[0126] Fusion polypeptides of the invention may be produced by any method known in the art, hi some embodiments, fusion polypeptides of the invention are produced by recombinant expression in a host cell of a nucleic acid molecule encoding the fusion polypeptide.

[0127] Methods known to those skilled in the art can be used to construct expression vectors containing the nucleic acid molecules of the invention. Suitable vectors include, for example, plasmid, phagemid, phage, or viral vectors.

[0128] Vectors containing the nucleic acid molecules of the present invention can be transferred into host cells by conventional techniques. Suitable host cells are known in the art. In some embodiments, the host cells are mammalian cells, such as HEK293 cells or CHO cells.

[0129] The transfected cells may be cultured by conventional techniques to produce the fusion polypeptide of the invention.

[0130] 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 invention provides isolated fusion polypeptides separated from cell culture, optionally by at least one purification step.

[0131] treatment method The fusion polypeptides of the invention may be provided in pharmaceutical compositions.

[0132] Pharmaceutical compositions of the invention may include one or more excipients. Pharmaceutically acceptable excipients are known in the art; see, for example, Remington's Pharmaceutical Sciences (by Joseph P. Remington, 18th ed., Mack Publishing Co., Easton, PA), which is incorporated herein in its entirety.

[0133] The present invention encompasses therapeutic methods comprising administering a fusion polypeptide of the present invention to an animal, particularly a mammal, such as a human, to prevent, treat, or ameliorate symptoms associated with a disease, disorder, or infection.

[0134] Thus, the fusion polypeptides or pharmaceutical compositions of the invention may be used in therapy, for example, to treat a disease or disorder. Also provided are methods for treating a disease or disorder, comprising administering to a subject or patient in need thereof a therapeutically effective amount of a fusion polypeptide of the invention. The use or method may comprise administering a therapeutically effective schedule of taking the fusion polypeptide of the invention less frequently than a therapeutically effective dosing schedule of a wild-type relaxin molecule.

[0135] It will be appreciated that the fusion polypeptides of the present invention may be used in the treatment of cardiovascular diseases, for example, in the treatment of heart failure.

[0136] 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).

[0137] Fusion polypeptides of the invention may also be used in the treatment of kidney disease, lung disease, and fibrotic disorders, such as fibrotic disorders of the kidney, heart, lung, and liver, as well as in wound healing (Sherwood OD (2004) Endocrine Reviews 25(2):205-234). Fusion polypeptides of the invention may also be used to reverse insulin resistance in diabetic patients (Bonner JS et al. (2013) Diabetes 62(9):3251-3260). Fusion polypeptides of the invention may also be used in various forms of pulmonary hypertension. Fusion polypeptides of the invention may also be used in diseases caused by or resulting from arteriosclerosis, decreased arterial elasticity, decreased arterial compliance and distensibility, including hypertension, kidney disease, peripheral arterial disease, carotid and cerebrovascular disease (i.e., stroke and dementia), diabetes, microvascular disease resulting in end-organ damage, coronary artery disease, and heart failure.

[0138] The fusion polypeptides and / or pharmaceutical compositions of the invention are suitable for parenteral administration to a subject or patient. In some embodiments, the subject or patient is a mammal, particularly a human.

[0139] Wild-type human relaxin-2 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 embodiments of the fusion polypeptides and / or pharmaceutical compositions of the present invention may be administered to a subject or patient by injection, such as by intravenous, subcutaneous, or intramuscular injection. In some embodiments, the fusion polypeptides and / or pharmaceutical compositions are 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 flexibility of administering to the subject or patient outside of a hospital. In some embodiments, the fusion polypeptide or pharmaceutical composition is administered by self-administration.

[0140] In some embodiments, the fusion polypeptides of the invention have an increased half-life compared to wild-type relaxin, which allows for lower overall exposure on a molar basis. For example, the fusion polypeptides of the invention may be administered less frequently than wild-type relaxin, thus providing a more convenient dosing schedule.

[0141] The present invention provides kits comprising the pharmaceutical compositions of the present invention. The kits may include a package containing the pharmaceutical compositions of the present invention and instructions. In some embodiments, the pharmaceutical compositions of the present invention are formulated in single-dose vials or container closure systems (e.g., pre-filled syringes). Optionally, such containers may be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice indicates approval by the agency for manufacture, use, or sale for human administration.

[0142] 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.

[0143] "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.

[0144] Embodiments described herein as "comprising" one or more features may also be considered to disclose corresponding embodiments "consisting of" such features.

[0145] As used herein, the term "pharmaceutically acceptable" 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.

[0146] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format, it being understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limits, but also all of the individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly recited.

[0147] The above-described embodiments should be understood as illustrative examples. Further embodiments are contemplated. It should be understood that any feature described with respect to any one embodiment may be used alone or in combination with other features described, and may also be used in combination with one or more features of any other embodiment or any combination of any other embodiments. Furthermore, equivalents and modifications not described above may also be used without departing from the scope of the invention as defined in the appended claims.

[0148] 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 this disclosure, and are within the scope of this disclosure, as set forth in the appended claims.

[0149] All documents cited herein are hereby incorporated by reference in their entirety, including all data, tables, figures, and text presented in the cited documents. [Example]

[0150] Example 1: Production 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 relaxin2 chains A and B.

[0151] More precisely, as shown in Figure 1, relaxin2 chains A and B are genetically fused to two complementary Fc chains (at the N- and / or C-termini of the 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 within 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.

[0152] Once the heterodimeric Fc-relaxin2 fusion protein is secreted into the supernatant, it is purified using an automated system by affinity chromatography, where the Fc region of the protein binds to a column matrix.

[0153] 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 heterodimers. 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 unit of enzyme per 50 μg of Fc-relaxin2 and incubated overnight at 37°C. For non-reduced analysis, samples were diluted to 0.05 mg / ml with water, and 20 μL was loaded into LC-MS-certified total collection vials with pre-slit caps (Waters part number: 186005663CV). For reduced analysis, 10 mM TCEP was added, and the samples were further incubated at 37°C for 30 minutes before analysis.

[0154] 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 containing 0.1% formic acid, and solvent B was acetonitrile containing 0.1% formic acid (both UPLC-MS grade, BioSolve). The UV detector was set to measure wavelengths of 220 nm and 280 nm, and the vial was placed in a sample chamber maintained at 4°C. A 1 μL volume was injected onto a reversed-phase ACQUITY UPLC Protein BEH C4 column, 300 Å pore size (Waters part number: 186004495), and the protein was eluted using a gradient of solvent B increasing from 5% to 75% over 6 minutes.

[0155] The mass spectrometer was calibrated from 500 to 5000 m / z by injecting 2 μg / μL sodium iodide in 50% 2-propanol and 200 pg / μL leucine enkephalin with lockspray. The instrument was operated in positive ionization mode and sensitivity analyzer mode with the following key settings: capillary voltage = 3.0 V; sample cone voltage = 40 V; source temperature = 120 °C; desolvation temperature = 450 °C; cone gas flow rate = 120 L / h; desolvation gas flow rate = 1000 L / h; mass range = 500 to 5000 m / z, scan time = 1.0 s.

[0156] Data were processed using UNIFI software. Spectra were aligned to the retention time of 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 the mass of theoretical sequences, which took into account disulfide bonds for non-reduced analysis and free cysteines for reduced analysis. After PNGasE F deglycosylation, asparagine deamidation (+1 Da) was also considered.

[0157] LC-MS analysis confirmed the formation of disulfide bonds between the complementary Fc chains and between chains A and B, reconstituting the native relaxin-2 structure. Figure 2A shows the LC-MS data for RELAX0019 and RELAX0023 as examples. Non-reduced analysis confirmed the formation of heterodimers with the expected masses of 58,932 Da and 59,361 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.

[0158] 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 and then incubating at room temperature for 20 minutes. 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 adding 40 μL of 8.0 M guanidine HCl and incubated at 37°C for 30 minutes. Dilution was achieved by adding 125 μL of 100 mM sodium phosphate buffer, pH 7.0, followed by 0.5 μL of 40 mM EDTA. Endoproteinase Lys-C (Wako Chemicals) was reconstituted in water at a concentration of 1 mg / ml, and 5 μL was added to Fc-relaxin 2. Digestion was carried out at 37°C for 2 hours, after which an additional 5 μL of Lys-C was added and incubation continued for another 2 hours. For peptide analysis, 42.5 μL of the sample was transferred to a UPLC vial and 2.5 μL of water was added. For disulfide bond reduction, 2.5 μL of 500 mM DTT was added to another 42.5 μL aliquot of the sample and left at room temperature for 15 minutes before LC-MS analysis.

[0159] Peptide analysis 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 containing 0.1% formic acid, and solvent B was acetonitrile containing 0.1% formic acid (both UPLC-MS grade, BioSolve). The UV detector was set to measure at a wavelength of 214 nm, and the vial was placed in a sample chamber maintained at 4 °C. A 10 μL volume was injected onto a reversed-phase ACQUITY BEH C18 300 Å pore size column (Waters part number: 186003687). Proteins were eluted using an increasing gradient of solvent B, increasing from 5% to 37% B 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.

[0160] The mass spectrometer was calibrated from 100 to 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 mode and sensitivity analyzer mode with the following key settings: capillary voltage = 3.0 V; sample cone voltage = 25 V; source temperature = 100 °C; desolvation temperature = 250 °C; cone gas flow rate = 0 L / h; desolvation gas flow rate = 500 L / h; mass range = 100 to 2600 m / z, scan time = 0.5 s.

[0161] 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.

[0162] As depicted at the top of Figure 3, we identified a peptide matching 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 containing three disulfide bonds of 6836.23 Da). Figure 3 (A-D) shows the identification of this peptide to RELAX0019 and confirmation that the peptide was no longer observed 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 corresponding mass spectrum of the peptide. Figure 3 (E-H) shows the identification of the same peptide for RELAX0023 and confirmation that the peptide disappeared 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 the corresponding mass spectra of the peptide. These data confirm that relaxin chains A and B interact via a disulfide bond within the heterodimers RELAX0019 and RELAX0023.

[0163] 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 method.

[0164] Stable cell lines expressing human or mouse receptors produced 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) Activation of these receptors results in the downstream production of the cAMP second messenger, which can be measured in a functional activity assay.

[0165] Routine cAMP assays were performed using a bovine serum albumin (BSA)-based assay buffer: Hank's Balanced Salt Solution (Sigma #H8264) supplemented with 0.1% BSA (Sigma #A9418) and 0.5 mM IBMX (Sigma #I7018), adjusted to pH 7.4 with 1 M NaOH. 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 minutes. Cells were cultured at an optimized concentration (e.g., 3.33 x 10 4 Cells were resuspended in cell culture medium at 1000 cells / ml hRXFP1, and 30 μL of the cell suspension was added to a poly-D-lysine-coated 384-well plate (Greiner #781946) and allowed to adhere overnight. The following day, the medium was gently flicked off the plate and replaced with 5 μL of assay buffer. Eleven 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 of assay buffer was added to each well, and the plate was incubated at room temperature for 30 minutes.

[0166] cAMP levels were measured using a commercially available cAMP dynamic G assay following a two-step protocol as recommended by the manufacturer. SMeasurements were performed using an HTRF kit (Cisbio, Cat #62AM4PEJ). Briefly, anti-cAMP cryptate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were prepared separately by diluting each 1 / 20 in the conjugate and lysis buffer provided in the kit. 5 μL of anti-cAMP cryptate was added to all wells of the assay plate, and 5 μL of cAMP-d2 was added to all wells except for the nonspecific binding (NSB) wells, which received the conjugate and lysis buffer. Plates were incubated for 1 hour at room temperature 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 and then to percent activation relative to the maximum natural agonist response, as described in the manufacturer's guidelines, and then to EC 50 The results were analyzed by a four-parameter logistic fit to determine the ΔΨ values. These results are compared to the corresponding results for recombinant hRelaxin-2 (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.

[0167] Data analysis was performed using statistical analysis software (GraphPad Prism, V6).

[0168] The biological activities of the constructs tested are listed in Table 4 and Figure 4. The mean EC50 measurements for both recombinant human relaxin2 and the fusion polypeptide from several assays are summarized in Table 4.

[0169] 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 described in WO 2018 / 138170.

[0170] [Table 19]

[0171] 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).

[0172] These results demonstrate that relaxin A and B chains can be fused to one or both ends of a heterodimeric Fc (connectors can be attached to the N or N terminus of a relaxin chain) and to either chain (X or Y) and retain biological activity. Thus, the heterodimeric Fc-relaxin fusion protein format described herein constitutes a robust format for producing active relaxin with a long half-life.

[0173] 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).

[0174] 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.

[0175] Example 4: Effect of connector composition and length in heterodimeric relaxin2 Fc fusion proteins The connector can be composed of glycine and serine residues (GS) or proline and alanine repeats (PA). The connectors used here ranged in length from 6 to 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 PAPAPAPAPAPAPAPAPAPAPAG (SEQ ID NO: 6) (21 amino acids). Connectors of different lengths and compositions can be placed on each Fc chain of a heterodimeric relaxin2 Fc fusion polypeptide.

[0176] Examples of heterodimeric relaxin-2 Fc 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 relaxin-2 Fc fusion protein after Protein A capture from cell culture supernatant) and biological activity.

[0177] [Table 20]

[0178] The length and composition of the connector affect generative aspects of the molecule. As shown in Table 5, heterodimeric relaxin2 Fc fusion polypeptides with PA connectors of 16 amino acids or less were not well expressed. In contrast, a 21-residue PA connector significantly increased expression yields. Expression yields of constructs with GS connectors were more consistent.

[0179] Heterodimeric relaxin2 Fc fusion proteins with short and asymmetric (different) connectors retained potency, and reduced biological activity was observed only with fusion proteins with low monomer content (RELAX0109, RELAX0110, and RELAX0111).

[0180] Example 5: Point mutations in the relaxin2 sequence Relaxin single point mutation analogs were generated as heterodimeric Fc-relaxin2 fusion proteins. Table 6 shows examples of such molecules that retain potency and favorable generative properties.

[0181] The targeted native residues are positively charged and may be prone to proteolysis, but were not involved in binding of relaxin to its receptor.

[0182] For example, heterodimeric Fc-relaxin2 fusion proteins of the R22X analog appear to have consistently improved generativity / manufacturability.

[0183] [Table 21]

[0184] 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 potency while retaining favorable generability.

[0185] 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) via either the subcutaneous (SC) or intravenous (IV) route. For the IV route, serum samples were collected at 5, 30, and 60 minutes, followed by 3, 6, 8, and 24 hours after drug administration, and then at minimum daily intervals for up to 21 days. For the SC route, a similar schedule was followed, but samples were collected less frequently within the first 8 hours; for example, the first sample was collected at 30 minutes, followed by 3, 8, 24, 30, and 48 hours, followed by at minimum daily intervals for up to 21 days. Samples were collected by cardiac puncture into serum tubes, held 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.

[0186] 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, HRP-conjugated), except for RELAX0010 (described in WO 2018 / 138170), which was tested by ELISA using anti-human Fc capture and anti-hRelaxin2 detection (using a polyclonal HRP-conjugated antibody from the Human Relaxin-2 ELISA kit, R&D Systems Cat# DRL200). For both assays, the capture antibody-coated plates 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. The samples were aspirated, and the wells were washed three 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-hRelaxin 2 detection. After a 1-hour incubation at room temperature and three washes, 50 μL of TMB (SureBlue Reserve KPL 53-00-03) was added per well, and upon color change, 50 μL of TMB stop solution (KPL 50-85-06) was added per well to stop the reaction.

[0187] 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 and assess the integrity of the Fc-relaxin2 fusion polypeptide. A stable cell line expressing the human RXFP1 receptor in CHO cells was purchased from DiscoverX. Activation of this receptor leads to downstream production of the cAMP second messenger, which can be measured in a functional activity assay.

[0188] cAMP assays were performed using a bovine serum albumin (BSA)-based assay buffer: Hank's balanced salt solution (Sigma #H8264) supplemented with 0.1% BSA (Sigma #A9418) and 0.5 mM IBMX (Sigma #I7018), adjusted to pH 7.4 with 1 M NaOH.

[0189] The dose solution of relaxin 2 fusion polypeptide or recombinant relaxin 2 peptide (R&D Systems Cat#6586-RN) was diluted with 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 manually added to the wells at twice the concentration required to allow for cell addition. Test samples were transferred from serum tubes to a 384-well source plate, which was used to set up four dilutions in assay buffer using a non-contact liquid dispenser (ECHO, Labcyte). All sample dilutions were performed in duplicate.

[0190] Frozen cryovials of cells expressing hRXFP1 were rapidly thawed in a water bath, transferred to preheated cell culture medium, and spun at 240 x g for 5 minutes. The cells were resuspended in 8 mL of cell culture medium, seeded into T75 flasks containing 10 mL of culture medium, and allowed to adhere overnight. The next day, cells were detached using Accutase and spun at 240 x g for 5 minutes. The resulting cell pellet was resuspended at the optimal concentration, and 2.5 μL of the cell suspension was added to each well of the assay plate using a Combidrop dispenser.

[0191] cAMP levels were measured using a commercially available cAMP dynamic 2 HTRF kit (Cisbio, Cat. #62AM4PEJ) according to the manufacturer's recommendations, following a two-step protocol. Briefly, anti-cAMP cryptate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were prepared separately by diluting them 1 / 20 with the conjugate and lysis buffer provided in the kit. 2.5 μL of anti-cAMP cryptate was added to all wells of the assay plate, and 2.5 μL of cAMP-d2 was added to all wells except the nonspecific binding (NSB) wells, which were then supplemented with conjugate and lysis buffer. Plates were incubated at room temperature for 1 hour and then read on an Envision (Perkin Elmer) instrument 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.

[0192] Results and Conclusions 5 shows a summary of data from a series of in vivo PK experiments in which Fc-relaxin2 polypeptide was administered IV to mice. Data are normalized to the 5 minute time point.

[0193] 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 et al. 1993). All recombinant relaxin Fc fusion polypeptides show improved half-lives compared to native relaxin-2. Fc-relaxin polypeptides in which the relaxin A and B chains are linked to different heterodimeric Fc chains (exemplified by RELAX0019, RELAX0023, RELAX0034, RELAX0046, and RELAX0117) have improved PK properties compared to 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 alone is not directly related to the rapid in vivo clearance of the Fc-relaxin polypeptide.

[0194] 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.

[0195] Example 7: Reversal of established hypertrophy and fibrosis by RELAX0019 and RELAX0023 Isoproterenol was infused into C57B6 mice via a minipump (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 the mice received either a new minipump containing rRelaxin2 (500 μg / kg / day) or the first of two weekly subcutaneous injections of RELAX0019 (20 mg / kg) or RELAX0023 (20 mg / kg). After the 14-day treatment period, the mice were sacrificed, and their hearts were harvested for hypertrophy and fibrosis analysis. 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. Isoproterenol infusion 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 per group. **p<0.01, ***p<0.001, ****p<0.0001

[0196] The recombinant relaxin Fc fusion proteins RELAX0019 and RELAX0023 were able to reverse hypertrophy and fibrosis, similar to native hRelaxin 2 (Fig. 6).

[0197] Example 8: Evaluation of non-specific binding of Fc-relaxin2 protein using Baculovirus ELISA. The RELAX protein was expressed in CHO cells and purified as previously described. A 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 in 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. After the color change, 50 μL / well of 0.5 M sulfuric acid was added to stop the reaction.Absorbance was measured at 450 nm to determine nonspecific binding for each sample. Nonspecific binding (fold binding over background) was defined as the ratio of nonspecific binding in the presence of Fc-relaxin2 protein to that 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.

[0198] [Table 22]

[0199] [Table 23]

[0200] [Table 24]

[0201] As shown in Table 7 and Figure 7, heterodimeric relaxin2 Fc fusion polypeptides exhibit lower nonspecific 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 nonspecific binding to both blank and BV-coated plates, particularly for the bivalent molecule (RELAX0117). Some Fc-relaxin proteins with particularly high nonspecific binding exhibit higher nonspecific binding to blank plates than to BV-coated plates at both high (100 nM) and low (10 nM) concentrations. The control molecules (linker-containing bivalent 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 in itself drives high non-specific binding.

[0202] Example 9: Stability in solution The stability of RELAX0023 was evaluated and compared with that of RELAX0127 and RELAX0128 using high-performance size-exclusion chromatography (HP-SEC) and liquid chromatography-mass spectrometry (LC-MS). HP-SEC, including detection by absorbance at 280 nm, can be used to measure purity, aggregation, and fragmentation. The molecule was buffer-exchanged into an optimized formulation and then concentrated to 10 mg / mL. All samples were subjected to stress temperature conditions (40°C) for up to 4 weeks. At 1, 2, and 4 weeks, samples were collected and injected onto a size-exclusion column and eluted isocratically with a constant flow rate of aqueous mobile phase. Larger molecules are excluded from the pores of the size-exclusion column in greater amounts than smaller molecules, resulting in earlier elution. 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 reported as percent purity; percent aggregates; and percent fragments, and are shown in Figure 8. RELAX0023 was the most stable molecule, with a purity loss rate of only 0.1% per month, compared to 7.7% and 9.3% for RELAX0128 and RELAX0127, respectively. While both RELAX0127 and RELAX0128 showed signs of aggregation, aggregate levels for RELAX0023 did not increase, indicating good physical solution stability. Fragmentation appeared to be the primary factor in purity loss, with RELAX0127 having 6.6% fragmentation per month and RELAX0128 having 6.8% fragmentation per month. RELAX0023 had a fragmentation rate of only 0.7% per month. Furthermore, after 4 weeks of storage at 40°C, the total peak area for RELAX0128 decreased from 22403 to 18216 (a 19% decrease), and that for RELAX0127 decreased from 22225 to 18823 (a 15% decrease). This significant loss of total peak area, along with the high fragmentation rate, indicated a 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 percent aggregates 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 decreased by only 0.03%, from 21828 to 21761, indicating a better stability profile compared to RELAX0128 and RELAX0127.

[0203] The fragmentation of the molecules was further verified by LC-MS using 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 remained unchanged after stress. Mass spectra under reducing conditions also showed changes over time for RELAX0127 and RELAX0128, evidenced by peak shifts to higher masses and peak broadening, indicating greater heterogeneity (Figure 9B). In contrast, the intact mass spectrum of RELAX0023 remained unchanged, indicating no changes had occurred. This study demonstrates that RELAX0023 has superior physical and chemical stability compared to RELAX0127 and RELAX0128.

[0204] 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 12 female cynomolgus monkeys, randomly assigned to four groups of three monkeys per group. Animals in groups 1, 2, and 3 received 0.1, 1, and 10 mg / kg of RELAX0023 SC, respectively. Animals in group 4 received a 10 mg / kg IV bolus of RELAX0023. Serum samples were collected at 0.25 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 96 hours, 7 days, 14 days, and 21 days after drug administration.

[0205] Assay plates were coated with goat anti-human IgG antibody and incubated with cynomolgus monkey serum from animals in groups 1 to 4. Plate-bound RELAX0023 was detected with an HRP-conjugated anti-relaxin antibody. Cynomolgus monkey serum was diluted 1:10 before addition to the plate. The lower limit of quantitation in 100% serum was 0.010 μg / mL, and the upper limit of quantitation was 0.300 μg / mL.

[0206] 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 exhibited 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. 0-last A dose-proportional increase in mean AUC values ​​was observed from the 0.1 mg / kg to 10 mg / kg SC groups. 0-last The 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%.

Claims

1. below: (i) a polypeptide set forth in SEQ ID NO: 11; (ii) a polypeptide set forth in SEQ ID NO: 20; and a heterodimeric fusion comprising: SEQ ID NO:11 comprises a first heterodimerization domain linked to a relaxin A chain polypeptide; SEQ ID NO:20 comprises a second heterodimerization domain linked to a relaxin B chain polypeptide; A heterodimeric fusion, wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and wherein the heterodimeric fusion has relaxin activity.

2. The heterodimeric fusion of claim 1, wherein the relaxin A chain polypeptide and the relaxin B chain polypeptide are covalently linked by at least one interchain disulfide bond.

3. The heterodimeric fusion of claim 1 or 2, wherein the relaxin A chain and the relaxin B chain are not covalently linked to each other by an amino acid linker.

4. The heterodimeric fusion of any one of claims 1 to 3, wherein said heterodimeric fusion further comprises one or more Fabs.

5. 5. The heterodimeric fusion of claim 4, wherein the heterodimeric fusion comprises a first Fab linked to the N-terminus of the first heterodimerization domain and a second Fab linked to the N-terminus of the second heterodimerization domain.

6. A nucleic acid molecule encoding the heterodimeric fusion of any one of claims 1 to 5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the vector of claim 7 or the nucleic acid molecule of claim 6.

9. 10. A method for producing the heterodimeric fusion of any one of claims 1 to 5, comprising culturing the host cell of claim 8 and harvesting the heterodimeric fusion.

10. A pharmaceutical composition comprising the heterodimeric fusion of any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

11. 11. The pharmaceutical composition of claim 10 for use in therapy.

12. 11. The pharmaceutical composition of claim 10 for use in the treatment of heart failure.

13. 13. The pharmaceutical composition for use according to claim 11 or 12, which is administered by subcutaneous injection.

14. The pharmaceutical composition for use according to any one of claims 11 to 13, which is administered by self-administration.

15. A kit comprising the pharmaceutical composition of claim 10.

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