Heterodimer relaxin fusions and their use

Abstract

This invention provides further recombinant relaxin that retains relaxin biological activity and offers advantages such as a long half-life and favorable drug administration. [Solution] The present invention relates to heterodimeric relaxin fusion polypeptides, particularly heterodimeric relaxin 2 fusion polypeptides, and their uses. Accordingly, the present invention provides relaxin fusion polypeptides, nucleic acid molecules, vectors, host cells, pharmaceutical compositions, and the use of pharmaceutical compositions, including kits and methods of treatment comprising the pharmaceutical compositions. The polypeptides and compositions of the present invention may be particularly useful in the treatment of cardiovascular diseases, for example, in the treatment of heart failure.

Description

[Technical Field]

[0001] Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy was created on 11 June 2021, named 201011(PCT)_SL.txt, and has a size of 236,203 bytes.

[0002] This invention relates to heterodimer relaxin fusions and their uses. In particular, this invention relates to relaxin-2 fusions and their uses. [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: relaxin 1, 2, and 3, as well as insulin-like peptides INSL3, INSL4, INSL5, and INSL6. Natural relaxin consists of A and B polypeptide chains covalently linked by two interchain disulfide bonds. The A chain further contains another intrachain disulfide bond. The relaxin gene encodes a prohormone in structure BCA (B and A polypeptide chains linked by a C peptide). The prohormone undergoes intracellular proteolytic cleavage by the PC1 and PC2 enzymes to remove the C peptide, after which it secretes mature relaxin.

[0004] Relaxin is a multifaceted hormone known to mediate adaptive changes in systemic hemodynamics and renal function during pregnancy. Relaxin also possesses anti-fibrotic properties and has been shown to have beneficial effects in heart failure, such as acute decompensated heart failure (ADHF). Heart failure is associated with severe morbidity and mortality. It is characterized by complex tissue remodeling accompanied by increased cardiomyocyte death and interstitial fibrosis. Relaxin activates numerous signaling cascades that have been shown to be beneficial in situations such as ischemia-reperfusion and heart failure. These signaling pathways include activation of the phosphoinositide 3-kinase pathway and the nitric oxide signaling pathway (Non-Patent Literature 1; Non-Patent Literature 2; Non-Patent Literature 3; Non-Patent Literature 4).

[0005] Clinical trials were conducted using seleraxin, which is unmodified recombinant human relaxin 2. Sequential intravenous administration of seleraxin to hospitalized patients improved markers of cardiac, renal, and hepatic damage, as well as congestion (Non-Patent Literature 5; Non-Patent Literature 6; Non-Patent Literature 7). However, the therapeutic effect was limited due to the rapid elimination of seleraxin from the patient's blood circulation, and the positive effect rapidly disappeared once intravenous injection was stopped. Furthermore, it was concluded that approximately one-third of patients experienced a severe drop in blood pressure (>40 mm Hg) after receiving seleraxin intravenously, requiring a dose reduction of 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 by a linker peptide. Patent document 1 describes recombinant relaxin containing a linker peptide of at least 5 amino acids and less than 15 amino acids. Patent document 2 describes recombinant relaxin containing a linker peptide of at least 15 amino acids. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2013 / 004607 Pamphlet [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 [Overview of the Initiative] [Means for solving the problem]

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

[0010] The present invention relates to a heterodimeric fusion having relaxin activity.

[0011] Thus, in one aspect, the present invention provides: (i) a first heterodimerization domain linked to at least one relaxin A-chain polypeptide or a variant thereof; (ii) a second heterodimerization domain linked to at least one relaxin B-chain polypeptide or a variant thereof, wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and the 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) inter-chain bonds, preferably one or more (e.g., two) inter-chain 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 preferred embodiments, the first and second heterodimerization domains are derived from the Fc region of an immunoglobulin, such as the Fc region of immunoglobulin G (IgG) (the "first Fc region" and the "second Fc region"). The first and second Fc regions may include the constant domains CH2 and / or CH3. Preferably, the first and second Fc regions include CH2 and CH3.

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

[0016] ​In yet another embodiment, the first and second heterodimerized domains heterodimerize to form parallel coiled coils.

[0017] In some embodiments, the relaxin A chain is ligated to a first heterodimerization domain (e.g., a first Fc region) via a connector, and the relaxin B chain is ligated 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 ligated 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 ligated 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 ligated 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 ligated 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) include 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 located in the CH3 domain. In preferred embodiments, the first Fc region includes an "Fc knob" mutation and the second Fc region includes 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 their conserved substitutions; and in the other CH3 domain, "Fc knob" mutations S354C and T366W, or their conserved substitutions, where amino acid numbering follows the Kabat EU index.

[0021] In any embodiment of the present invention, the relaxin 2A chain polypeptide comprises the sequence described in SEQ ID NO: 1 or a variant thereof, and the relaxin 2B chain polypeptide comprises the sequence described in SEQ ID NO: 2 or a variant thereof. In some embodiments, the relaxin 2A chain polypeptide comprises the amino acid mutation K9H.

[0022] Furthermore, the present invention provides the following: (i) FcX-con-A fusion polypeptide and; (ii) FcY-con-B fusion polypeptide and A heterodimer fusion containing the following is provided: Here, A is the relaxin A chain or its variant, for example, the relaxin 2A chain or its variant; B is the relaxin B chain or its variant, for example, the relaxin 2B chain or its variant; FcY is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and / or modification, preferably comprising a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V or a conservative substitution thereof; FcX comprises an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and / or modification, preferably a CH3 domain having the amino acid mutation S354C:T366W or a conservative substitution thereof; con is a connector, for example, preferably a connector polypeptide having the sequence GGGGSGGGGSGGGGSGGGGGS[SEQ ID NO: 5], Here, the amino acid numbering is based on the Kabat EU index, and FcX heterodimerizes with FcY, and the heterodimer fusion product has relaxin activity.

[0023] In a particularly preferred embodiment, the heterodimer fusion product 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 present invention, the heterodimer fusion further comprises one or more Fabs, optionally comprising one Fab ligated to the N-terminus of a first heterodimerization domain (e.g., a first Fc region) and a second Fab ligated to the N-terminus of a second heterodimerization domain (e.g., a second Fc region).

[0025] In some embodiments of any aspect of the present invention, the heterodimer fusion further comprises a second relaxin A chain polypeptide or a variant thereof tethered to the N-terminus of a first heterodimerization domain (e.g., a first Fc region), and a second relaxin B chain polypeptide or a variant thereof tethered to the N-terminus of the second heterodimerization domain (e.g., a second Fc region), wherein optionally, the second relaxin A chain is tethered to the first heterodimerization domain (e.g., a first Fc region) via a connector polypeptide, and the second relaxin B chain is tethered to the second heterodimerization domain (e.g., a second Fc region) via a connector polypeptide.

[0026] In another embodiment, the present invention is as follows: (i) FcX-BLA and FcY, optionally FcY-BLA; or (ii) FcY-BLA and FcX, and optionally FcX-BLA; The present invention provides a heterodimer fusion product containing, Here, FcY is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and / or modification, preferably comprising a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V, or a conservative substitution thereof; FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and / or modification, preferably comprising a CH3 domain having the amino acid mutation S354C:T366W, or a conservative substitution thereof; B is the relaxin B chain or its variant, for example, the relaxin 2B chain or its variant; A is the relaxin A chain or its variant, for example, the relaxin 2A chain or its variant; L is preferably a linker polypeptide having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60]. Here, the amino acid numbering is according to the Kabat EU index, and FcX heterodimerizes with FcY, and the heterodimer fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerized domains as described herein. In some embodiments, the relaxin B chain is linked to FcX and / or FcY via a connector polypeptide, optionally having a length of 6 to 40 amino acids, for example, 21 amino acids.

[0027] In yet another embodiment, the present invention is as follows: (i) FcX-ALB and FcY, optionally FcY-ALB; or (ii) FcY-ALB and FcX, and optionally FcX-ALB; The present invention provides a heterodimer fusion product containing, Here, FcY is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and / or modification, preferably comprising a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V, or a conservative substitution thereof; FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and / or modification, preferably comprising a CH3 domain having the amino acid mutation S354C:T366W, or a conservative substitution thereof; A is the relaxin A chain or its variant, for example, the relaxin 2A chain or its variant; B is the relaxin B chain or its variant, for example, the relaxin 2B chain or its variant; L is preferably a linker polypeptide having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60], Here, amino acid numbering is based on the EU index similar to that of Kabat, and FcX heterodimerizes with FcY, and the heterodimer fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerized domains as described herein. In some embodiments, the relaxin A chain is linked to FcX and / or FcY via a connector polypeptide, optionally having a length of 6 to 40 amino acids, for example, 21 amino acids.

[0028] In some embodiments of any aspect of the present invention, the ratio of the relaxin activity of the heterodimer fusion to the relaxin activity of the reference relaxin protein is about 0.001 to about 10.

[0029] In related embodiments, the present invention provides a nucleic acid molecule (e.g., a DNA molecule) encoding the heterodimer fusion of the present invention, a vector containing the nucleic acid molecule, a host cell containing the vector or nucleic acid, and a method for producing the heterodimer fusion of the present invention by culturing the host cell and collecting the fusion protein.

[0030] In another embodiment, the present invention provides a pharmaceutical composition comprising the heterodimer fusion of the present invention, a kit comprising the same, and the use of the heterodimer fusion in therapies such as a method for treating a subject having heart failure.

[0031] Aspects and embodiments of the present invention are described in the appended claims. These and other aspects and embodiments of the present invention are also described herein.

[0032] Brief description of the drawings and arrangement list [Brief explanation of the drawing]

[0033] [Figure 1]Figure 1 shows exemplary formats of heterodimer fusions according to several embodiments of the present invention. The format of each fusion polypeptide of the heterodimer fusion is given with respect to 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 the heavy chains of the antibody, LC is the light chain of the antibody, Hinge is the hinge region of the antibody, and Fab is the Fab fragment of the antibody. [Figure 2] Figure 2 shows the LC-MS analysis of RELAX0019 and RELAX0023. A) The deglycosylation and non-reductive analysis of RELAX0019 and RELAX0023 shows the mass of the intact molecule, and B) The deglycosylation and reductive analysis of RELAX0019 and RELAX0023 shows the mass 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-reducing peptide mapping using LC-MS. The amino acid sequence of the C-terminal peptide with a predicted disulfide bond, represented by a line, is shown in the upper panel. Panels A and E - Extracted ion chromatogram of the C-terminal peptide in the absence of a reducing agent (-DTT). Panels C and G - Deconvolution mass spectrum of the C-terminal peptide in the absence of a reducing agent. Panels B and F - Extracted ion chromatogram in the presence of a reducing agent (+DTT), and Panels D and H - Deconvolution mass spectrum in the presence of a reducing agent. Figure 3 discloses sequence numbers 75, 77, and 76 in order of appearance. [Figure 4] Figure 4 shows the in vitro biological activity of several heterodimer fusions of the present invention, measured by cAMP induction in cells expressing recombinant human RXFP1. [Figure 5]Figure 5 shows the in vivo pharmacokinetic (PK) profiles from a series of ELISA experiments in which the heterodimer fusion of the present invention was administered intravenously to mice. The data are normalized as %cMax at 5 minutes (T1). [Figure 6] Figure 6 shows the recovery of isoproterenol-induced myocardial fibrosis and hypertrophy in mice treated with RELAX0019 and RELAX0023. The levels of fibrosis and hypertrophy are shown for (1) vehicle (baseline), (2) isoproterenol, (3) isoproterenol + relaxin 2, (4) isoproterenol + RELAX0019, and (5) isoproterenol + RELAX0023. [Figure 7] Figure 7 shows the in vitro nonspecific binding of the heterodimer fusion of the present invention in a baculovirus (BV) ELISA assay. [Figure 8] Figure 8 shows the percentage of purity loss, aggregation, and fragmentation of RELAX0023, RELAX0127, and RELAX0128 in solution during storage. [Figure 9] Figure 9 shows the time-dependent stability of RELAX0023, RELAX0127, and RELAX0128 in solution as evaluated by reducing LC-MS analysis. A) Total ion chromatogram, B) Mass spectrum of the reducing molecule [Figure 10] Figure 10 shows the PK profiles of RELAX0023 in cynomolgus monkeys after intravenous and subcutaneous injection. [Figure 11] Figure 11 shows the base sequences encoding a portion of the polypeptide of the present invention (SEQ ID NOs. 80-140, 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] [Modes for carrying out the invention]

[0049] Relaxin The present invention is at least in part based on the finding that the heterodimer fusion described herein may exhibit relaxin activity when the relaxin A and relaxin B chains are not covalently linked to each other via an amino acid linker. This is surprising, according to the disclosures in International Publications 2013 / 004607 and 2018 / 138170, which describe recombinant relaxins in which relaxin A and relaxin B are fused into a single chain. The inventors have further found that heterodimerization of the heterodimerization domain also induces the correct folding and heterodimerization of the relaxin A and relaxin B chains (see Example 2). In addition, unlike wild-type relaxin protein, the fusion polypeptide of the present invention does not require internal proteolytic processing for biological activity.

[0050] As used herein, the term “heterodimer fusion” means a heterodimer of a fusion polypeptide, wherein one fusion polypeptide comprises a first heterodimerization domain linked to a first subunit of the heterodimer protein (e.g., relaxin A chain), and the other fusion polypeptide comprises a second heterodimerization domain linked to a second subunit of the heterodimer protein (e.g., relaxin B chain).

[0051] The heterodimer fusion of the present 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 present invention is relaxin 2 A-chain polypeptide or a variant thereof; the relaxin B-chain polypeptide of the present invention is relaxin 2 B-chain polypeptide or a variant thereof. In specific embodiments, the relaxin A-chain polypeptide comprises human relaxin 2 A-chain polypeptide or a variant thereof and 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 together via peptide bonds.

[0053] In some embodiments, the relaxin 2A chain polypeptide has the sequence described in SEQ ID NO: 1 or a variant thereof, and the relaxin 2B chain polypeptide has the sequence described in SEQ ID NO: 2 or a variant thereof. The variants may include one or more amino acid substitutions, deletions, and / or insertions. In some embodiments, the relaxin 2A chain polypeptide includes one or more amino acid mutations selected from K9E, K9H, K9L, K9M, R18E, R18H, R22A, R22I, R22M, R22Q, R22S, R22Y, F23E, F23A, and F23I. In preferred embodiments, the relaxin 2A chain includes the amino acid mutation K9H.

[0054] Mutants of relaxin A and B chains are known in the art. Furthermore, guidance for designing relaxin A and B chain mutants 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, relaxin 2 B chain mutants 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). Mutants may contain one or more amino acid substitutions and / or insertions. For example, relaxin-2 B chain mutants may have one or more additional amino acids compared to SEQ ID NO: 62, such as K30 and R31, N-terminal V-2, A-1 and M-1. Alternatively, or in addition, the variant may contain one or more amino acid derivatives. For example, the first amino acid of the relaxin-2 B chain variant may be pyroglutamic acid.

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

[0056] The relaxin family of peptides transmits their biological effects, at least partially, through the activation of G protein-coupled receptors (GPCRs) and subsequent stimulation or inhibition of the cAMP signaling pathway by Gs or Gi protein subunits, respectively. Relaxin-2 is known to activate GPCR RXFP1 (also known as LGR7) and, to a lesser extent, GPCR RXFP2 (also known as LGR8), thereby 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 the relaxin receptor and / or initiate a signaling cascade within a cell. In embodiments where relaxin activity is relaxin 2 activity, relaxin activity may also 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 the binding of relaxin molecules to relaxin receptors and / or by measuring downstream events of binding to relaxin receptors.

[0059] Relaxin activity may be determined in vitro and / or in vivo. In 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 the 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 the relaxin-mediated activation of the receptor. For example, relaxin activity may be determined by measuring the activation of p42 / 44 MAPK.

[0061] Alternatively, or even better, relaxin activity may be determined by measuring the activation of known relaxin target genes. For example, relaxin activity may be determined by measuring the activation of transcription of a known relaxin target gene, VEGF, in THP-1 cells. Methods for determining the activation of gene transcription 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 even moreover, relaxin activity may be determined by measuring one or more downstream effects of relaxin. For example, a 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 embodiment, relaxin activity may be determined by measuring a reduction in fibrosis by Masson's tricolor staining method. In yet another embodiment, relaxin activity may be determined by measuring changes in connective tissue metabolism such as inhibition of profibrotic factors (e.g., 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 recovery of isoproterenol-induced cardiac hypertrophy (measured as cardiac weight relative to tibia length) and fibrosis (measured as collagen content relative to cardiac weight), as performed, for example, in Example 7.

[0064] The activity of the heterodimer fusion of the present invention can be determined by comparison with a reference relaxin protein. In some embodiments, the reference relaxin protein is a recombinant protein. In preferred embodiments, the reference relaxin protein is a relaxin protein having an array of relaxin A and B chains from a mature relaxin protein. Recombinant relaxins having an array of relaxin A and B chains from 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 and relaxin B chains as the heterodimer fusion of the present invention, or differs from the relaxin A and relaxin B chains of the heterodimer fusion of the present invention by 10 or fewer amino acids, for example, one or two amino acids. In one embodiment, the first amino acid of the B chain of the reference relaxin-2 is D, which is deleted in the relaxin B chain of the heterodimer fusion of the present invention.

[0066] The reference relaxin protein is as follows: (i) Recombinant human relaxin 2 (referred to herein as RELAX0013); (ii) Recombinant mouse relaxin 1 (referred to herein as RELAX0014); (iii) Recombinant Fc-fused relaxin 2 (referred to herein as RELAX0010, described in International Publication No. 2018 / 138170), wherein relaxin A and relaxin B are fused in a single chain and Fc is a half-life extension Fc region); (iv) Recombinant Fc-fused relaxin 2 (referred to herein as RELAX0009, described in International Publication No. 2018 / 138170), wherein relaxin A and relaxin B are fused in a single chain and Fc is a half-life extension Fc region); (v) Recombinant Fc-fused relaxin 2, in which relaxin A and relaxin B are fused in a single chain (referred to herein as RELAX0126, and described in International Publication No. 2013 / 004607); (vi) Recombinant Fc-fused relaxin 2 (referred herein to as RELAX0127, described in International Publication No. 2013 / 004607), in which relaxin A and relaxin B are fused in a single chain; and (vii) Recombinant Fc-fusion relaxin in which relaxin A and relaxin B are fused in a single chain (referred to herein as RELAX0128, and described in International Publication No. 2013 / 004607) You can choose from these options.

[0067] In a particularly preferred embodiment, the reference relaxin protein is a relaxin 2 protein having an array of relaxin 2 A and relaxin 2 B chains of a mature relaxin 2 protein, as disclosed in UniProtKB / Swiss-Prot accession number P04090.1.

[0068] The heterodimer fusions of the present invention may be considered to have relaxin activity if they exhibit at least a portion of the activity of a reference relaxin protein. For example, a fusion polypeptide may be considered to have relaxin activity if it exhibits at least about half of the activity of a reference relaxin protein. The heterodimer fusions of the present invention have a ratio of the activity of the fusion polypeptide to the activity of a reference relaxin protein of about 10 -5 And for about 1, about 10 -4 And for about 1, about 10 -3 And for about 1, about 10 -2 The heterodimer fusion of the present 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 approximately 1 and approximately 1, between approximately 1 / 50 and approximately 1, between approximately 1 / 20 and approximately 1, between approximately 1 / 15 and approximately 1, between approximately 1 / 10 and approximately 1, between approximately 1 / 5 and approximately 1, and between approximately 1 / 2 and approximately 1. Alternatively, the heterodimer fusion of the present 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 approximately 1 and approximately 10.5 , approximately 1 to approximately 10 4 , approximately 1 to approximately 10 3 If the values ​​are approximately 1 to 100, 1 to 50, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 2, it may be considered to have relaxin activity.

[0069] In some embodiments, the relaxin activity of the heterodimer 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” (semi-effective concentration) refers to the effective concentration of a therapeutic compound that elicits an intermediate response between baseline and a maximum value after a particular exposure time.

[0071] Heterodimizing domain The heterodimer fusion of the present invention comprises a first heterodimer domain and a second heterodimer domain. In preferred embodiments, the first and second heterodimer domains are derived from immunoglobulin Fc regions.

[0072] The term "Fc region" defines the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally contains two constant domains, the CH2 and CH3 domains, and sometimes the CH4 domain.

[0073] The first and second Fc regions may include immunoglobulin domains CH2 and / or CH3. In preferred embodiments, the first and second Fc regions include 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 where the Fc region is derived from IgG, the Fc region may be derived from any subclass of IgG (e.g., IgG1, IgG2, IgG3, IgG4), preferably IgG1. Preferably, the first and second Fc regions are derived from human IgG1 immunoglobulin. In other embodiments, the first and second Fc regions are derived from human IgG4 immunoglobulin.

[0075] In preferred embodiments, the first and second Fc regions include heterodimerization-promoting amino acid mutations and / or modifications. Such modifications may include the introduction of asymmetric complementary modifications to each of the first and second Fc regions, resulting in both chains becoming compatible with each other and thus capable of forming a heterodimer, but each chain being unable to dimerize with itself. Such modifications may include insertions, deletions, conservative and non-conservative substitutions, and rearrangements. Incorporation of such modifications provides a method for increasing the yield of heterodimers produced by recombinant cell culture compared to other unwanted end products such as homodimers.

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

[0077] In the "knob-in-hole" method, heterodimerization can be promoted by introducing steric hindrance between contacting residues. A "protrusion" is generated by substituting 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 larger side chain is formed at the interface of another Fc region ("Fc hole") by substituting the amino acid with the larger side chain with an amino acid with a smaller side chain (e.g., alanine or valine). The "knob-in-hole" modification is described in detail, for example, 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 may be used to generate heterodimers include, but are not limited to, modifications that generate favorable electrostatic interactions between two Fc regions. For example, one or more positively charged amino acids may be introduced into one Fc region, and one or more uncharged amino acids may be introduced into the corresponding positions of the other Fc region. Alternatively or in addition, the Fc region may be modified to include mutations that introduce cysteine ​​residues capable of forming disulfide bonds. Alternatively or in addition, the Fc region may include one or more modifications to hydrophilic and hydrophobic residues at the interchain interface to make heterodimer formation more entropically and enthalpy favorable than homodimer formation.

[0079] Accordingly, in some embodiments, heterodimerization-promoting amino acid mutations and / or modifications introduce a cysteine ​​residue that can create steric hindrance between contact residues (e.g., by a "knob-in-hole"), generate a favorable electrostatic interaction between two Fc regions to form a disulfide bond, 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 located in the CH3 domain.

[0081] In some embodiments, the first and second Fc regions include "FcX" and "FcY" derived from human IgG1 immunoglobulin and having mutations in the CH3 domain, where the "FcX" and "FcY" mutations are selected from the combinations (or their conservative substitutions) listed in Table 2.

[0082] [Table 16]

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

[0084] The term "Kabat EU Index" refers to the numbering scheme for human IgG1 EU antibodies described in Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service. National Institutes of Health. Bethesda, MD. All amino acid positions 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 include other amino acid modifications compared to the wild-type Fc region. The Fc region can be modified, for example, to increase the affinity of the IgG molecule to FcRn. International Publication No. 02 / 060919 discloses modified immunoglobulins containing Fc regions having one or more amino acid modifications, which are incorporated herein by reference in their entirety. Methods for producing Fc regions having one or more amino acid modifications are known in the art.

[0087] In some embodiments, the first and / or second Fc region may include one or more amino acid modifications to reduce or eliminate the effector function of the Fc region. In some embodiments, the amino acid modifications reduce or avoid cytotoxicity, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0088] In some embodiments, the first and / or second Fc region may include one or more amino acid modifications to increase the half-life of the heterodimer fusion.

[0089] In some embodiments, the first and / or second Fc region is the following combination of amino acid mutations: (i) M252Y, S254T, and T256E, or their conservative substitutes; (ii) L234F, L235Q, and K322Q, or their conservative substitutions; (iii) L234F, L235E, and P331S, or their conservative substitutes; (iv) M252Y, S254T, T256E, L234F, L235Q and K322Q, or their conservative substitutions; or (v) M252Y, S254T, T256E, L234F, L235E and P331S, or their conservative substitutions It contains at least one of the following, where the amino acid numbering is based on the Kabat EU Index.

[0090] In some embodiments, the first and / or second Fc region may include amino acid mutations L234F, L235E, and P331S, or their conserved substitutions, where amino acid numbering is based on the Kabat EU index.

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

[0092] In some embodiments, the Fc region includes the SEQ ID NO: 3 mutant having the amino acid mutation Y349C, which is reversed to Y349, and the SEQ ID NO: 4 mutant having the amino acid mutation S354C, which is reversed to S354, and as a result, the Fc region is unable to form a stabilizing disulfide bond.

[0093] In some embodiments, the Fc region includes the SEQ ID NO: 3 variant and / or the SEQ ID NO: 4 variant, where the first five residues DKTHTCPPC (SEQ ID NO: 69) are modified. In some embodiments, this region is replaced with the sequence DKTHTACPPC (SEQ ID NO: 70). In another embodiment, this region is replaced with the sequence GGAGGACPPC (SEQ ID NO: 71). In yet another embodiment, this region is replaced with the sequence ACPPC (SEQ ID NO: 72).

[0094] In another embodiment, the first and second heterodimerization domains are derived from the immunoglobulin Fab region. In some embodiments, the heterodimerization domain includes the CH1 and CL regions. The Fab region, including the L and Fd chains, has been found to mediate efficient heterodimerization (Schoonjans R et al. (2000) J.Immunol. 165(12):7050-7057). Therefore, in another embodiment, the heterodimerization domain includes the L and Fd chains. In some embodiments, the L and Fd chains heterodimerize to form a disulfide-bridged stabilized heterodimer.

[0095] In yet another embodiment, the first and second heterodimerizing domains heterodimerize to form parallel coiled coils. Heterodimerated coiled coils are described, for example, in Aronsson et al. (2015) Sci. Rep. 5:14063. In some embodiments, the heterodimerizing domains include amino acid mutations and / or modifications to prevent the formation of undesirable 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 half-life extension regions. Therefore, in some embodiments, the heterodimer fusions of the present invention have a longer half-life compared to standard relaxin.

[0097] As used herein, the term “half-life” is used to refer to the time it takes for the concentration of a fusion protein in plasma to 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, its turnover rate, in vivo proteolysis, and the rate of absorption 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 inventors have demonstrated that the heterodimer fusion of the present invention, having first and second heterodimerizing domains derived from immunoglobulin Fc, has a half-life of at least 5 hours in a mouse model (see Example 6). In comparison, the half-life of human relaxin 2 after IV administration is approximately 0.09 ± 0.04 hours, or 5.4 ± 2.4 minutes in humans (Chen SA et al. (1993) Pharm. Res. 10(6):834-838).

[0099] A long half-life is considered advantageous because it allows therapeutic proteins to be administered according to a safe and convenient dosing schedule, for example, by enabling lower doses at lower frequencies. Furthermore, achieving lower doses may result in additional benefits such as providing an improved safety profile and / or activation of multiple mechanisms of action in vivo.

[0100] connector Either or both of the relaxin A and B chains may be ligated to their respective heterodimerization domains by connector polypeptides. In some embodiments, the relaxin A chain is ligated to a first heterodimerization domain (e.g., a first Fc region) via a connector polypeptide, and the relaxin B chain is ligated to a second heterodimerization domain (e.g., a second Fc region) via a connector polypeptide.

[0101] The connector polypeptide may have any suitable length, for example, a length of about 6 to 40 amino acids, preferably about 6 to 21 amino acids. In some embodiments, the connector polypeptide has a length of at least 6 amino acid residues, preferably at least 11 amino acids, preferably at least 16 amino acids. In some embodiments, the connector polypeptide has a length of less than 40 amino acids. Connector polypeptides of different or the same length can be used in each arm of the heterodimer fusion of the present invention. In some embodiments, at least one connector polypeptide has a length of 21 amino acids. In preferred embodiments, both connector polypeptides have a length of 21 amino acids. The connector polypeptide may have any amino acid sequence. Connector polypeptides of different or the same amino acid composition can be used in each arm of the heterodimer fusion of the present invention.

[0102] In some embodiments, one or preferably both connector polypeptides comprise a proline and alanine repeat (PA)x (SEQ ID NO: 73), preferably x is 3 to 15, preferably the connector polypeptide has a length of more than 16 amino acids, and preferably the connector polypeptide consists of a 21-amino acid sequence PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO: 6).

[0103] In some embodiments, one or preferably both connector polypeptides include 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 include the motif (GGGGS)n (SEQ ID NO: 74), where n may be 1 to 8, for example, n is 4. In some embodiments, one or more connector polypeptides consist of a 21-amino acid sequence GGGGSGGGGSGGGGGSGGGGGS (SEQ ID NO: 5). In certain embodiments, both connector polypeptides consist of a 21-amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5).

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

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

[0106] stability The inventors have revealed that the heterodimer fusions of the present invention possess unexpectedly excellent physical and chemical stability. Therefore, in some embodiments, the heterodimer fusions of the present invention exhibit superior physical and / or chemical stability compared to reference relaxin proteins.

[0107] The physical stability of relaxin can be determined by measuring its purity and aggregation, for example, by HP-SEC, as in Example 9. The chemical stability of relaxin can be determined by measuring molecular fragmentation and modification, for example, by LC-MS, as in Example 9.

[0108] Surprisingly, the inventors have demonstrated that the heterodimer fusions of the present invention exhibit superior physical and chemical stability compared to recombinant Fc-fusion relaxins in which relaxin A and relaxin B are fused to a single chain (rather than relaxin A and B in individual fusion polypeptides). International Publication No. 2013 / 004607 describes recombinant single-chain relaxin fusion polypeptides fused to an immunoglobulin Fc region, for example, fusion polypeptides referred to herein as RELAX0127 and RELAX0128. Accordingly, in some embodiments, the heterodimer fusions of the present invention exhibit superior physical and / or chemical stability compared to RELAX0127 and RELAX0128.

[0109] The heterodimer fusion may include a half-life extension portion in addition to the first and second heterodimerization domains. In some embodiments, the half-life extension portion is a proteinaceous half-life extension portion. The proteinaceous half-life extension portion can be selected from the group consisting of the Fc region of immunoglobulins, albumin-binding domains, and serum albumin. In further embodiments, the half-life extension portion is a chemical substance that is not a protein or peptide, such as a polyethylene glycol (PEG) polymer chain.

[0110] The half-life extension portion can be attached to the N-terminus or C-terminus of the first or second heterodimerization domain. In some embodiments, the half-life extension portion is attached to the N-terminus of the first or second heterodimerization domain. In other embodiments, the half-life extension portion is attached to the C-terminus of the first or second heterodimerization domain. Methods for attaching the half-life extension portion to the heterodimer fusion are known in the art. For example, the half-life extension portion can be attached by chemical bonding or recombination techniques. The half-life extension portion can be attached directly to the heterodimer fusion or via a connector (e.g., a connector polypeptide). The use of a connector polypeptide may be particularly appropriate when the fusion polypeptide contains a proteinaceous half-life extension portion, such as an Fc region.

[0111] Exemplary Embodiments The heterodimer fusions of the present invention may have various formats and / or sequences.

[0112] The terms “fusion polypeptide of the present invention” and “fusion polypeptide of the present invention” may be used to refer to a first heterodimerized domain fused to the relaxin A chain and / or a second heterodimerized domain fused to the relaxin B chain. The fusion polypeptide of the present invention may be a recombinant fusion polypeptide, i.e., one produced by recombinant DNA technology.

[0113] In preferred embodiments, the C-terminus of the first heterodimerization domain (e.g., the first Fc region) is ligated 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 ligated 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 the first heterodimerization domain (e.g., the first Fc region) is ligated 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 ligated to the C-terminus of the relaxin B chain. In some embodiments, the relaxin A chain polypeptide and / or the relaxin B chain polypeptide have a free N-terminus.

[0115] The heterodimer fusion of the present invention may further include one or more Fabs. In some embodiments, the heterodimer fusion includes one Fab ligated to the N-terminus of a first heterodimerization domain (e.g., a first Fc region) and a second Fab ligated to the N-terminus of a second heterodimerization domain (e.g., a second Fc region).

[0116] The heterodimer fusion of the present invention may further comprise a second relaxin A chain polypeptide or a variant thereof, and a second relaxin B chain polypeptide or a variant thereof. In some embodiments, the second relaxin A chain polypeptide or a variant thereof is ligated to the N-terminus of the first heterodimerization domain (e.g., the first Fc region), and the second relaxin B chain polypeptide or a variant thereof is ligated to the N-terminus of the second heterodimerization domain (e.g., the second Fc region), and optionally, in this case, the second relaxin A chain is ligated 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 ligated to the second heterodimerization domain (e.g., the second Fc region) via a connector (e.g., a connector polypeptide).

[0117] Therefore, in some embodiments, the format of the heterodimer fusion is as follows: (i) FcX-con-A / FcY-con-B (see Figure 1, for example); (ii) FcX-con-B / FcY-con-A (see Figure 1, for example); (iii) A-con-FcX / B-con-FcY (see Figure 1, for example); (iv) B-con-FcX / A-con-FcY (see Figure 1, for example); (v)Fab-FcX-con-A / Fab-FcY-con-B (see Figure 1, for example); (vi)Fab-FcX-con-B / Fab-FcY-con-A; (vii) A-con-FcX-con-A / B-con-FcY-con-B (see Figure 1, for example); (viii)B-con-FcX-con-B / A-con-FcY-con-A; (ix) FcX-con-BLA and FcY, and optionally FcY-con-BLA (see Figure 1, for example); (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 Selected from, Here, FcY is an immunoglobulin Fc region having an "Fc hole" amino acid mutation and / or modification, preferably comprising a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V, or a conservative substitution thereof; FcX is an Fc region having an "Fc knob" amino acid mutation and / or modification, preferably comprising a CH3 domain having the amino acid mutation S354C:T366W, or a conservative substitution thereof; "con" is a connector polypeptide; B is the relaxin B chain or a variant thereof; A is the 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 embodiment, the present invention is as follows: (i) XBLA and Y, and YBLA if selected; or (ii) YBLA and X, and optionally XBLA The present invention provides a heterodimer fusion product containing, Here, X and Y are heterodimerization domains as described herein; B is the relaxin B chain or its variant, for example, the relaxin-2B chain or its variant; A is the relaxin A chain or its variant, for example, the relaxin-2A chain or its variant; L is preferably a linker polypeptide having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60), Here, X heterodimerizes with Y, and the heterodimer fusion has relaxin activity.

[0119] In yet another aspect, the present invention is as follows: (i) XALB and Y, optionally YALB or (ii) YALB and X, and optionally XALB Provides a heterodimer fusion containing; Here, X and Y are heterodimerization domains as described herein; A is the relaxin A chain or its variant, for example, the relaxin 2A chain or its variant; B is the relaxin B chain or its variant, for example, the relaxin 2B chain or its variant; L is preferably a linker polypeptide having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60), Here, X heterodimerizes with Y, and the heterodimer fusion has relaxin activity.

[0120] In a particularly preferred embodiment, the heterodimer fusion comprises the fusion polypeptide Rlx011DD described in SEQ ID NO: 11 and Rlx014DD described in SEQ ID NO: 20. In another preferred embodiment, the heterodimer fusion comprises the fusion polypeptide Rlx013DD described in SEQ ID NO: 17 and Rlx012DD described in SEQ ID NO: 14.

[0121] In aspects of the present invention, a heterodimer fusion product is provided that includes a combination of fusion polypeptides selected from the combinations of FcX and FcY listed in Table 3.

[0122] [Table 17]

[0123] [Table 18]

[0124] In one embodiment, a heterodimer fusion product comprising the fusion polypeptide described in Sequence ID No. 11 and Sequence ID No. 20 is provided.

[0125] In another embodiment, a heterodimer fusion product comprising the fusion polypeptide described in Sequence ID No. 17 and Sequence ID No. 14 is provided.

[0126] The fusion polypeptide of the present invention may be produced by any method known in the art. In some embodiments, the fusion polypeptide of the present invention is produced by recombinant expression of a nucleic acid molecule encoding the fusion polypeptide in a host cell.

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

[0128] The vector containing the nucleic acid molecule of the present invention may be transferred to a host cell by conventional techniques. Suitable host cells are known in the art. In some embodiments, the host cell is a mammalian cell such as HEK293 cells or CHO cells.

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

[0130] Once the fusion polypeptide of the present invention is produced, for example, by recombinant expression, it may be purified by any method known in the art. Exemplary protein purification techniques include chromatography (e.g., ion exchange, affinity, and / or sizing column chromatography), centrifugation, and differential solubility. The present invention provides isolated fusion polypeptides separated from cell cultures by at least one optional purification step.

[0131] treatment The fusion polypeptide of the present invention may be provided in a pharmaceutical composition.

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

[0133] The present invention encompasses therapies comprising the step of administering the fusion polypeptide of the present invention to an animal, particularly a mammal, such as a human, for the purpose of preventing, treating, or relieving symptoms associated with a disease, disorder, or infection.

[0134] Therefore, the fusion polypeptide or pharmaceutical composition of the present invention may be used in therapy, for example, to treat a disease or disorder. A method for treating a disease or disorder is also provided, comprising the step of administering a therapeutically effective amount of the fusion polypeptide of the present invention to a subject or patient in need thereof. The use or method may include the step of implementing a therapeutically effective schedule in which the fusion polypeptide of the present invention is administered at a lower frequency than a therapeutically effective dosing schedule for wild-type relaxin molecule.

[0135] It will be understood that the fusion polypeptide 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] The fusion polypeptide of the present invention may also be used in the treatment of renal disease, pulmonary disease, and fibrous disorders, such as fibrous disorders of the kidneys, heart, lungs, and liver, as well as in wound healing (Sherwood OD (2004) Endocrine Reviews 25(2):205-234). The fusion polypeptide of the present invention may also be used in the restoration of insulin resistance in diabetic patients (Bonner JS et al. (2013) Diabetes 62(9):3251-3260). The fusion polypeptide of the present invention may also be used in various forms of pulmonary hypertension. The fusion polypeptide of the present invention may also be used in diseases resulting from or causing arteriosclerosis, decreased arterial elasticity, decreased arterial compliance and extensibility, such as hypertension, renal disease, peripheral artery disease, carotid and cerebrovascular disease (i.e., stroke and dementia), diabetes, microvascular disease resulting in peripheral organ damage, coronary artery disease, and heart failure.

[0138] The fusion polypeptide and / or pharmaceutical composition of the present invention is suitable for parenteral administration to subjects or patients. In some embodiments, the subjects or patients are mammals, particularly humans.

[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 polypeptide and / or pharmaceutical composition of the present invention may be administered to subjects or patients by injection, such as by intravenous, subcutaneous, or intramuscular injection. In some embodiments, the fusion polypeptide and / or pharmaceutical composition is administered by subcutaneous injection. Administration by injection, such as by subcutaneous injection, has the advantage of being more comfortable for the subject or patient and provides the freedom to administer to subjects or patients outside of a hospital. In some embodiments, the fusion polypeptide or pharmaceutical composition is administered by self-administration.

[0140] In some embodiments, the fusion polypeptide of the present invention has an increased half-life compared to wild-type relaxin, which allows for lower overall exposure based on molar concentration. For example, the fusion polypeptide of the present invention may be administered at a lower frequency than wild-type relaxin, thus providing a more convenient dosing schedule.

[0141] The present invention provides a kit comprising the pharmaceutical composition of the present invention. The kit may include a package containing the pharmaceutical composition of the present invention and instructions. In some embodiments, the pharmaceutical composition of the present invention is formulated in a single-dose vial or container dispensing system (e.g., a pre-filled syringe). Optionally, such containers may be accompanied by a notice in the form directed by a government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, which represents agency approval for manufacture, use, or sale for human administration.

[0142] As used herein, the articles “a” and “an” may refer to one or more than one (for example, at least one) grammatical object of the article.

[0143] "Approximately" may generally mean the degree of error that is permissible in relation to the quantity being measured, given the nature or precision of the measurement method. Exemplary degrees of error include being within a percentage (%) of a given value or range of values, typically within 10%, and more typically within 5%.

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

[0145] As used herein, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory body for use in animals, and more specifically in humans, or that it is listed in the United States Pharmacopoeia, the European Pharmacopoeia, or any other generally recognized pharmacopoeia.

[0146] Concentrations, quantities, volumes, percentages, and other numerical values ​​may be expressed herein in range form. Such range forms are used solely for convenience and brevity, and should also be understood to be flexibly interpreted to include not only the numerical values ​​explicitly detailed as limits of the range, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly detailed.

[0147] The embodiments described above should be understood as illustrative examples. Further embodiments are conceivable. Any feature described in any one embodiment may be used alone or in combination with other features described, or 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] In connection with this disclosure, other examples and variations of the fusion polypeptides and methods described herein will be apparent to those skilled in the art. Other examples and variations are within the scope of this disclosure, as set forth in the appended claims.

[0149] All references cited herein, including all data, tables, figures, and original texts presented therein, are incorporated herein in their entirety by reference. [Examples]

[0150] Example 1: Preparation of recombinant heterodimer Fc relaxin 2 fusion protein The Fc relaxin 2 fusion proteins described herein are designed using the heterodimerizing properties of the knob-in-hole Fc domain (Fc knob and Fc hole) to induce the correct folding and heterodimerization of relaxin 2 chains A and B.

[0151] More precisely, as shown in Figure 1, relaxin 2 chains A and B are genetically fused to two complementary Fc (the N-terminus and / or C-terminus of the Fc) via connectors. Next, CHO cells were co-transfected with two expression vectors, each containing a single Fc-relaxin chain (A and / or B). The two complementary Fc portions assemble within the CHO cells, facilitating the assembly and correct folding of relaxin 2. Subsequently, as shown in Example 2 below, disulfide bonds are formed between the complementary Fc chains and between chain A and chain B, reconstructing the native relaxin 2 structure.

[0152] Once the heterodimer Fc relaxin 2 fusion protein is secreted into the supernatant, it is purified using an automated affinity chromatography system, where the Fc region of the protein binds to the column matrix.

[0153] Example 2: LC-MS analysis of Fc relaxin 2 knob-in-hole heterodimer LC-MS analysis was performed for both non-reducible and reductively deglycosylated Fc-relaxin 2 heterodimers. For deglycosylation, the sample was diluted to 1 mg / ml and buffered with 10 mM Tris-Cl at pH 7.80. PNGase F (Roche) was added to the sample at a concentration of 1 unit of enzyme per 50 μg of Fc-relaxin 2, and incubated overnight at 37°C. For non-reducible analysis, the sample was diluted to 0.05 mg / ml with water, and 20 μL was packed into an LC-MS certified total recovery vial with a pre-slit cap (Waters part number: 186005663CV). For reductive analysis, 10 mM TCEP was added, and the sample was further incubated at 37°C for 30 minutes before analysis.

[0154] The experiments were conducted using an ACQUITY I-Class UPLC connected to a Xevo G2-XS Q-TOF instrument (Waters, Milford, MA), and all operations were performed using the UNIFI Scientific Information System. In the case of the LC system, solvent A was water containing 0.1% formic acid, and solvent B was acetonitrile containing 0.1% formic acid (both ULC-MS grade, BioSolve). The UV detector was set to measure at wavelengths of 220 nm and 280 nm, and the vials were placed in a sample chamber maintained at a temperature of 4°C. 1 μL volume was injected into a reversed-phase ACQUITY UPLC Protein BEH C4 column, 300 Å pore size column (Waters part number: 186004495), and the protein was eluted using a solvent B gradient 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 into 50% 2-propanol, and the lockspray was 200 pg / μL leucine enkephalin. The instrument was operated in positive ionization mode and sensitivity analyzer mode using 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 sec.

[0156] Data were processed using UNIFI software. Spectra were matched from retention times in chromatograms where the target protein was eluted. Raw data were subtracted from the background and deconvoluted using the MaxEnt1 algorithm for macromolecules. Experimental data were compared to the masses of theoretical sequences that accounted for disulfide bonds for non-reductive analysis and free cysteine ​​for reductive analysis. Deamidation of asparagine (+1Da) was also considered after PNGasE F deglycosylation.

[0157] LC-MS analysis confirmed the formation of disulfide bonds between complementary Fc chains and between chain A and chain B, reconstructing the native relaxin-2 structure. Figure 2A shows LC-MS data for RELAX0019 and RELAX0023 as examples. Non-reducible analysis confirmed the formation of heterodimers with expected masses of 58932Da and 59361Da for RELAX0019 and RELAX0023, respectively; no homodimers were detected. Reduced analysis (Figure 2B) confirmed the sequence identity of both chains and revealed that they were unmodified.

[0158] Non-reducing peptide mapping for identifying disulfide bonds Heterodimer Fc-relaxin (50 μg) was placed in a clean sample tube and diluted with 17 μL of 100 mM sodium phosphate pH 7.0. Free cysteine ​​alkylation was achieved by adding 0.5 μL of 5 mg / ml iodoacetamide and incubating at room temperature for 20 minutes. After alkylation, 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 5 μL of Lys-C was added and incubation was 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. To reduce the disulfide bonds, 2.5 μL of 500 mM DTT was added to another 42.5 μL aliquot of the sample, left at room temperature for 15 minutes, and then LC-MS analysis was performed.

[0159] Peptide analysis was performed using an ACQUITY I-Class UPLC connected to a Xevo G2-XS Q-TOF instrument (Waters, Milford, MA), and all operations were performed using the UNIFI Scientific Information System. In the case of 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 a temperature of 4°C. 10 μL was injected into a reversed-phase ACQUITY BEH C18 300 Å pore size column (Waters part number: 186003687), and proteins were eluted using an increasing gradient of solvent B, increasing from 5% to 37% over 73.5 minutes, and then to 60% B over a further 2.5 minutes. After 77.5 minutes, the column was held at 95% B for 5 minutes.

[0160] The mass spectrometer was calibrated from 100 to 2600 m / z by injecting 2 μg / μL sodium iodide into 50% 2-propanol, and the lockspray was 200 pg / μL leucine enkephalin. The instrument was operated in positive ionization mode and sensitivity analyzer mode using 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 sec.

[0161] The data were processed using UNIFI software by importing sequences containing the expected disulfide bonds and performing a search for matching Lys-C-producing peptides. Chromatograms obtained in the absence and presence of the reducing agent were superimposed to confirm that the identified disulfide-bonded peptides were no longer observed after reduction.

[0162] As shown at the top of Figure 3, a peptide was identified that matched the predicted mass of the disulfide-bonded relaxin 2 peptide incorporating both chains A and B (SLSLSPGGGGGSGGGGSGGGGSGGGGGSQLYSALANKCCHVGCTK=LCGRELVRAQIAICGMSTWS=RSLARFC (SEQ ID NOs. 75-77, respectively), with a predicted mass containing three disulfide bonds of 6836.23 Da). Figure 3(A-D) shows the identification of this peptide for RELAX0019 and confirmation that the peptide was no longer observed upon addition of a reducing agent: Panels A and B show the extracted ion chromatograms in the absence and presence of DTT, and panels C and D show the corresponding mass spectra of the peptide. Figure 3(E-H) shows the identification of the same peptide for RELAX0023 and confirmation that the peptide was no longer observed when a reducing agent was added: Panels E and F show the 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 support the idea that relaxin chains A and B interact via disulfide bonds within the heterodimers RELAX0019 and RELAX0023.

[0163] Example 3: In vitro activity of Fc-relaxin 2 fusion protein (cell-based cAMP activity assay) The relaxin 2 fusion polypeptide produced as described above was tested for biological activity, such as stimulation of one or more cell receptor responses, by the following method.

[0164] We purchased stable cell lines expressing human or mouse receptors, created from CHO cells, from DiscoverX. - cAMP Hunter (trademark) CHO-K1 RXFP1 Gs cell line (DiscoverX catalog number 95-0127C2) - cAMP Hunter (trademark) CHO-K1 RXFP2 Gs cell line (DiscoverX catalog number 95-0140C2) - cAMP Hunter (trademark) CHO-K1 mRXFP1 Gs cell line (DiscoverX catalog number 95-0180C2) Activation of these receptors results in the production downstream of the cAMP second messenger, which can be measured in functional activity assays.

[0165] Normal cAMP assays were performed using Hank's solution (Sigma #H8264) supplemented with bovine serum albumin (BSA)-based assay buffer: 0.1% BSA (Sigma #A9418) and 0.5 mM IBMX (Sigma #I7018) and adjusted to pH 7.4 with 1 M NaOH. Cryovials of cells expressing the receptor of interest were quickly thawed in a water bath, transferred to pre-warmed cell culture medium, and rotated at 240 xg for 5 minutes. The cells were resuspended in cell culture medium at an optimized concentration (e.g., 3.33 x 10 4 cells / ml of 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 next day, the medium was gently flicked off the plate and replaced with 5 μL of assay buffer. Serial dilutions in 11 steps of the test recombinant peptide or Fc fusion sample were added to the cells using a non-contact liquid dispenser (ECHO (trademark), Labcyte). Two replicates of all sample dilutions were made. 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 according to a two-step protocol as recommended by the manufacturer SMeasurements were performed using the HTRF kit (Cisbio, Cat #62AM4PEJ). Briefly, anti-cAMP cryptotate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were prepared separately by diluting each to 1 / 20 in the conjugate and lysis buffer provided in the kit. 5 μL of anti-cAMP cryptotate was added to all wells of the assay plate, and 5 μL of cAMP-d2 was added to all wells except the nonspecific binding (NSB) wells, to which conjugate and lysis buffer were added. The plate was incubated at room temperature for 1 hour, and then read using Envision (Perkin Elmer) with an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The data were converted to %Delta F, then to activation percentage relative to the maximum native agonist response, and EC2. 50 The values ​​were analyzed using a four-parameter logistic fit. These results were compared with 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 activity of the tested constructs is described in Table 4 and Figure 4. Mean EC50 measurements for both recombinant human relaxin 2 and fusion polypeptides 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-stranded fusion protein comprising chain A, a 15-amino acid linker, chain B, a 15-amino acid connector, and Fc, and contains the amino acid sequence of SEQ ID NO: 8 as described in International Publication No. 2018 / 138170.

[0170] [Table 19]

[0171] Based on the results shown in Table 4, we can conclude that the tested heterodimer Fc relaxin fusion proteins were less potent than single-strand fusion RELAX0010 or recombinant human relaxin 2 peptide, but still retained high levels of biological activity (approximately 10 pM to 80 pM in the human RXFP1 cell line).

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

[0173] The presence of disulfide bonds to stabilize the heterodimer Fc did not affect the efficacy of the fusion protein (comparing 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 (comparing RELAX0023 with RELAX0019, and RELAX0024 with RELAX0020). The exact amino acid sequence of the upper hinge is not important for the activity of the fusion protein.

[0175] Example 4: Effect of connector composition and length on heterodimer relaxin 2 Fc fusion protein The connectors may consist of glycine and serine residues (GS) or proline and alanine repeats (PA). The connectors used here had lengths of 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 PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO: 6) (21 amino acids). Connectors of different lengths and compositions can be placed on each Fc chain of the heterodimeric relaxin 2 Fc fusion polypeptide.

[0176] Table 5 shows examples of heterodimeric relaxin-2 Fc fusion proteins with various connectors. This table also includes information on development / manufacturability (expression yield and percentage of monomer / non-aggregated relaxin-2 Fc fusion proteins after protein A capture from cell culture supernatant) and biological activity.

[0177] [Table 20]

[0178] The length and composition of the connector affect the generative aspects of the molecule. As shown in Table 5, heterodimer relaxin 2 Fc fusion polypeptides with PA connectors of 16 amino acids or less were not well expressed. In contrast, PA connectors with a length of 21 residues significantly increased the expression yield. The expression yield of constructs with GS connectors was more consistent.

[0179] Heterodimeric relaxin 2 Fc fusion proteins with short, asymmetric (different) connectors retained their potency. Reduced biological activity was observed only in fusion proteins with low monomer content (RELAX0109, RELAX0110, and RELAX0111).

[0180] Example 5: Point mutation of relaxin 2 sequence Relaxin single-point mutant analogs were constructed as heterodimer Fc relaxin 2 fusion proteins. Table 6 shows examples of such molecules that retained potency and favorable development.

[0181] The targeted native residues are positively charged and may be prone to proteolytic degradation, but they were not involved in relaxin's binding to its receptor.

[0182] For example, the R22X analog heterodimer Fc relaxin 2 fusion protein appears to consistently exhibit improved developmental / 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 is acceptable without loss of efficacy, while maintaining desirable development.

[0185] Example 6: PK profile of Fc-relaxin 2 fusion protein The pharmacokinetic (PK) profile of relaxin 2 fusion polypeptide was determined using relaxin ELISA and / or cAMP assays. Relaxin 2 fusion polypeptide was administered at a dose of 6 mg / kg to 6-10 week old male C57BL / 6J (Jax) mice (Jackson Laboratories) via either subcutaneous (SC) and / or intravenous (IV) routes. For IV administration, serum samples were collected at 5, 30, and 60 minutes post-administration, followed by 3 and / or 6 and / or 8 and 24 hours, and then at minimum daily intervals up to day 21. A similar schedule was followed for SC administration, but with less frequent collection within the first 8 hours; for example, the first sample was collected at 30 minutes, followed by collections at 3, 8, 24, 30, and 48 hours, and then at minimum daily intervals up to day 21. Samples were collected in serum tubes by cardiac puncture, held at room temperature for 15–30 minutes, and then centrifuged at 10,000 rpm for 10 minutes within 30 minutes of collection. Aliquot samples were stored at <-80°C and subsequently examined by ELISA or cAMP activity assay.

[0186] For most molecules, PK samples were examined by ELISA using anti-h relaxin-2 capture (using the pre-coated Human Relaxin-2 Quantikine ELISA Kit, R&D Systems Cat# DRL200) and anti-human Fc detection antibody (HRP-labeled AU003), with the exception of RELAX0010 (described in International Publication No. 2018 / 138170), which was examined by ELISA using anti-human Fc capture and anti-hRelaxin-2 detection (using the polyclonal HRP-labeled antibody from the Human Relaxin-2 ELISA Kit, R&D Systems Cat# DRL200). In both assays, plates coated with capture antibodies were blocked with 100 μL of RD1-19 assay diluent at room temperature for 1 hour. 50 μL of standard or sample was added to each well and incubated at room temperature for 2 hours. Samples were aspirated and the wells were washed three times with assay washing buffer. 50 μL of HRP-labeled detection antibody was added per well. For anti-human Fc specific detection, the antibody was diluted 1:1000 with PBS / 1% BSA, or used undiluted for anti-h relaxin 2 detection. After incubation at room temperature for 1 hour, followed by three washes, 50 μL of TMB (SureBlue Reserve KPL 53-00-03) was added per well. Once a color change occurred, 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 cell-based cAMP activity assays As outlined above, serum samples collected from animals were tested for biological activity to assess the integrity of the Fc-relaxin 2 fusion polypeptide by measuring functional relaxin 2. A stable cell line expressing the human RXFP1 receptor, prepared from CHO cells, was purchased from DiscoverX. Activation of this receptor leads to the downstream production of the cAMP second messenger, which can be measured by functional activity assays.

[0188] The cAMP assay was performed using a Hanks equilibrium salt solution (Sigma#H8264) prepared by adding bovine serum albumin (BSA)-based assay buffer: 0.1% BSA (Sigma#A9418) and 0.5 mM IBMX (Sigma#I7018), and adjusting the pH to 7.4 with 1 M NaOH.

[0189] The administration solution of relaxin 2 fusion polypeptide or recombinant relaxin 2 peptide (R&D Systems Cat#6586-RN) was diluted in assay buffer, and an 11-point standard curve was created at four matrix concentrations using a non-contact liquid dispenser (ECHO, Labcyte). The matrix used was blank serum from simulated animals, which was manually added to the wells at twice the concentration required to allow cell addition. The test samples were transferred from serum tubes to a 384-well source plate, which was then used with a non-contact liquid dispenser (ECHO, Labcyte) to set up four dilutions in assay buffer. All sample dilutions were repeated in double succession.

[0190] Frozen cryovials of cells expressing hRXFP1 were rapidly thawed in a water bath, transferred to preheated cell medium, and spun at 240×g for 5 minutes. The cells were resuspended in 8 mL of cell culture medium and seeded into a T75 flask containing 10 mL of culture medium, and allowed to adhere overnight. The following day, the cells were detached using an oxidizer and spun at 240×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) following a two-step protocol as recommended by the manufacturer. Briefly, anti-cAMP cryptotate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were prepared separately by diluting each to 1 / 20 with the conjugate and lysis buffer provided in the kit. 2.5 μL of anti-cAMP cryptotate 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) well, followed by the addition of conjugate and lysis buffer. After incubating the plate at room temperature for 1 hour, readings were taken using an Envision (Perkin Elmer) with an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The 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 Figure 5 shows a summary of data from a series of in vivo pharmacokinetic (PK) experiments in which Fc-relaxin 2 polypeptide was administered intravenously to mice. The data were normalized at 5 minutes.

[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-life compared to natural relaxin-2. Fc-relaxin polypeptides in which relaxin A and B chains are linked to different heterodimeric Fc chains (exemplified by RELAX0019, RELAX0023, RELAX0034, RELAX0046, and RELAX0117) have improved PK properties compared to Fc-relaxin polypeptides in which the relaxin chains are linked by a linker (exemplified by RELAX0010 and RELAX0009). However, since both RELAX0088 and RELAX0122, which are molecules containing linkers, exhibit good in vivo stability, the presence of a linking linker between relaxin chain A and relaxin chain B is not alone directly related to the rapid in vivo elimination of Fc-relaxin polypeptide.

[0194] Unexpectedly in this study, all heterodimeric Fc-relaxin fusion polypeptides (RELAX0019, RELAX0023, RELAX0034, RELAX0046, RELAX0117, RELAX0088, and RELAX0122) exhibit significantly improved pharmacokinetic properties compared to Fc-relaxin fusion polypeptides RELAX0010 and RELAX0009.

[0195] Example 7: Established recovery of hypertrophy and fibrosis using RELAX0019 and RELAX0023 Isoproterenol was injected into C57B6 mice via a minipump (15 mg / kg / day) for 10 days to induce cardiac hypertrophy and fibrosis. Mice injected with the vehicle for the same period were used as baseline controls. After 10 days, the minipump was removed, and the mice were administered either a new minipump containing r-relaxin 2 (500 ug / kg / day) or the first of two weekly (QW) subcutaneous injections of RELAX0019 (20 mg / kg) or RELAX0023 (20 mg / kg). After a 14-day treatment period, the mice were sacrificed, and their hearts were harvested for analysis of hypertrophy and fibrosis. Hearts were harvested from baseline control mice after removal of the vehicle minipump. 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 injection significantly induced both hypertrophy and fibrosis in this model. QW administration of RELAX0019 and RELAX0023, as well as continuous infusion of r-relaxin 2, reduced isoproterenol-induced hypertrophy to baseline levels. All relaxin treatments also reduced myocardial fibrosis by more than 50%. N=8 for each group. **p<0.01, ***p<0.001, ****p<0.0001

[0196] Recombinant relaxin Fc fusion proteins RELAX0019 and RELAX0023 were able to restore hypertrophy and fibrosis, similar to native h-relaxin 2 (Figure 6).

[0197] Example 8: Evaluation of nonspecific binding of Fc-relaxin 2 protein using baculovirus ELISA. The RELAX protein was expressed in CHO cells and purified as described above. The baculovirus ELISA (Ref: Hotzel et al 2012 mAbs 4:6,753-760), developed to evaluate the nonspecific binding of monoclonal antibodies, was modified to determine the nonspecific binding of Fc-relaxin polypeptide. In this case, instead of calculating the "BV score" (absorbance of baculovirus plate / absorbance of blank plate), nonspecific binding was calculated separately for baculovirus plates and blank plates as a signal against the background (in this case, the background being Fc-relaxin polypeptide). This measurement was introduced to reflect the increased nonspecific binding of certain Fc peptides to both coated and uncoated (blank) plates compared to monoclonal antibodies. Each protein preparation was prepared at 100 nM or 10 nM in PBS (Gibco 14190-086) + 0.5% BSA (Sigma A9576), and used twice in an ELISA assay on a 96-well Nunc Maxisorp F plate coated overnight at 4°C with either 50 μL / well of 1% baculovirus extract in 50 mM sodium carbonate (BV plate) or 50 mM sodium carbonate (blank plate). After washing with PBS, the plate was blocked with 300 μL / well of PBS + 0.5% BSA at room temperature for 1 hour, and washed three times with PBS. Either 50 μL / well of PBS + 0.5% BSA (background) or RELAX protein dilution was added, and the plate was 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. The samples were incubated at room temperature for 1 hour, and the plates were washed three times with PBS. Next, 50 μL / well of the HRP substrate TMB (SureBlue Reserve KPL 53-00-03) was added, and after a color change, the reaction was stopped by adding 50 μL / well of 0.5 M sulfuric acid.Absorbance was measured at 450 nm to determine nonspecific binding for each sample. Nonspecific binding (binding ratio relative to background) was defined as the ratio of nonspecific binding in the presence of Fc relaxin 2 protein and in the absence of Fc relaxin 2 protein (background). Table 7 shows the data for Fc relaxin 2 protein tested at two different concentrations, either 100 nM or 10 nM.

[0198] [Table 22]

[0199] [Table 23]

[0200] [Table 24]

[0201] As shown in Table 7 and Figure 7, heterodimeric relaxin 2 Fc fusion polypeptides exhibit lower nonspecific binding when the relaxin chain is ligated to the C-terminus using a GS connector. Some asymmetric and PA connectors, specific point mutations, and the placement of the relaxin chain at the N-terminus increase nonspecific binding to both blank and BV-coated plates, particularly with respect to the divalent 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. All control molecules (divalent RELAX0009, RELAX0010, RELAX0126, RELAX0127, and RELAX0128, which contain linkers) exhibit high nonspecific binding. However, as can be demonstrated by the low nonspecific binding of RELAX0122, neither the presence of a linker between relaxin chains A and B nor its divalent nature itself drives high nonspecific binding.

[0202] Example 9: Stability in solution The stability of RELAX0023 was evaluated using high-speed size exclusion chromatography (HP-SEC) and liquid chromatography-mass spectrometry (LC-MS), and compared with RELAX0127 and RELAX0128. Purity, aggregation, and fragmentation could be measured using HP-SEC, including detection by absorbance at 280 nm. Molecules were concentrated to 10 mg / mL after buffer exchange in an optimized formulation composition. All samples were placed under stress temperature conditions (40°C) for up to 4 weeks. At 1, 2, and 4 weeks, samples were collected, injected into a size exclusion column, and eluted to a uniform concentration using a constant flow rate of aqueous mobile phase. Larger molecules elute faster than smaller molecules because they are excluded more readily from the pores of the size exclusion column. Peaks that elute before the monomer peak are recorded as aggregates. Peaks that elute after the monomer peak (excluding buffer-related peaks) are recorded as fragments. The results were recorded as purity percent; aggregate percent; and fragment percent, and are shown in Figure 8. RELAX0023 is the most stable molecule, with a purity loss rate (%) of only 0.1% per month, compared to RELAX0128 and RELAX0127, which had rates of 7.7% and 9.3% respectively. While both RELAX0127 and RELAX0128 showed signs of aggregation, the aggregate level of RELAX0023 did not increase, indicating good physical solution stability. For RELAX0127, which had a fragmentation rate of 6.6% per month, and RELAX0128, which had 6.8%, fragmentation appeared to be the main cause of purity loss. RELAX0023 had a fragmentation rate of only 0.7% per month. Furthermore, after storage at 40°C for 4 weeks, the total peak area of ​​RELAX0128 decreased from 22403 to 18216 (a 19% decrease), and RELAX0127 decreased from 22225 to 18823 (a 15% decrease). This significant loss of total peak area, along with a high fragmentation rate, indicated the potential for high chemical degradation associated with these two molecules. It should be noted that this loss of total area strongly affected the chromatographic profiles of these two molecules.This explains why RELAX0128 and RELAX0127 showed lower aggregate percentages at 4 weeks compared to previous 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, demonstrating a better stability profile compared to RELAX0128 and RELAX0127.

[0203] Molecular fragmentation was further verified by LC-MS using reduced 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 peak of RELAX0023 remained unchanged after stress. The mass spectra under reducing conditions also showed changes over time for RELAX0127 and RELAX0128, as evidenced by a shift of the peaks to a larger mass and a broadening of the peaks showing greater heterogeneity (Figure 9B). In contrast, the intact mass spectrum of RELAX0023 remained unchanged, indicating that no changes occurred. This test 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 a total of 12 female cynomolgus monkeys, randomly assigned to four groups of three monkeys each. Animals in groups 1, 2, and 3 received RELAX0023 SC at doses of 0.1, 1, and 10 mg / kg, 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 1-4. RELAX0023 bound to the plates was detected by HRP-conjugated anti-relaxin antibody. Cynomolgus monkey serum was diluted 1:10 before being added to the plates. The lower limit of quantification in 100% serum was 0.010 μg / mL, and the upper limit of quantification 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 via SC, RELAX0023 showed a linear pharmacokinetic profile in the dose range of 0.01–10 mg / kg. max A dose-proportional increase was observed. Mean C max The values ​​were 0.400, 4.69, and 34.8 μg / mL for the SC dose groups of 0.1, 1, and 10 mg / kg, respectively. Also, the AUC 0-last A dose-proportional increase in the value was observed from the 0.1 mg / kg to 10 mg / kg SC group. Mean AUC 0-last The values ​​were 2.01, 25.5, and 193 μg·day / mL for the 0.1, 1, and 10 mg / kg SC dose groups, respectively. Overall, RELAX0023 PK was linear in the range of 0.1 mg / kg to 10 mg / kg, with a mean CL / F of 51.0 mL / day / kg and an mean t 1 / 2 The average lifespan is 3.07 days. The SC bioavailability of RELAX0023 was estimated at 88.2%.

Claims

1. below: (i) a first heterodimer domain linked to at least one relaxin A chain polypeptide or a variant thereof; (ii) A second heterodimerization domain linked to at least one relaxin B chain polypeptide or a variant thereof, A heterodimer fusion product containing, The first heterodimerizing domain heterodimerizes with the second heterodimerizing domain, and the heterodimer fusion is a heterodimer fusion having relaxin activity.

2. The heterodimer fusion product according to 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 heterodimer fusion according to 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 heterodimer fusion product according to any one of claims 1 to 3, wherein the relaxin A chain is a relaxin-2A chain and the relaxin B chain is a relaxin-2B chain.

5. The heterodimer fusion according to any one of claims 1 to 4, wherein the relaxin A chain is linked to the first heterodimer domain via a connector, and the relaxin B chain is linked to the second heterodimer domain via a connector, and optionally one or preferably both connectors are polypeptides.

6. The heterodimer fusion according to claim 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.

7. The heterodimer fusion according to any one of claims 1 to 6, wherein the first and second heterodimerizing domains are derived from immunoglobulin Fc regions ("first Fc region" and "second Fc region," respectively), and optionally the first and second Fc regions include constant domains CH2 and CH3.

8. The heterodimer fusion according to claim 7, wherein the C-terminus of the first Fc region is connected to the N-terminus of the relaxin A chain, and the C-terminus of the second Fc region is connected to the N-terminus of the relaxin B chain.

9. The heterodimer fusion according to claim 7 or 8, wherein the first and second Fc regions include heterodimerization-promoting amino acid mutations and / or modifications, and optionally, the heterodimerization-promoting amino acid mutations are "Fc knob" and "Fc hole" mutations, for example, "Fc knob" and "Fc hole" mutations located in the CH3 domain.

10. The heterodimer fusion according to any one of claims 7 to 9, wherein the first and second Fc regions are derived from human IgG1 immunoglobulin.

11. The aforementioned heterodimer-promoting amino acid mutations are as follows: a. “Fc hole” mutations in one CH3 domain Y349C, T366S, L368A and Y407V; and b. Fc knob mutations S354C and T366W in the other CH3 domain. Includes, The heterodimer fusion according to claim 10, wherein the numbering of the amino acids is based on the EU index of Kabat.

12. a. The first Fc region contains the "Fc knob" mutation, and the second Fc region contains the "Fc hole" mutation; or b. The heterodimer fusion according to claim 11, wherein the second Fc region includes the "Fc knob" mutation and the first Fc region includes the "Fc hole" mutation.

13. The heterodimer fusion according to any one of claims 10 to 12, wherein the first and / or second Fc region comprises the amino acid mutations L234F, L235E, and P331S, where the numbering of the amino acids is according to the Kabat EU index.

14. The heterodimer fusion according to any one of claims 4 to 13, wherein the relaxin-2A chain polypeptide comprises the sequence described in SEQ ID NO: 1 or a variant thereof, and the relaxin-2B chain polypeptide comprises the sequence described in SEQ ID NO: 2 or a variant thereof.

15. The heterodimer fusion product according to claim 14, wherein the relaxin-2A chain polypeptide comprises amino acid mutations K9H, K17M, or K17I.

16. The heterodimer fusion according to any one of claims 5 to 15, wherein both connectors have the sequence GGGGGSGGGGGSGGGGGGGS [Sequence ID 5].

17. below: (i) FcX-con-A fusion polypeptide and; (ii) FcY-con-B fusion polypeptide and A heterodimer fusion product containing, A is the relaxin A chain or its variant, for example, the relaxin-2A chain or its variant; B is the relaxin B chain or its variant, for example, the relaxin-2B chain or its variant; FcY is an Fc region containing the constant domains CH2 and CH3 of human IgG1 immunoglobulin, and includes "Fc hole" amino acid mutations and / or modifications, preferably amino acid mutations Y349C:T366S:L368A:Y407V; FcX is preferably an Fc region having an "Fc knob" amino acid mutation and / or modification, which includes the constant domains CH2 and CH3 of human IgG1 immunoglobulin, and preferably includes the amino acid mutation S354C:T366W; con is preferably a connector polypeptide having the sequence GGGGGSGGGGGSGGGGGGGS[SEQ ID NO: 5], Here, the numbering of the amino acids is based on the EU index of Kabat, and FcX heterodimerizes with FcY, and the heterodimer fusion is a heterodimer fusion having relaxin activity.

18. The heterodimer fusion according to any one of claims 1 to 17, wherein the heterodimer fusion comprises a fusion polypeptide having the amino acid sequence of SEQ ID NO: 11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO:

20.

19. The heterodimer fusion according to any one of claims 8 to 18, wherein the heterodimer fusion further comprises one or more Fabs, optionally comprising one Fab attached to the N-terminus of the first Fc region and a second Fab attached to the N-terminus of the second Fc region.

20. The heterodimer fusion according to any one of claims 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, wherein optionally, 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.

21. below: (i) FcX-B-L-A and FcY, optionally FcY-B-L-A; or (ii) FcY-B-L-A and FcX, and optionally FcX-B-L-A; A heterodimer fusion product containing, FcY is preferably an immunoglobulin Fc region having an "Fc hole" amino acid mutation and / or modification, including a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V; FcX is preferably an immunoglobulin Fc region having an "Fc knob" amino acid mutation and / or modification, including a CH3 domain having the amino acid mutation S354C:T366W; B is the relaxin B chain or a variant thereof, for example, the relaxin 2B chain or a variant thereof; A is the relaxin A chain or a variant thereof, for example, the relaxin 2A chain or a variant thereof; L is preferably a linker polypeptide having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60], Here, the numbering of the amino acids is based on the EU index, similar to that of Kabat. In this case, FcX heterodimerizes with FcY, and the heterodimer fusion is a heterodimer fusion having relaxin activity.

22. The heterodimer fusion according to claim 21, wherein the relaxin B chain is linked to FcX and / or FcY via a connector, optionally a connector polypeptide having a length of 6 to 40 amino acids, for example, 21 amino acids.

23. The heterodimer fusion according to any one of claims 1 to 22, wherein the ratio of the relaxin activity of the heterodimer fusion to the relaxin activity of a reference relaxin protein is about 0.001 to about 10.

24. A nucleic acid molecule encoding a heterodimer fusion according to any one of claims 1 to 23.

25. A vector comprising the nucleic acid molecule described in claim 24.

26. A host cell comprising the vector according to claim 25 or the nucleic acid molecule according to claim 24.

27. A method for producing a heterodimer fusion according to any one of claims 1 to 23, comprising the steps of culturing the host cells according to claim 26 and collecting the fusion protein.

28. A pharmaceutical composition comprising a heterodimer fusion according to any one of claims 1 to 23 and a pharmaceutically acceptable excipient.

29. A heterodimer fusion according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 28 for use in therapy.

30. The heterodimer fusion or pharmaceutical composition described above is administered to the subject for use in the treatment of a subject having heart failure, as described in any one of claims 1 to 23 or claim 28.

31. The heterodimer fusion or pharmaceutical composition for use according to claim 29 or 30 is administered to the subject by subcutaneous injection.

32. The fusion polypeptide or pharmaceutical composition is administered by autoadministration, and is a heterodimer fusion for use according to any one of claims 29 to 31 or a pharmaceutical composition for use according to any one of claims 29 to 31.

33. A kit comprising the pharmaceutical composition of claim 28.

34. A method for treating a subject having a disease or disorder, comprising the step of administering to the subject a heterodimer fusion according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 28.

35. A method for treating a subject having heart failure, comprising the step of administering to the subject a heterodimer fusion according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 28.

36. The method according to claim 34 or 35, wherein the heterodimer fusion or pharmaceutical composition is administered to the subject by subcutaneous injection.

37. The method according to any one of claims 34 to 36, wherein the heterodimer fusion or pharmaceutical composition is administered by self-administration.

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