Manipulated relaxin and its method of use

Abstract

The present invention provides novel recombinant relaxin-2 compositions and methods for producing them.The method of treating relaxin-related disorders or diseases using the compositions of the present invention is also disclosed herein.The compositions and methods disclosed herein are particularly advantageous in that they use various fusion proteins and polypeptides disclosed herein, which provide excellent properties.For example, compared with natural relaxin-2 protein, the fusion proteins and polypeptides of the present invention have improved pharmacokinetics, for example, longer circulating half-life, or improved activity, for example, enhanced activation of RXFP1.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 021,814, filed 8 May 2020, the entire contents of which are expressly incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing, submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on 6 May 2021, named 117823-19620_SL.txt, and has a size of 79,204 bytes.

[0003] Technical field The present invention relates to compositions and methods for modulating relaxin-2 activity. [Background technology]

[0004] Background of the Invention Relaxin is a small protein hormone distantly related to insulin. They regulate various biological functions through their four receptors: RXFP1, RXFP2, RXFP3, and RXFP4. Of these, the first, RXFP1, is of particular interest as a therapeutic target due to its anti-fibrotic effects and ability to enhance cardiac output. Its ligand, relaxin-2, has been evaluated in large-scale clinical trials for the treatment of heart failure. The relaxin receptor may also be an effective target for the treatment of pulmonary arterial hypertension and various fibrotic diseases.

[0005] Relaxin and its receptors are both biochemically difficult molecules to process. Relaxin consists of two chemically distinct chains, and existing methods for their production are slow, costly, and cumbersome. Furthermore, relaxin-2 produced using currently available methods has a short in vivo half-life. Therefore, there is a need in this field for recombinant relaxin-2 proteins that possess high levels of biological activity, a long cyclic half-life, and are cost-effective to produce. [Overview of the Initiative] [Means for solving the problem]

[0006] Summary of the Invention Novel relaxin-2 compositions and methods of use thereof are disclosed herein for modulating, for example, enhancing, relaxin-2 activity in subjects, such as human subjects. The compositions and methods disclosed herein provide means for treating and / or preventing relaxin-2-related diseases in subjects, such as subjects who may benefit from modulated, for example, increased or decreased levels of relaxin-2.

[0007] The compositions and methods disclosed herein are particularly advantageous in that they utilize various fusion proteins and polypeptides disclosed herein that offer superior properties. For example, the fusion proteins and polypeptides of the present invention have improved pharmacokinetics, e.g., a longer circulating half-life, or improved activity, e.g., enhanced activation of RXFP1, compared to the natural relaxin-2 protein. The fusion proteins and polypeptides of the present invention have an EC of about 0.085 nM to about 465 nM 50 This has been shown to provide improved activation of RXFP1 on cells and exhibit an enhanced circulating half-life of at least approximately 77.5 hours to at least approximately 130 hours.

[0008] Therefore, in one embodiment, the present invention features a fusion protein. The fusion protein comprises a first peptide containing an amino acid sequence from the N-terminus to the C-terminus that is at least approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical to the entire amino acid sequence of SEQ ID NO: 2; a peptide linker containing an amino acid sequence containing at least approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical to the entire amino acid sequence of an amino acid sequence selected from the group consisting of DAASSHSHSSAR (SEQ ID NO: 14) and DAAGANANAGAR (SEQ ID NO: 16); and a second peptide containing an amino acid sequence containing at least approximately 85% identical to the entire amino acid sequence of SEQ ID NO: 1; the first peptide, the peptide linker, and the second peptide are operably linked.

[0009] In one embodiment, the fusion protein has the activity of the natural relaxin-2 protein. In another embodiment, the fusion protein has at least about 50% of the activity of the natural relaxin-2 protein. In yet another embodiment, the fusion protein has at least about 90% of the activity of the natural relaxin-2 protein. In yet another embodiment, the fusion protein has at least about 100% of the activity of the natural relaxin-2 protein. In one embodiment, the fusion protein has at least about 150% of the activity of the natural relaxin-2 protein.

[0010] In another embodiment, the peptide linker includes an amino acid sequence that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the entire amino acid sequence of an amino acid sequence selected from the group consisting of DAASSHSHSSAR (SEQ ID NO: 14), DAASSHSHSSAA (SEQ ID NO: 15), and DAAGNANANAGAR (SEQ ID NO: 16). In yet another embodiment, the peptide linker includes the amino acid sequence of DAASSHSHSSAR (SEQ ID NO: 14), DAASSHSHSSAA (SEQ ID NO: 15), or DAAGNANANAGAR (SEQ ID NO: 16).

[0011] In another embodiment, the first peptide has an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the entire amino acid sequence of SEQ ID NO: 2, and the second peptide has an amino acid sequence that is at least about 95% identical to the entire amino acid sequence of SEQ ID NO: 1, and the fusion protein has innate relaxin-2 activity. In yet another embodiment, the amino acid sequence of the first peptide is selected from the group consisting of SEQ ID NOs: 2, 7, 8, 9, and 10, and the amino acid sequence of the second peptide is selected from the group consisting of SEQ ID NOs: 1 and 6. In one embodiment, the first peptide includes substitutions selected from the group consisting of M4K, M25K, W28A, and combinations thereof. In another embodiment, the first peptide includes substitutions M4K, M25K, and W28A.

[0012] In another embodiment, the present invention provides a fusion protein comprising a first peptide, a peptide linker, and a second peptide, wherein the amino acid sequence of the fusion protein is at least about 85% identical to the entire amino acid sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, and 55.

[0013] In another embodiment, the present invention provides a fusion protein comprising a first peptide, a peptide linker, and a second peptide, wherein the amino acid sequence of the fusion protein is at least about 85% identical to the entire amino acid sequence shown in SEQ ID NO: 55. In one embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 55.

[0014] In various embodiments of the above-described aspects of the present invention or any other aspects thereof, as detailed herein, the fusion protein further comprises a first detectable label. In one embodiment, the first detectable label is operably ligated to the N-terminus of the first peptide or the C-terminus of the second peptide. In another embodiment, the first detectable label is an immunoglobulin G (IgG)Fc peptide containing an amino acid sequence that is at least about 85% identical to the entire amino acid sequence of SEQ ID NO: 20. In yet another embodiment, the first detectable label has the amino acid sequence of SEQ ID NO: 20 or 21. In yet another embodiment, the first detectable label is operably ligated to the N-terminus of the first peptide.

[0015] In one embodiment, the fusion protein further comprises a second linker, which is operably ligated to the C-terminus and N-terminus of the first detectable label. In another embodiment, the second linker is selected from the group consisting of Gly-Gly-Ser, Ala-Ala-Ala, Pro-Pro-Pro, Gly-Ser-Gly, (Gly-Ser-Gly)2 (SEQ ID NO: 57), and (Gly-Gly-Ser)4 (SEQ ID NO: 17).

[0016] In one embodiment, the fusion protein has an in vivo circulating half-life longer than approximately 10 hours. In another embodiment, the fusion protein has an in vivo circulating half-life of approximately 130 hours.

[0017] In another embodiment, the first detectable label is a polyhistidine tag having an amino acid sequence that is at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an entire amino acid sequence selected from the group consisting of SEQ ID NOs. 18 and 19. In another embodiment, the first detectable active substance includes the amino acid sequence of SEQ ID NO. 18 or 19.

[0018] In one embodiment, the fusion protein further comprises a second detectable label. In another embodiment, the first detectable label is operably ligated to the N-terminus of the first peptide, and the second detectable label is operably ligated to the C-terminus of the second peptide. In yet another embodiment, both the first and second detectable labels are operably ligated to the N-terminus of the first peptide. In yet another embodiment, the first and second detectable labels are different peptides.

[0019] In various embodiments of the above-described model, the fusion protein further comprises a cleavable linker. In one embodiment, the cleavable linker is a peptide that undergoes specific digestion by a protease. In another embodiment, the protease is HRV 3C protease or thrombin. In yet another embodiment, the cleavable linker is a peptide having the sequence of SEQ ID NO: 23 or a variant thereof.

[0020] In various embodiments of the above-described model, the fusion protein further comprises a signal peptide at its N-terminus.

[0021] In one aspect, the present invention provides a fusion protein. The fusion protein comprises a detectable label, a second linker, a first peptide, a peptide linker and a second peptide, and the amino acid sequence of the fusion protein is at least about 85% identical to the entire amino acid sequence shown in SEQ ID NO: 41, SEQ ID NO: 60 or SEQ ID NO: 61. In one embodiment, the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 41, SEQ ID NO: 60 or SEQ ID NO: 61.

[0022] In one aspect, the present invention provides a fusion protein. The fusion protein comprises a detectable label, a second linker, a first peptide, a peptide linker and a second peptide, and the amino acid sequence of the fusion protein is at least about 85% identical to the entire amino acid sequence shown in SEQ ID NO: 41.

[0023] In another aspect, the present invention provides a peptide linker comprising an amino acid sequence having at least about 85% amino acid identity to the entire amino acid sequence of an amino acid selected from the group consisting of DAASSHSHSSAR (SEQ ID NO: 14) and DAAGANANAGAR (SEQ ID NO: 16).

[0024] In another aspect, the present invention provides a fusion protein comprising, from N-terminus to C-terminus, a first peptide comprising a relaxin B amino acid sequence; a peptide linker; and a second peptide comprising a relaxin A amino acid sequence, wherein the fusion protein has the activity of native relaxin-2 protein and has an EC of about 4.2 nM or less 50 for activating the relaxin-2 receptor RXFP1 on the cell surface; (ii) a melting temperature of at least about 57°C; (iii) a circulation half-life of at least about 77.5 hours; and (iv) a property selected from the group consisting of any combination thereof.

[0025] In another aspect, the present invention provides a fusion protein comprising, from N-terminus to C-terminus, a first peptide comprising an amino acid sequence at least about 90% identical to the entire amino acid sequence of SEQ ID NO: 10; a peptide linker; and a second peptide comprising a relaxin A amino acid sequence.

[0026] In one embodiment, the amino acid in the first peptide corresponding to amino acid 4 of SEQ ID NO: 10 is K; the amino acid in the first peptide corresponding to amino acid 25 of SEQ ID NO: 10 is K; and the amino acid in the first peptide corresponding to amino acid 28 of SEQ ID NO: 10 is A.

[0027] In another embodiment, the peptide linker includes the amino acid sequence of SEQ ID NO: 16.

[0028] In another embodiment, the present invention provides a fusion protein comprising a first peptide containing a relaxin B amino acid sequence from the N-terminus to the C-terminus; a peptide linker containing the amino acid sequence of SEQ ID NO: 16; and a second peptide containing a relaxin A amino acid sequence.

[0029] In one embodiment, the first peptide contains the amino acid sequence of SEQ ID NO: 10; the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 16; the second peptide contains an amino acid sequence that is at least about 85% identical to the entire amino acid sequence of SEQ ID NO: 1; the second peptide contains the amino acid sequence of SEQ ID NO: 1; the first peptide contains an amino acid sequence that is at least about 90% identical to the entire amino acid sequence of SEQ ID NO: 10; the peptide linker contains the amino acid sequence of SEQ ID NO: 16; and the second peptide contains an amino acid sequence that is at least about 85% identical to the entire amino acid sequence of SEQ ID NO: 1; or the first peptide contains the amino acid sequence of SEQ ID NO: 10; the peptide linker contains the amino acid sequence of SEQ ID NO: 16; and the second peptide contains the amino acid sequence of SEQ ID NO: 1.

[0030] In another aspect, the present invention provides a polypeptide having an amino acid sequence that is at least about 90% identical to the entire amino acid sequence of SEQ ID NO: 10.

[0031] In one embodiment, the amino acid corresponding to amino acid 4 in SEQ ID NO: 10 is K; the amino acid corresponding to amino acid 25 in SEQ ID NO: 10 is K; and the amino acid corresponding to amino acid 28 in SEQ ID NO: 10 is A.

[0032] In another embodiment, the polypeptide comprises an amino acid sequence including SEQ ID NO: 10. In yet another embodiment, the amino acid sequence of the polypeptide consists of SEQ ID NO: 10.

[0033] In yet another embodiment, the polypeptide further comprises an amino acid sequence that is at least about 85% identical to the entire amino acid sequence of SEQ ID NO: 16. In yet another embodiment, the polypeptide further comprises an amino acid sequence comprising SEQ ID NO: 16.

[0034] In yet another embodiment, the polypeptide further comprises an amino acid sequence that is at least about 85% identical to the entire amino acid sequence of SEQ ID NO: 1. In yet another embodiment, the polypeptide further comprises an amino acid sequence comprising SEQ ID NO: 1.

[0035] In another embodiment, the present invention provides a polypeptide comprising amino acid sequences including SEQ ID NO: 1 and SEQ ID NO: 16.

[0036] In another embodiment, the present invention provides a polypeptide comprising an amino acid sequence comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 16, wherein the amino acid sequence of SEQ ID NO: 16 is inserted between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 10.

[0037] In another embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding a fusion protein or polypeptide of any embodiment of the above embodiments. In one embodiment, the polynucleotide is an RNA molecule.

[0038] In yet another embodiment, the present invention provides an expression vector comprising a polynucleotide as described above. In one embodiment, the expression vector is a plasmid. In another embodiment, the expression vector is a viral vector.

[0039] In one embodiment, the present invention provides recombinant cells. Recombinant cells comprise the polynucleotide or expression vector described above. In one embodiment, the cells are prokaryotic cells or eukaryotic cells. In another embodiment, the cells are prokaryotic cells selected from the group consisting of E. coli cells and Bacillus cells. In yet another embodiment, the cells are eukaryotic cells selected from the group consisting of yeast cells, insect cells and mammalian cells. In yet another embodiment, the cells are mammalian cells selected from the group consisting of CHO cells, HeLa cells and 293 cells. In one embodiment, the cells are Expi293 cells.

[0040] In one embodiment, the present invention provides a method for producing the fusion protein of the above embodiment, comprising the steps of culturing recombinant cells of the above embodiment and purifying the fusion protein.

[0041] In another embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of a fusion protein or polynucleotide of any one of the above embodiments or an expression vector of any one of the above embodiments.

[0042] In another embodiment, the present invention provides a method for enhancing relaxin-2-related activity in cells, comprising the step of contacting cells with a fusion protein according to any of the above embodiments, thereby enhancing relaxin-2-related activity in the cells. In one embodiment, the fusion protein activates the relaxin-2 receptor RXFP1 on the cell surface. In another embodiment, the method increases cAMP levels in cells to induce vasodilation, induce the expression of angiogenic factors, induce the expression of MMPs, and induce collagen degradation. In yet another embodiment, the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts.

[0043] In one embodiment, the cells are within the subject. In another embodiment, the subject has a relaxin-2 related disorder. In yet another embodiment, the relaxin-2 related disorder is selected from the group consisting of kidney disease, fibrous disease, and cardiovascular disease. In yet another embodiment, the disorder is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, hepatorenal syndrome, scleroderma, idiopathic pulmonary fibrosis, renal fibrosis, cardiac fibrosis, NASH, dilated cardiomyopathy, diastolic heart failure, pulmonary arterial hypertension, chronic heart failure, acute heart failure, congestive heart failure, coronary artery disease, hypertension, and pre-eclampsia.

[0044] In one embodiment, the present invention provides a method for treating relaxin-2-related disorders in subjects requiring such treatment. The method comprises the step of administering an effective amount of a fusion protein of any one of the above embodiments, a polynucleotide of any one of the above embodiments, an expression vector of any one of the above embodiments, or a pharmaceutical composition of any one of the above embodiments to a subject, thereby treating the relaxin-2-related disorder. In one embodiment, the relaxin-2-related disorder is selected from the group consisting of kidney disease, fibrous disease, and cardiovascular disease. In another embodiment, the disorder is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, hepatorenal syndrome, scleroderma, idiopathic pulmonary fibrosis, renal fibrosis, cardiac fibrosis, NASH, dilated cardiomyopathy, diastolic heart failure, pulmonary arterial hypertension, chronic heart failure, acute heart failure, congestive heart failure, coronary artery disease, hypertension, and preeclampsia. In another embodiment, the method involves reducing arterial pressure, increasing renal artery blood flow, increasing diastolic cardiac filling, resolving established fibrosis, or suppressing the development of new fibrosis.

[0045] In another embodiment, the present invention provides a kit comprising an effective amount of any one of the above embodiments of a fusion protein, any one of the above embodiments of a polynucleotide, any one of the above embodiments of an expression vector, or any one of the above embodiments of a pharmaceutical composition, and Instructions for use Includes. [Brief explanation of the drawing]

[0046] [Figure 1] Figures 1A-1C show images of electrophoresis and Coomassie blue staining of recombinant relaxin-2 proteins SE001, SE201, SE202, SE203, SE204, SE205, SE206, SE207, and SE301.

[0047] [Figure 2] Figure 2 is a graph showing the size exclusion chromatography of SE301.

[0048] [Figure 3] Figure 3 is a graph showing the determination of Tm of SE301 using differential scanning fluorescence quantification.

[0049] [Figure 4] Figure 4 is a graph showing the activity of two recombinant relaxin-2 proteins, SE001 and SE004, compared to natural relaxin-2.

[0050] [Figure 5] Figure 5 is a graph showing the activity of three recombinant relaxin-2 proteins, SE101, SE102, and SE103, compared to natural relaxin-2.

[0051] [Figure 6] Figure 6 is a graph showing the activity of three recombinant relaxin-2 proteins, SE201, SE202, and SE203, compared to natural relaxin-2.

[0052] [Figure 7] Figure 7 is a graph showing the activity of four recombinant relaxin-2 proteins, SE204, SE205, SE206, and SE207, compared to natural relaxin-2.

[0053] [Figure 8] Figure 8 is a graph showing the activity of one recombinant relaxin-2 protein, SE301, compared to natural relaxin-2.

[0054] [Figure 9] Figure 9 is a graph showing the activity of one recombinant relaxin-2 protein, SE302, compared to natural relaxin-2.

[0055] [Figure 10] Figure 10 is a graph showing the activity of two recombinant relaxin-2 proteins, SE303 and SE304, compared to natural relaxin-2.

[0056] [Figure 11] Figure 11 is a graph showing the activity of one recombinant relaxin-2 protein, SE305, compared to natural relaxin-2.

[0057] [Figure 12] Figure 12 is a graph showing the activity of one recombinant relaxin-2 protein, SE401, compared to natural relaxin-2.

[0058] [Figure 13] Figure 13 is a graph showing pharmacokinetic data for SE301.

[0059] [Figure 14] Figure 14 is a graph showing the activity of two recombinant relaxin-2 proteins, SE501 and SE502, compared to natural relaxin-2.

[0060] [Figure 15] Figure 15 is a graph showing flow cytometry data for SE301. [Modes for carrying out the invention]

[0061] Detailed description of the invention The present invention is at least in part based on the discovery that recombinant single-chain relaxin-2 protein, for example, a fusion protein comprising relaxin B chain, linker, and relaxin A chain, maintains a high level of biological activity compared to natural relaxin-2. In some embodiments, the recombinant single-chain relaxin-2 protein includes an immunoglobulin G constant region (Fc domain) operably linked thereto with little or no reduction in biological activity. Accordingly, novel recombinant relaxin-2 compositions and methods for producing them are disclosed herein. Methods for treating relaxin-2-related disorders or diseases using the compositions of the present invention are also disclosed herein. Recombinant single-chain relaxin-2 proteins according to the present invention have several excellent properties. For example, recombinant single-chain relaxin-2 proteins have improved pharmacokinetics, e.g., a longer circulating half-life, or improved activity, e.g., enhanced maximal activation of RXFP1. Furthermore, producing recombinant single-chain relaxin-2 proteins according to the present invention is simple and cost-effective.

[0062] I. Definition Certain terms are defined first so that the present invention may be more easily understood.

[0063] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have meanings generally understood by those skilled in the art. The meaning and scope of terms shall be clear, but in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions.

[0064] The use of the terms “a,” “an,” and “the,” as well as similar references, relating to describing the present invention (in particular to the following claims), should be interpreted as covering both singular and plural forms (i.e., one or more), unless otherwise indicated herein or unless clearly inconsistent with the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including but not limited to”) unless otherwise noted herein. The enumeration of value ranges herein is intended only as abbreviation for referring individually to each separate value enumerated or falling within its range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were enumerated individually.

[0065] The terms "about" or "approximately" mean within 5% of a given value or range, or more preferably within 1%.

[0066] As used herein, the term “substantially” refers to a qualitative condition indicating the extent or degree of the desired feature or characteristic. Those skilled in the art will understand that biological and chemical phenomena rarely proceed to completion and / or completeness, or to achieve or avoid absolute results, if any. Therefore, in some embodiments herein, the term “substantially” may be used to capture the inherent potential lack of completeness in many biological and chemical phenomena.

[0067] When used herein, “therapeutic dose” is intended to include an amount of a drug or composition sufficient to treat a subject having relaxin-2-related disease when administered to a patient (for example, by ameliorating, improving or maintaining, one or more symptoms of the pre-existing disease or one or more of the disease or its associated comorbidities). “Therapeutic dose” may vary depending on the drug or composition, the method of administration, the disease and its severity, as well as the patient’s medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by relaxin-2, the type of preceding or accompanying treatment, if any, and other individual characteristics of the patient being treated.

[0068] Generally, the term “treatment” or “doing treatment” is defined as the application or administration of a therapeutic agent to a patient, or to a tissue or cell line isolated from a patient, with the aim of curing, healing, reducing, alleviating, modifying, remedying, improving, or influencing a disease, symptoms of a disease, or predisposition to a disease, the patient having the disease, symptoms of a disease, or predisposition to a disease. Therefore, treatment may include suppressing, inhibiting, preventing, treating, or a combination thereof. Treatment, in particular, refers to increasing the time to progression of persistence, accelerating remission, inducing remission, improving remission, accelerating recovery, increasing the effectiveness of an alternative treatment and / or reducing resistance to an alternative treatment, or a combination thereof.

[0069] "Suppressing" or "inhibiting" means, in particular, delaying the onset of symptoms, preventing disease relapses, reducing the number or frequency of relapse episodes, increasing the incubation period between symptomatic episodes, reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the incubation period of symptoms, improving symptoms, reducing secondary symptoms, reducing secondary infections, extending patient survival, or a combination thereof.

[0070] In one embodiment, the symptoms are primary, while in another embodiment, the symptoms are secondary.

[0071] "Primary" refers to symptoms that are a direct result of a disorder, such as diabetes, while "secondary" refers to symptoms that originate from or result from a primary cause. Symptoms can be any manifestation of a disease or pathological condition.

[0072] Therefore, as used herein, the terms “treatment” or “to treat” include any administration of the compositions described herein and include: (i) preventing the development of disease in a subject who may be predisposed to the disease but has not yet experienced or shown any symptoms of the disease; (ii) inhibiting the disease in a subject who is experiencing or showing symptoms of the disease (i.e., halting further development of the pathology and / or symptoms); or (iii) improving the disease in a subject who is experiencing or showing symptoms of the disease (i.e., reversing the pathology and / or symptoms).

[0073] "Treatment," "prevention," or "improvement" of a disease or disorder means delaying or preventing the onset of such disease or disorder, reversing, reducing, improving, inhibiting, slowing, or stopping the progression, exacerbation, or worsening of any condition associated with such disease or disorder. In one embodiment, the symptoms of the disease or disorder are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0074] The effectiveness of the treatment is determined in relation to any known method for diagnosing the disorder. Alleviation of one or more symptoms of the disorder indicates that the composition provides a clinical benefit. Any of the above treatment methods may be applied to any suitable subject, including, for example, mammals such as dogs, cats, cattle, horses, rabbits, monkeys, and most preferably humans.

[0075] As used herein, the term “Subject” includes any subject that may benefit from being administered the hydrogel or implantable drug delivery device of the present invention. The term “Subject” includes animals, e.g., humans and primates, e.g., chimpanzees, monkeys, etc., vertebrates, amphibians, fish, mammals, and non-human animals. In one embodiment of the present invention, the subject is a human.

[0076] The term "subject" includes agriculturally productive livestock, such as cattle, sheep, goats, horses, pigs, donkeys, camels, buffalo, rabbits, chickens, turkeys, ducks, geese, and bees; as well as domestic pets, such as dogs, cats, caged birds, and ornamental fish; and also so-called test animals, such as hamsters, guinea pigs, rats, and mice.

[0077] II. Compositions of the present invention A. Relaxin-2 Human relaxin-2 is a peptide hormone with multiple multifaceted effects. Initially considered merely a reproductive hormone involved in promoting neonatal delivery, more recent studies have demonstrated that relaxin-2 plays a crucial role in inflammatory and matrix remodeling processes and possesses potent vasodilatory, angiogenic, and other cardioprotective effects. The vasodilatory effect of relaxin-2 is thought to be involved in the vasoconstrictive action of endothelin-1 and angiotensin II, as well as the promotion of nitric oxide and gelatinase, matrix metalloproteinase-2, and matrix metalloproteinase-9. This leads to systemic and renal vasodilation, increased arterial compliance, and other vascular changes. These findings have led to the evaluation of relaxin-2 as a drug for the treatment of patients with acute heart failure (AHF) and other diseases. Furthermore, relaxin-2's matrix remodeling action enhances its reputation as a rapidly acting yet safe antifibrotic agent, which is further supported by its ability to successfully inhibit and / or reverse fibrosis in all preclinical models of the experimental disease evaluated to date.

[0078] The action of relaxin-2 is thought to be mediated through its native receptor RXFP1 (originally named LGR7), a leucine-rich repeat-containing G protein-coupled receptor characterized by an unusually large external domain. Human relaxin-2 can also bind to and activate the related receptor RXFP2, the native receptor for insulin-like peptide 3 (INSL3), suggesting that potential cross-reactivity may be related to its diverse effects.

[0079] Natural relaxin-2 has an insulin-like core structure comprising two chains (relaxin A and relaxin B) and three disulfide bonds. As used herein, the term “natural relaxin-2” refers to any relaxin-2 produced naturally in the subject, e.g., human relaxin-2. Naturally occurring orthologues of human relaxin-2, e.g., mouse relaxin-1, are also intended as natural relaxin-2 in the present invention. Natural relaxin-2 also includes relaxin-2 produced using any recombinant method, which has substantially the same structure as naturally occurring relaxin-2, i.e., primary, secondary, and tertiary structures, as well as substantially the same biological activity, e.g., binding to RXFP1.

[0080] Human relaxin A and B chains are derived from a single gene product (GenBank accession number CAA25460.1). The human precursor relaxin-2 protein is typically proteolytically degraded post-translation to yield the mature A / B forms. In some embodiments, exemplary human natural relaxin-A has the amino acid sequence shown in SEQ ID NO: 1 (QLYSALANKCCHVGCTKRSLARFC). In some embodiments, exemplary human natural relaxin-B has the amino acid sequence shown in SEQ ID NO: 2 (DSWMEEVIKLCGRELVRAQIAICGMSTWS). The mouse counterpart of human relaxin-2 is mouse relaxin-1, which is similarly derived from a single precursor protein (GenBank accession number CAA81611.1). In some embodiments, exemplary mouse natural relaxin-A has the amino acid sequence shown in SEQ ID NO: 3 (ESGGLMSQQCCHVGCSRRSIAKLYC). In some embodiments, exemplary mouse native relaxin-B has the amino acid sequence shown in SEQ ID NO: 4 (RVSEEWMDGFIRMCGREYARELIKICGASVGRLAL).

[0081] B. Recombinant relaxin-2 1. Relaxin A and Relaxin B The present invention provides recombinant relaxin-2 proteins, e.g., recombinant human relaxin-2, that have higher levels of biological activity compared to natural relaxin-2 while allowing modification for an enhanced serum half-life. The term “recombinant” indicates that a material (e.g., nucleic acid or polypeptide) has been artificially or synthetically (i.e., non-naturally) modified by human intervention. Modification may be carried out on material that is in or extracted from its natural environment or state. For example, “recombinant nucleic acid” is a nucleic acid produced by recombining nucleic acids, e.g., during cloning, DNA shuffling or other well-known molecular biological procedures. A “recombinant DNA molecule” consists of DNA segments linked together by such molecular biological techniques. The terms “recombinant protein” or “recombinant polypeptide,” as used herein, refer to a protein molecule expressed using a recombinant DNA molecule. A “recombinant host cell” is a cell that contains and / or expresses recombinant nucleic acid. The terms recombinant relaxin-2 and engineered relaxin-2 may be used interchangeably.

[0082] Recombinant relaxin-2 proteins include natural relaxin A, e.g., human relaxin A or its variants, and natural relaxin B, e.g., human relaxin B or its variants. As used herein, “relaxin A,” “relaxin B,” “relaxin-2,” and other proteins or peptides refer to natural or variant proteins or peptides when the name of the protein or peptide is used independently of the terms “natural” or “variant.” As used herein, the term “variant” refers to a protein or peptide derived from one or more amino acid insertions, substitutions, or deletions from a precursor protein or peptide (e.g., a “parent” protein or peptide). In certain embodiments, a variant includes at least one modification, including a change in charge compared to the precursor protein or peptide. In certain preferred embodiments, the precursor protein or peptide is a parent protein or peptide, which is natural or a peptide.

[0083] In certain embodiments, a variant protein or peptide, for example, variant human relaxin-A or relaxin-B, has at least about 85% sequence identity with the native protein or peptide, e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity. The term “sequence identity,” as used herein, refers to the comparison between pairs of nucleic acids or amino acid molecules, i.e., the relationship between two amino acid sequences or two nucleotide sequences. Generally, sequences are aligned to obtain a top-level match. Methods for determining sequence identity are known and can be determined by commercially available computer programs that can calculate the percentage of identity between two or more sequences. A typical example of such a computer program is CLUSTAL. As an example, a polynucleotide having a nucleotide sequence with at least, for example, 90% sequence identity to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to that of the reference nucleotide sequence, except that the polynucleotide sequence may contain up to 10 point mutations on average per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 90% identical to the reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 10% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between those terminal positions, and may be scattered individually between nucleotides in the reference sequence or in one or more consecutive groups within the reference sequence. Similarly, a polypeptide having an amino acid sequence that is at least, for example, 90% sequence-identical to a reference amino acid sequence is intended to have an amino acid sequence identical to the reference sequence, except that the polypeptide sequence may contain up to 10 amino acid changes on average per 100 amino acids of the reference amino acid sequence.In other words, to obtain a polypeptide having an amino acid sequence that is at least 90% identical to a reference amino acid sequence, up to 10% of the amino acid residues in the reference sequence may be deleted or replaced with other amino acids, or up to 10% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These modifications to the reference sequence may occur at the amino or carboxyl terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, and may be scattered individually between residues in the reference sequence or in one or more consecutive groups within the reference sequence.

[0084] A preferred method for determining identity is designed to give the greatest possible match between the sequences being tested. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid. Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wis., USA), BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLASTManual, Altschul et al. NCB / NLM / NIH Bethesda, Md., USA; Altschul et al., above). The well-known Smith-Waterman algorithm can also be used to determine identity. For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis., USA), two proteins whose percentage sequence identity is determined are aligned for the optimal matching of their respective amino acids ("matched spans" determined by the algorithm). A gap start penalty (which is calculated as 3 times the average diagonal; the "average diagonal" is the average of the diagonals of the comparison matrix used; the "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (which is typically 1 / 10 of the gap start penalty), as well as a comparison matrix, e.g., PAM 250 or BLOSUM 62, are used in conjunction with the algorithm.Standard comparison matrices are also used by the algorithm (see Dayhoffet al., 1978, Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, (1978) for the PAM 250 comparison matrix, and Henikoffet al., 1992, Proc. Natl. Acad. Sci USA 89: 10915-10919 for the BLOSUM 62 comparison matrix).

[0085] In certain embodiments, variant human relaxin A includes substitution Q1D (SEQ ID NO: 5). As used herein, the form "L1NL2" indicates a substitution at position N. "L1" is a single letter symbol indicating an amino acid at position N of a native protein or peptide. "N" is a number indicating the position of the substitution, counting from the first amino acid of a native protein or peptide, for example, the first amino acid of human natural relaxin A having the sequence shown in SEQ ID NO: 1, or the first amino acid of human natural relaxin B having the sequence shown in SEQ ID NO: 2. "L2" is a single letter symbol indicating the amino acid that replaces L1.

[0086] In certain embodiments, variant human relaxin B comprises a truncated peptide (SEQ ID NO: 6), in which the first five amino acids (DSWME (SEQ ID NO: 59)) are deleted from human natural relaxin B. In certain embodiments, variant human relaxin B comprises one or more substitutions selected from the group consisting of M4K, R13A, R13D, R17A, R17D, I20A, I20D, M25K, and W28A.

[0087] In a particular embodiment, variant human relaxin B is selected from the group consisting of SEQ ID NOs: 7, 8, 9, and 10.

[0088] In some embodiments, variant human relaxin B has the sequences shown in SEQ ID NOs: 11, 12, and 13.

[0089] In some embodiments, the recombinant relaxin-2 protein is a single-chain protein, e.g., a fusion protein. In a single-chain recombinant relaxin-2 protein, relaxin A and relaxin B of recombinant relaxin-2 are operably linked via a linker, e.g., covalently linked. The terms “operably linked,” “in an operable combination,” and “in an operable order” refer to the linking of nucleic acid sequences in such a manner that a nucleic acid molecule is produced that can direct the transcription of a given gene and / or the synthesis of a desired protein molecule. The terms also refer to the linking of amino acid sequences in such a manner that a functional protein is produced. In certain embodiments, relaxin A, relaxin B, and the linker are covalently linked in the following operable order: Relaxin B-linker-relaxin A.

[0090] In certain embodiments, recombinant relaxin-2 includes a linker or a variant thereof having the amino acid sequence DAASSHSHSSAR (SEQ ID NO: 14). In some embodiments, the linker has the sequence DAASSHSHSSAA (SEQ ID NO: 15). In some embodiments, recombinant relaxin-2 includes a linker or a variant thereof having the amino acid sequence DAAGANANAGAR (SEQ ID NO: 16). The linker having the amino acid sequence DAASSHSHSSAA (SEQ ID NO: 15) has been reported in a publication on methods for producing natural relaxin-3 (Luo et al., A simple approach for the preparation of mature humanrelaxin-3, Peptides, 2010).

[0091] 2. Linker In some embodiments, recombinant relaxin-2 includes a linker. The linker covalently links at least two components of recombinant relaxin-2 in an operable order. The term “linker,” as used herein, refers to a chemical group or molecule that connects two molecules or parts (e.g., two peptides, e.g., relaxin A and relaxin B). Typically, a linker is located between or adjacent to two groups, molecules, or other parts, and is connected to one another via covalent bonds, thus the two are linked. In some embodiments, the linker includes one or more amino acids (e.g., a peptide or protein). In some embodiments, the linker includes a cleavable site. For example, the linker includes a peptide that can be cleaved by the HRV3C protease. In some embodiments, the linker is any stretch of amino acids, and is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50 or 51 or more amino acids.

[0092] In some embodiments, the peptide linker comprises a tripeptide Gly-Gly-Ser repeat (repeat) or a variant thereof, for example, a sequence (GGS) n The formula includes, where n is at least 1, 2, 3, 4, 5, 6, or 7, and represents 8, 9, 10 or 11 or more repeats. In some embodiments, the linker includes sequence (GGS)4 (sequence number 17). In some embodiments, the peptide linker includes a repeat (repeat) or variant of the tripeptide Gly-Ser-Gly, for example, sequence (GSG) nThe formula includes, where n is at least 1, 2, 3, 4, 5, 6, 7, and represents 8, 9, 10 or 11 or more repeats. In some embodiments, the linker includes sequence (GSG)2 (SEQ ID NO: 57). In some embodiments, the peptide linker includes a tripeptide Ala-Ala-Ala repeat (repeat), for example, sequence (AAA) n The formula includes, where n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or more repeats. In some embodiments, the peptide linker includes a tripeptide Pro-Pro-Pro repeat (repeat), for example, a sequence (PPP) n The formula includes, where n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or more repeats.

[0093] 3. Detectable signs In some embodiments, recombinant relaxin-2 of the present invention further includes a detectable label, such as an enzymatic label, a fluorescent label, or an affinity label, to enable the detection and isolation of the protein. Such detectable labels may include, but are not limited to, polyhistidine tags, immunoglobulin Fc tags, myc tags, HA tags, glutathione S-transferase, fluorescent tags, or variants thereof. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase. In some embodiments, the detectable label is a polyhistidine tag, such as 6×His (SEQ ID NO: 18) or a variant thereof, or 8×His (SEQ ID NO: 19) or a variant thereof. In some embodiments, a detectable label, such as a protein C tag, may be used for antibody affinity chromatography or detection. Examples of protein C tags include, but are not limited to, a peptide having the amino acid sequence EDQVDPRLIDGKGS (SEQ ID NO: 24) or a variant thereof.

[0094] In some embodiments, the detectable label is an immunoglobulin Fc domain, e.g., the IgG1 Fc domain (SEQ ID NO: 20) or a variant thereof, e.g., the IgG1 Fc domain containing the N77Q substitution (SEQ ID NO: 21). The detectable label may also have other functions. For example, an immunoglobulin Fc domain tag may increase the half-life of recombinant relaxin-2 in a subject. The Fc fragment also promotes immunoeffector functions, including complement activation and cytotoxicity via Fc gamma receptor binding. In some embodiments, the Fc fragment of recombinant single-chain relaxin-2 may contain one or more substitutions that attenuate immunoeffector function, e.g., the substitution of Asn297 with Gln in the IgG1 Fc region (referred to as N77Q in the recombinant relaxin-2 protein). Exemplary effector-attenuating substitutions of the Fc fragment include, but are not limited to, N297G(NG) and D265A, N297G, L234A, L235A, and P329G. An exemplary effector-attenuating substitution is described in Lo et al., Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice, J. Biol. Chem., 292, 3900-08 (2017), which is thus incorporated herein by reference.

[0095] In certain embodiments, the detectable label is serum albumin, for example, human serum albumin or mouse serum albumin. Examples of mouse serum albumin include the protein or a variant thereof having the amino acid sequence of SEQ ID NO: 22.

[0096] The detectable label can be operably coupled to the N-terminus or C-terminus of relaxin A or relaxin B, for example, by covalent bonding. The detectable label can be operably coupled directly to relaxin A or relaxin B. The detectable label can also be operably coupled to relaxin A or relaxin B via a linker, for example, a GGS or GSG linker.

[0097] The detectable label can also be operably linked to relaxin A or relaxin B via a cleavable linker, such as a protease-cleavable peptide. Exemplary proteases that specifically cleave the cleavable linker include, but are not limited to, thrombin, HRV3C protease, factor Xa, and TEV protease. Examples of HRV3C sites include, but are not limited to, peptides or variants having the amino acid sequence LEVLFQGP (SEQ ID NO: 23) or GSLEVLFQGPG (SEQ ID NO: 58) or peptides or variants having the amino acid sequence LVPRGS (SEQ ID NO: 56). Examples of thrombin sites include, but are not limited to, peptides or variants having the amino acid sequence LVPRGS (SEQ ID NO: 56).

[0098] 4. Biological activity of recombinant relaxin-2 In some embodiments, recombinant relaxin-2 of the present invention has a higher level of bioactivity compared to natural relaxin-2. For example, recombinant relaxin-2 may have at least about 50% to at least about twice the bioactivity of natural relaxin-2. In some embodiments, recombinant relaxin-2 has at least about 50%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about twice the bioactivity of natural relaxin-2. In certain embodiments, recombinant relaxin-2 has more than about twice the bioactivity of natural relaxin-2.

[0099] The biological activity may be any biological activity of native relaxin-2. For example, the biological activity may be the ability of recombinant relaxin-2 to bind to RXFP1, the receptor for native relaxin-2. The binding of relaxin-2 to RXFP1 can be measured by any well-known method in the art, such as radioligand binding. In some embodiments, recombinant relaxin-2 binds to RXFP1 on the cell surface.

[0100] In some embodiments, the biological activity may be the ability of recombinant relaxin-2 to activate RXFP1 on the cell surface. While we do not wish to be bound by any theory, the present invention is at least in part based on the surprising discovery that some exemplary recombinant relaxin-2 proteins exhibit higher maximal activation of RXFP1 compared to native double-chain relaxin-2. Activation of RXFP1 by recombinant relaxin-2 may be determined by an increase in cAMP, using any method known in the art, such as measuring the activity of a cAMP-driven reporting gene, e.g., β-galactosidase. Activation of RXFP1 by recombinant relaxin-2 in cells may also be determined by measuring the expression of certain genes, e.g., angiogenic factors, e.g., VEGF, or MMP, using methods known in the art. In some embodiments, the biological activity may be the physiological activity, biochemical activity, or any other effect-inducing activity of relaxin-2. Exemplary biological activities include, but are not limited to, vasodilation, collagen degradation, angiogenesis, decreased arterial blood pressure, increased renal artery blood flow, increased diastolic cardiac filling, resolution of established fibrosis, and suppression of the development of new fibrosis.

[0101] In certain embodiments, the present invention provides a protein or peptide, such as recombinant relaxin-2, that has a high level of activity, for example, in one embodiment, at least about 50% to about 2 times the bioactivity of natural relaxin-2, but in another embodiment, has a low level of activity, for example, less than 50% of the bioactivity of natural relaxin-2. In some embodiments, recombinant relaxin-2 has, in one embodiment, about 50%, about 40%, about 30%, about 20%, about 10%, or less than about 5% of the bioactivity of natural relaxin-2. For example, recombinant relaxin-2 may bind to RXFP1 with high affinity, for example, at least about 50% to about 2 times the affinity, but have low activity in activating RXFP1, for example, less than 50% of the ability to activate RXFP1. Such recombinant relaxin-2 may be a dominant-negative variant that reduces relaxin-2 activity in the target where it is needed.

[0102] In some embodiments, the present invention provides a protein or peptide having an improved pharmacokinetic profile, such as recombinant relaxin-2. While not wishing to be bound by any theory, the present invention is at least partly based on the surprising discovery that some exemplary recombinant relaxin-2 proteins exhibit a much longer circulating half-life compared to natural double-chain relaxin-2. For example, the recombinant single-chain relaxin-2 of the present invention may have a circulating half-life longer than about 5 hours, e.g., longer than about 10 hours, longer than about 20 hours, longer than about 50 hours, longer than about 75 hours, longer than about 100 hours, longer than about 125 hours, or longer than about 150 hours. Intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of the present invention. In certain embodiments, the recombinant single-chain relaxin-2 of the present invention has a circulating half-life of about 130 hours. Surprisingly, the single-chain relaxin-2 of the present invention may have a longer circulating half-life than natural double-chain relaxin-2. For example, the circulating half-life of natural double-chain relaxin-2 may be less than approximately 5 hours (see, for example, Chen et al., The Pharmacokinetics of Recombinant Human Relaxin in Non-Pregnant Women after Intravenous, Intravaginal, and Intracervical Administration, Pharm. Res. 10: 834038 (1993), which is incorporated herein by reference).

[0103] As used herein, "circulating half-life" refers to the time it takes for the plasma concentration of a drug, such as natural relaxin-2 or recombinant single-chain relaxin-2, to halve its steady state when circulating in the whole blood of an organism. The circulating half-life of a particular drug may vary depending on a number of factors, including but not limited to the dosage, formulation, and / or route of administration of the drug. Those skilled in the art can determine the circulating half-life of a drug, such as a protein, such as recombinant relaxin-2, using methods well known in the art, for example, the method described in Example 3 or above.

[0104] 5. Nucleic acid molecules encoding recombinant relaxin-2 The present invention also provides nucleic acid molecules encoding any of the proteins or peptides described herein, for example, recombinant relaxin-2. In some embodiments, the nucleic acid molecule of the present invention is a DNA molecule. In some embodiments, the nucleic acid molecule of the present invention is an RNA molecule.

[0105] Individual strands (one or more) of a DNA molecule encoding either a protein or a peptide, such as recombinant relaxin-2, can be transcribed from a promoter in an expression vector. If two separate proteins or peptides are expressed to produce, for example, relaxin A and relaxin B, two separate expression vectors can be co-introduced into target cells (e.g., by transfection or infection).

[0106] Expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably vertebrate cells, may be used to produce the recombinant relaxin-2 described herein. The production and purification of recombinant proteins are well known in the art, such as the methods described in "Molecular Cloning: A Laboratory Manual", Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press. While we do not wish to be bound by any theory, the present invention is at least in part based on the surprising discovery that some exemplary recombinant relaxin-2 proteins can be produced in high yield (see, for example, Example 3, Table 2).

[0107] The proteins described herein, for example, nucleic acids encoding recombinant relaxin-2, can be incorporated into a vector.

[0108] The expression of natural or synthetic nucleic acids is typically achieved by operably ligating the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. A typical cloning vector contains transcription and translation terminators, start sequences, and promoters useful for the expression of the desired nucleic acid sequence.

[0109] Further promoter elements, such as enhancement sequences, regulate the frequency of transcription initiation. Typically, these are located in a region 30–110 bp upstream of the initiation site, although it has recently been shown that some promoters also include functional elements downstream of the initiation site. The spacing between promoter elements is often flexible, and as a result, promoter function is preserved even if the elements are inverted or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased up to 50 bp, after which activity begins to decline. Depending on the promoter, individual elements appear to be able to function to activate transcription either cooperatively or independently.

[0110] One example of a suitable promoter is the very early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably ligated to it. Another example of a suitable promoter is elongation growth factor-la (EF-la). However, other constitutive promoter sequences may also be used, including but not limited to the monkey virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long-term repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, Rous sarcoma virus promoter, and human gene promoters such as, for example, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0111] Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also intended as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence to which it is operably linked when such expression is desired, and turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothioneine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0112] The expression vector may also include either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and reporter genes may be flanked by appropriate transcriptional regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0113] Reporter genes can be used to identify potentially transfected cells and to evaluate the functionality of transcriptional regulatory sequences. Generally, a reporter gene is a gene encoding a polypeptide that is not present in the recipient source and is not expressed by it, but whose expression is indicated by several readily detectable characteristics, such as enzymatic activity. Reporter gene expression is assayed at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct with the smallest 5' facile region exhibiting the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0114] In certain embodiments, the expression vector is a plasmid vector, such as a prokaryotic plasmid vector or a eukaryotic plasmid vector. Exemplary prokaryotic plasmid vectors include, but are not limited to, the pET expression series plasmids and the pGEX expression series plasmids. Exemplary eukaryotic expression plasmids include, but are not limited to, yeast expression plasmids, plant cell expression plasmids, insect cell expression plasmids, bird cell expression plasmids, and mammalian expression plasmids. Exemplary mammalian expression plasmids include, but are not limited to, pRc / CMV, pcDNA3.1, pcDNA4, pcDNA6, pGene / V5, pFUSE-hIgG1-Fc2, pTT, and pED.dC. In certain embodiments, the expression plasmid includes one or more inductive elements for controlling the expression of recombinant single-chain relaxin-2. Exemplary plasmids containing inductive elements include, but are not limited to, pcDNA3.1-Zeo-tetO, a modified pcDNA3.1 plasmid for tetracycline-inducible protein expression and Zeocin antibiotic resistance.

[0115] In certain embodiments, the expression vector of the present invention can be delivered to a host cell for the in vitro production of a protein or peptide, for example, recombinant relaxin-2. The present invention also provides recombinant cells comprising a nucleic acid molecule encoding any of the proteins or peptides of the present invention, or a vector comprising such a nucleic acid molecule. Methods for introducing nucleic acid molecules into cells, including but not limited to transformation, transfection, viral infection, or electroporation, are well known in the art.

[0116] Examples of host cells include, but are not limited to, prokaryotic and eukaryotic cells selected from any kingdom of organism. Examples of eukaryotic cells include, but are not limited to, protists, fungi, plant and animal cells. Non-limiting examples of host cells include, but are not limited to, the prokaryotic cell E. coli; mammalian cell lines CHO, HEK 293, HeLa, Expi293F and COS; insect cell lines Spodoptera frugiperda Sf9 and Trichoplusia ni HighFive; and fungal cell Saccharomyces cerevisiae.

[0117] In certain embodiments, expression vectors may be used to deliver and / or express in vivo a gene encoding any protein or peptide of the present invention into cells for gene therapy. Vectors containing retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow for long-term stable integration of the transgene and its expansion and proliferation in daughter cells. Examples of vectors include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors. Expression vectors may be supplied to cells in the form of viral vectors. Viral vector technology is well known in the art and is described in various virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector includes a functional origin of replication, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers in at least one organism.

[0118] Viral vector systems that may be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentivirus vectors and Moloney's mouse leukemia virus; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, e.g., orthopox, e.g., vaccinia virus vector, or tripox, e.g., canary pox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not be incorporated into the cell genome. The constructs may optionally contain viral sequences for transfection. Alternatively, the constructs may be incorporated into episomal replication-capable vectors, e.g., EPV and EBV vectors. Constructs for the recombinant expression of disruptors generally require regulatory elements, such as promoters and enhancers, to ensure the expression of the disruptor in target cells. Other aspects of vectors and constructs are known in the art.

[0119] Methods for delivering viral vectors into cells in vivo are well known in the art. Viral vectors may be administered by any means known in the art, including but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration.

[0120] RNA molecules comprising genes encoding any protein or peptide of the present invention may be used for gene therapy to deliver and / or express genes in vivo. Methods for formulating RNA molecule genes and delivering them in vivo are well known in the art, such as those described in U.S. Patent Application Publication 2016 / 0038612A1, which is thus incorporated herein by reference.

[0121] C. Pharmaceutical Compositions and Administration The present invention provides pharmaceutical compositions comprising the protein or peptide of the present invention, for example, recombinant relaxin-2 or nucleic acid molecules or expression vectors. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, resistance, etc. Numerous suitable formulations can be found in the formula collection known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LlPOFECTIN®, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) JPharm Sci Technol 52:238-311.

[0122] The dose of the protein, peptide, or nucleic acid molecule of the present invention administered to a patient may vary depending on the patient's age and size, target disease, condition, route of administration, etc. Preferred doses are typically calculated according to body weight or body surface area. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective dosages and schedules for administering recombinant relaxin-2 may be determined experimentally; for example, patient progression may be monitored by periodic assessments, and the dose may be adjusted accordingly. Furthermore, interspecies scaling of dosages may be carried out using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0123] Various delivery systems are known and may be used to administer the pharmaceutical composition of the present invention, such as liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. BioI. Chem. 262:4429-4432). Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example, by injection or bolus injection, by absorption via the epithelium or inner layers of the skin mucosa (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or topical.

[0124] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, with respect to subcutaneous delivery, a pen delivery device is readily applicable when delivering the pharmaceutical composition of the present invention. Such a pen delivery device may be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0125] Numerous reusable pen and auto-injector delivery devices have applications in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, to name a few, AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, and OPTIPEN®. Examples of disposable pen delivery devices having applications in subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, STARLET® and OPTICLIK® (Sanofi-Aventis, Frankfurt, Germany).

[0126] In certain circumstances, pharmaceutical compositions may be delivered by controlled-release systems. In one embodiment, a pump may be used (see Langer, above; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida). In yet another embodiment, the controlled-release system may be placed proximal to the target of the composition and therefore require only a small fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, above, vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0127] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, as well as intravenous infusion. These injectable preparations may be prepared by known methods. For example, an injectable preparation may be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include saline, isotonic solutions containing glucose and other adjuvants, which may be used in combination with a suitable solubilizer, such as alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), or nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil, soybean oil, etc., which may be used in combination with a solubilizer, such as benzyl benzoate or benzyl alcohol. The injectable preparations thus prepared are preferably filled into suitable ampoules.

[0128] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared into dosage forms in unit doses suitable for adapting the dosage of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in unit doses; in particular, it is preferable that the antibody be contained in about 5 to about 100 mg in the injection form and about 10 to about 250 mg in the other dosage forms.

[0129] III. Therapeutic use of recombinant relaxin-2 A. Method using recombinant relaxin-2 The present invention includes a method comprising the step of administering a therapeutic composition comprising the recombinant relaxin-2 of the present invention to a subject in need thereof. The therapeutic composition may comprise any of the proteins or peptides disclosed herein and a pharmaceutically acceptable carrier or diluent. Where used herein, the expression “subject in need thereof” means a human or non-human animal exhibiting one or more symptoms or signs of a relaxin-2-related disorder or disease, or otherwise benefiting from an increase or decrease in relaxin-2 activity. The proteins or peptides of the present invention (and therapeutic compositions comprising them) are useful, among other things, for treating any disease or disorder in which activation or deactivation of RXFP1 is beneficial.

[0130] In certain embodiments, the present invention provides a method for activating RXFP1 on a cell surface, comprising the step of administering an effective amount of the present invention's protein or peptide, for example recombinant relaxin-2, to a subject requiring it, thereby activating RXFP1 on the cell surface. Activation of RXFP1 on the cell surface may result in cellular responses including, but not limited to, increased cAMP levels, vasodilation, expression of angiogenic factors including VEGF, expression of MMPs, and collagen degradation. In some embodiments, the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts.

[0131] In some embodiments, the present invention provides methods for treating various relaxin-2 related diseases. As used herein, the term “relaxin-2 related disease” refers to a disease or disorder caused by or associated with relaxin-2 protein production or relaxin-2 protein activity. The term “relaxin-2 related disease” includes diseases, disorders or conditions that benefit from increased relaxin-2 protein activity. Non-limiting examples of relaxin-2 related diseases include, for example, kidney diseases including but not limited to focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, and hepatorenal syndrome; fibrotic diseases including but not limited to scleroderma, idiopathic pulmonary fibrosis, renal fibrosis, cardiac fibrosis, and NASH; and cardiovascular diseases including dilated cardiomyopathy, diastolic heart failure, pulmonary arterial hypertension, chronic heart failure, acute heart failure, congestive heart failure, coronary artery disease, hypertension, and pre-eclampsia. Further details regarding the signs and symptoms of various diseases or conditions are provided herein and are well known in the art.

[0132] Administration of compositions according to the method of the present invention may result in a reduction of the severity, signs, symptoms, or markers of relaxin-2-related disease or disorder in patients with such disease or disorder. In this context, "reduction" means a statistically significant decrease at such a level. The reduction (absolute reduction, or reduction of the difference between the elevated level and the normal level in the subject) may be, for example, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the detection level of the assay used.

[0133] B. Combination Therapies and Formulations The present invention includes compositions and therapeutic formulations comprising, in combination with one or more further therapeutically active components, any of the exemplary proteins or peptides described herein, for example, recombinant relaxin-2 protein or nucleic acid molecules, as well as methods of treatment comprising the step of administering such combination to a subject in need.

[0134] Exemplary further therapeutic agents include any therapeutic agent that may be used for the treatment of any relaxin-2 related disorder described herein. Exemplary further therapeutic agents that may be combined with or administered in combination with the recombinant relaxin-2 protein or nucleic acid molecule of the present invention include angiotensin II receptor blockers, e.g., azilsartan, candesartan, eprosartan, losartan; ACE inhibitors, e.g., lisinopril, benazepril, captopril, enalapril, moexipril, perindopril, quinapril, trandolapril; calcium channel blockers, e.g., amlodipine, amlodipine and benazepril, amlodipine and valsartan, diltiaze This includes, but is not limited to, felodipine, isradipine, nicardipine, nimodipine, nisoldipine, verapamil, or diuretics such as chlorthalidone, hydrochlorothiazide, metrazone, indapamide, torsemide, furosemide, bumetanide, amiloride, triamterene, spironolactone, eplerenone, aldosterone antagonists such as spironolactone, eplerenone, digoxin such as lanoxine, and beta-blockers such as carvedilol, metoprolol, and bisoprolol.

[0135] In some embodiments, further therapeutic agents are drugs for fibrosis, including but not limited to small molecule drugs and antibodies. Exemplary anti-fibrotic drugs include, but are not limited to, TGF-β inhibitors, e.g., small molecules such as hydronidone and distiertide, or antibodies, e.g., fresolimmab, PDGF or VEGF antagonists, e.g., small molecules such as imatinib and nilotinib, or any drugs that target extracellular factors involved in the pathogenesis of fibrosis. A description of exemplary drugs for fibrosis can be found, for example, in Li et al., Drugs and Targets in Fibrosis, Frontiers in Pharm., 8:Article 855 (2007), which is thus incorporated herein by reference.

[0136] Further therapeutically active components(s) may be administered prior to, concurrently with, or immediately after the administration of the antigen-binding molecule of the present invention (for the purposes of this disclosure, such administration regimens are considered to be administrations of recombinant relaxin-2 "in combination with" further therapeutically active components).

[0137] The present invention comprises a pharmaceutical composition in which recombinant relaxin-2 of the present invention is co-formulated with one or more of the further therapeutically active components described elsewhere in this specification.

[0138] C. Dosage regimen According to certain embodiments of the present invention, multiple doses of the protein or peptide of the present invention, for example recombinant relaxin-2, may be administered to a subject over a defined period of time. A method according to this aspect of the present invention includes the step of sequentially administering multiple doses of recombinant relaxin-2 of the present invention to a subject. As used herein, “sequentially administering” means that each dose of the protein or peptide of the present invention is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., several hours, several days, several weeks, or several months). The present invention includes a method comprising the step of sequentially administering to a patient a single initial dose of recombinant relaxin-2, one or more subsequent secondary doses of recombinant relaxin-2, and optionally one or more subsequent tertiary doses of recombinant relaxin-2.

[0139] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the chronological order of administration of recombinant relaxin-2 in the present invention. Thus, the “initial dose” is the dose administered at the start of the treatment regimen (also called the “baseline dose”); the “secondary dose” is the dose administered after the initial dose; and the “tertiary dose” is the dose administered after the secondary dose. While the initial, secondary, and tertiary doses may all contain the same amount of recombinant relaxin-2, they may generally differ from one another in terms of the frequency of administration. However, in certain embodiments, the amounts of recombinant relaxin-2 contained in the initial, secondary, and / or tertiary doses may differ from one another during the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, two or more doses (e.g., two, three, four, or five) may be administered as a “loading dose” at the start of the treatment regimen, followed by subsequent doses administered at a lower frequency (e.g., “maintenance doses”).

[0140] In an exemplary embodiment of the present invention, each secondary and / or tertiary dose is 1 to 26 times the preceding dose (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13 and It is administered after 1 / 2, 14, 14 and 1 / 2 weeks, 15, 15 and 1 / 2 weeks, 16, 16 and 1 / 2 weeks, 17, 17 and 1 / 2 weeks, 18, 18 and 1 / 2 weeks, 19, 19 and 1 / 2 weeks, 20, 20 and 1 / 2 weeks, 21, 21 and 1 / 2 weeks, 22, 22 and 1 / 2 weeks, 23, 23 and 1 / 2 weeks, 24, 24 and 1 / 2 weeks, 25, 25 and 1 / 2 weeks, 26, 26 and 1 / 2 weeks or longer. The phrase “immediately preceding dose,” as used herein, means the dose of recombinant relaxin-2 administered to the patient in a sequence of multiple doses, without any intervening doses, immediately before the next dose in the sequence.

[0141] A method according to this aspect of the present invention may include the step of administering to a patient any number of secondary and / or tertiary doses of a protein or peptide (e.g., recombinant relaxin-2). For example, in one particular embodiment, only a single secondary dose is administered to the patient. In another embodiment, two or more (e.g., two, three, four, five, six, seven, eight or more) secondary doses are administered to the patient. Similarly, in one particular embodiment, only a single tertiary dose is administered to the patient. In another embodiment, two or more (e.g., two, three, four, five, six, seven, eight or more) tertiary doses are administered to the patient.

[0142] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency at which secondary and / or tertiary doses are administered to the patient may vary throughout the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment, depending on the individual patient's needs after clinical examinations.

[0143] In one embodiment, recombinant relaxin-2 is administered to a subject as a body weight-based dose. A "body weight-based dose" (e.g., a dose in mg / kg) is a dose of protein or peptide that varies depending on the subject's body weight.

[0144] In another embodiment, a protein or peptide, such as recombinant relaxin-2, is administered to a subject as a fixed dose. “Fixed dose” (e.g., dose in mg) means that one dose of the protein or peptide, such as recombinant relaxin-2, is used for all subjects, regardless of any specific subject-related factors, such as body weight. In a particular embodiment, the fixed dose of recombinant relaxin-2 of the present invention is based on a given body weight or age.

[0145] Generally, appropriate doses of the protein or peptide of the present invention may range from about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient, and more generally, from about 1 to 50 mg per kilogram of body weight. For example, a protein or peptide, such as recombinant relaxin-2, may be administered in single doses of about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, and about 50 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0146] In some embodiments, the protein or peptide of the present invention, for example, recombinant relaxin-2, is administered as a fixed dose between about 10 mg and about 2500 mg. In some embodiments, recombinant relaxin-2 of the present invention is administered in doses of about 10 mg, about 15 mg, about 20 mg, 25 mg, about 30 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg It is administered as a fixed dose of mg, approximately 525 mg, approximately 550 mg, approximately 575 mg, approximately 600 mg, approximately 625 mg, approximately 650 mg, approximately 675 mg, approximately 700 mg, approximately 725 mg, approximately 750 mg, approximately 775 mg, approximately 800 mg, approximately 825 mg, approximately 850 mg, approximately 875 mg, approximately 900 mg, approximately 925 mg, approximately 950 mg, approximately 975 mg, approximately 1000 mg, approximately 1500 mg, approximately 2000 mg, or approximately 2500 mg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0147] IV. Kit Any of the compositions described herein may be included in a kit. In a non-limiting example, the kit includes recombinant relaxin-2.

[0148] The kit may further include reagents or instructions for using recombinant relaxin-2 in the subject. The kit may also include one or more buffers.

[0149] The components of the kit may be packaged either in an aqueous medium or in a lyophilized form. The kit's container means generally include at least one vial, test tube, flask, bottle, syringe or other container means in which the components can be placed, preferably appropriately aliquoted. If there are more than one component in the kit (labeled reagents and labels may be packaged together), the kit also generally includes a second, third or other additional container in which the additional components can be placed separately. The kit may also include a second container means for containing sterile, pharmaceutically acceptable buffers and / or other diluents. However, various combinations of components may be contained in vials. The kit of the present invention also typically includes means for containing the composition of the present invention, e.g., recombinant relaxin-2, and any other tightly sealed reagent containers for commercial use.

[0150] If the components of the kit are provided in one and / or more liquid solutions, those liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly preferred. However, the components of the kit may be provided as dry powder(s). If the reagents and / or components are provided as dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is assumed that the solvent may also be provided in a separate container.

[0151] The present invention is further illustrated by the following embodiments, which should not be construed as limiting. The entire contents of all references cited throughout this application are thus expressly incorporated herein by reference. [Examples]

[0152] (Example 1) Recombinant relaxin-2 protein We designed an engineered form of relaxin-2 protein that enables simple production in mammalian cells. Briefly, we designed a single-chain recombinant relaxin-2 protein containing relaxin B-linker-relaxin A in an operable order from N-terminus to C-terminus. The recombinant relaxin-2 protein further includes a second linker and / or a detectable label, as desired. The components, structure, and sequence of an exemplary single-chain recombinant relaxin-2 protein are listed in Table 1 below. The single-chain recombinant relaxin-2 protein has several advantages, including, but not limited to, requiring no downstream processing or modification steps. Table 1. Single-chain recombinant relaxin-2 protein [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0153] (Example 2) Synthesis of recombinant single-chain relaxin-2 protein

[0154] The recombinant relaxin-2 protein of the present invention was generated using standard molecular biology techniques. Briefly, the DNA encoding one of the proteins listed in Table 1 was operably ligated and cloned into an inducible pcDNA3.1-Zeo-tetO expression plasmid or a pFUSE-hIgG1-Fc2 plasmid. The recombinant plasmids were transfected into Expi293F cells using ExpiFectamine or polyethyleneimine (PEI). For some recombinant plasmids, tetracycline-inducible stable cell lines were generated using Expi293F cells for the expression of recombinant relaxin-2 protein. Cell culture and transfection were carried out according to the manufacturer's manual. Cells were harvested and total protein was collected using techniques well known in the art. The recombinant relaxin-2 protein was purified using affinity chromatography, or affinity chromatography followed by size exclusion chromatography. Immobilized metal affinity chromatography (IMAC) and size exclusion chromatography were used for recombinant relaxin-2 proteins containing 6×His tags (SEQ ID NO: 18) or 8×His tags (SEQ ID NO: 19). Protein G antibody affinity chromatography was used for recombinant relaxin-2 proteins containing IgG1 Fc tags.

[0155] (Example 3) Biochemical data on single-chain relaxin-2 protein The molecular weight and purity of purified recombinant single-chain relaxin-2 proteins were determined by SDS-PAGE electrophoresis and Coomassie blue staining. As shown in Figures 1A-1C, the exemplary recombinant single-chain protein SE001 had a predicted molecular weight of approximately 8 kDa; the exemplary recombinant single-chain proteins SE201, SE202, SE203, SE204, SE205, SE206, SE207, and SE301 had a predicted molecular weight of approximately 32 kDa. Coomassie blue staining also demonstrated that the purified recombinant single-chain relaxin-2 proteins were substantially free of impurity proteins.

[0156] For SE301, size exclusion chromatography of the protein after affinity chromatography was monitored by measuring the absorbance of the eluted fraction at 280 nm. Figure 2 demonstrates that SE301 purified by affinity chromatography was substantially free of impurity proteins.

[0157] Melting temperature of SE301 (T m The T of SE301 was determined using differential scanning fluorescence quantification. As shown in Figure 3, m The temperature is approximately 57°C.

[0158] (Example 4) Activity of recombinant relaxin-2 protein The bioactivity of recombinant relaxin-2 protein was tested using a cAMP-driven reporting gene assay. In the reporter gene assay, recombinant relaxin-2 protein binds to the RXFP1 receptor expressed by transient transfection in HEK293T cells. Binding of recombinant relaxin-2 protein activates RXFP1, leading to an increase in cAMP levels in the cells. The cAMP signaling cascade results in promoter activation by the cAMP response element (CRE). The promoter controls the transcription of the reporter gene for enzyme-secreted embryonic alkaline phosphatase (SEAP). As a result, SEAP is produced and secreted into the cell culture medium by HEK293T cells. Then, 4-methylumbelliferyl phosphate (MUP), a substrate of SEAP, is mixed with the medium. Depending on the amount of SEAP present in the medium, the reaction may result in the enzymatic creation of a fluorescent product detectable by a plate reader. Therefore, the fluorescence readout used to detect the level of the SEAP enzyme acts as a readout for the recombinant relaxin-2-induced activation of RXFP1 in the cells. Cell culture and transfection were performed according to the manufacturer's manual. The cAMP-driven reporting gene assay is described herein by reference in Durocher et al., A Report Gene Assay for High-Throughput Screen of G-protein-coupled Receptors Stably or Transiently Expressed in HEK293 EBNACells Grown in Suspension Culture, Anal. Biochem., 284(2):316-26 (2000) and Liberles & Buck, A Second Class of Chemosensory Receptors in the Olfactory Epithelium, Nature, 442(7103): 645-50 (2006).

[0159] Activity and EC of single-chain recombinant relaxin-250 are summarized in Table 2 and Figures 4 to 12. Table 2. Activity of Recombinant Relaxin-2 Protein [Table 2-1] [Table 2-2] *: Modified pcDNA3.1 plasmid: pcDNA3.1-Zeo-tetO inducible expression plasmid

[0160] (Example 5) Pharmacokinetic Study of Recombinant Relaxin-2 Protein To determine the serum pharmacokinetics of SE301, a pharmacokinetic study was conducted after a single intraperitoneal injection administration to male CD-1 mice. A stock preparation of purified SE301 was prepared at 10 mg / mL in sterile phosphate-buffered saline and stored at -80°C.

[0161] On the day before dosing, the stock preparation of SE301 was diluted according to Table 3 below. The diluted preparation for injection was aliquoted under a laminar flow hood for dosing as needed. Dose formulation analysis was performed the day before dosing using an unvalidated method. The stability (24 hours at room temperature) of the test article (SE301 formulation) was established prior to the start of the study. The test article was warmed to room temperature at least 30 minutes before dosing, but within 3 hours if not used.

[0162] Nine male CD-1 mice were used in this study. Each mouse was between approximately 7 to approximately 10 weeks of age on the dosing day, with body weight between approximately 29 grams and approximately 40 grams. Animal housing and clinical observations were performed at the test facility in accordance with established protocols. The experimental design is shown in Table 3 below. Table 3 [Table 3]

[0163] Intraperitoneal injection (IP) doses were administered via the lower abdominal region. Animals were weighed before dose administration, and the dose volume was adjusted based on body weight. Blood samples from test mice were collected before administration and at 2, 24, 72, and 168 hours after administration.

[0164] For control serum, blood samples from male animals were collected from the inferior vena cava. Whole blood was collected from available CD-1 mice in commercially available tubes containing polymer silica activator. The vacuum tubes containing the blood samples were left at room temperature for 30 minutes, and then centrifuged (after serum appeared). The samples were centrifuged at 2,500 × g for 15 minutes at 4°C within 1 hour of collection. The serum was transferred to pre-labeled polyethylene microcentrifuge tubes. Approximately 5 mL of total male serum was collected. The serum was stored immediately at -60°C or lower until bioanalysis or shipment. The serum served as control serum for bioanalysis.

[0165] To prepare serum samples for PK analysis, at least 0.6 mL of blood sample was collected from each animal in the test compound treatment group at the time of sample collection. For samples collected within the first hour of administration, ±1 minute was considered acceptable. For the remaining time points, samples obtained within 5% of the planned time were considered acceptable and were not considered a deviation from the protocol. All blood samples were collected in commercially available tubes containing polymer silica activator. After blood collection, the tubes containing the blood samples were left at room temperature for approximately 30 minutes, and then centrifuged (after serum appeared). The samples were centrifuged at 2,500 × g for 15 minutes at 4°C within one hour of collection. The serum was then collected after centrifugation, and one aliquot (at least 30 μL) was prepared for PK analysis. The samples were then rapidly frozen on dry ice and maintained at -60°C or lower until analysis. The amount of SE301 was analyzed by performing a qualified ELISA.

[0166] Serum concentration-time data and derived pharmacokinetic parameters were analyzed using a non-compartmental approach with the WinNonlin software program.

[0167] The results of the pharmacokinetic study are shown in Figure 13 and Table 4 below. As shown in Figure 13 and Table 4, the circulating half-life of SE301 is approximately 77.5 hours at a dose of 10 mg / kg, approximately 90.7 hours at a dose of 1 mg / kg, and approximately 130 hours at a dose of 0.2 mg / kg. Table 4 [Table 4]

[0168] (Example 6) Activity of recombinant relaxin-2 protein

[0169] The biological activity of two recombinant relaxin-2 proteins, SE501 and SE502, was tested using a cAMP-driven reporting gene assay according to the method described in Example 4.

[0170] The amino acid sequence of single-chain recombinant relaxin-2 is summarized in Table 1.

[0171] Activity and EC of single-chain recombinant relaxin-2 50 This is summarized in Table 5 and Figure 14. Table 5. Activity of recombinant relaxin-2 protein [Table 5] *: Modified pcDNA3.1 plasmid: pcDNA3.1-Zeo-tetO inducible expression plasmid

[0172] (Example 7) Flow cytometry-coupled assay for recombinant relaxin-2 protein

[0173] The binding affinity of SE301 was determined by flow cytometry assay using Expi293F cells transiently transfected with either RXFP1 with an N-terminal FLAG tag or an empty vector plasmid. Cell culture and transfection were performed according to the manufacturer's manual. Cells transfected with RXFP1 or an empty vector control were incubated at 4°C for 30 minutes in a buffer of 20 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM calcium chloride, and 1% fetal bovine serum. Different concentrations of SE301 were added to the cells and incubated at 4°C for 1 hour. The cells were washed twice with buffer, and M1 antibody labeled with Alexa 488 (M1-488) and secondary anti-human Fc antibody labeled with Alexa 647 (anti-human Fc-647) were incubated with the cells at 4°C for 30 minutes. The cells were washed once, resuspended in 100 μL of buffer, and analyzed by flow cytometry. Cells were gated by forward scattering area versus lateral scattering area and forward scattering area versus forward scattering height. Cells were then gated according to receptor expression, indicated by the binding of M1-488 antibody to the receptor's FLAG tag. Cells in the final gated state were plotted according to the mean fluorescence intensity of the anti-human Fc-647 antibody to calculate the Kd of SE301.

[0174] The results of the flow cytometry study are shown in Figure 15. As shown in Figure 15, the binding affinity (Kd) of SE301 to RXFP1 is 122 nM.

[0175] Embedding by reference All publications, patents, and patent applications referenced herein are thus incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application were specifically and individually incorporated by reference. In case of any conflict, this application shall prevail, including any definitions herein.

[0176] Equal parts Those skilled in the art can recognize or confirm, by mere conventional experimentation, many equivalents to specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. The present invention provides, for example, the following items: (Item 1) From the N-terminus to the C-terminus, A first peptide containing an amino acid sequence that is at least approximately 85% identical to the entire amino acid sequence of SEQ ID NO: 2; A peptide linker containing an amino acid sequence that is at least approximately 85% identical to the entire amino acid sequence of an amino acid sequence selected from the group consisting of DAASSHSHSSAR (SEQ ID NO: 14) and DAAGANANAGAR (SEQ ID NO: 16); and A second peptide containing an amino acid sequence that is at least approximately 85% identical to the entire amino acid sequence of SEQ ID NO: 1. A fusion protein containing, A fusion protein in which the first peptide, the peptide linker, and the second peptide are operably linked. (Item 2) A fusion protein as described in item 1, possessing the activity of the natural relaxin-2 protein. (Item 3) A fusion protein as described in item 1 or 2, having at least about 50% of the activity of the natural relaxin-2 protein. (Item 4) A fusion protein as described in item 3, having at least about 90% of the activity of the natural relaxin-2 protein. (Item 5) A fusion protein as described in item 4, having at least approximately 100% of the activity of the natural relaxin-2 protein. (Item 6) A fusion protein as described in item 5, having at least about 150% of the activity of the natural relaxin-2 protein. (Item 7) The fusion protein according to any one of items 1 to 6, wherein the peptide linker contains an amino acid sequence that is at least approximately 90% identical to the entire amino acid sequence of an amino acid sequence selected from the group consisting of DAASSHSHSSAR (SEQ ID NO: 14), DAASSHSHSSAA (SEQ ID NO: 15), and DAAGANANAGAR (SEQ ID NO: 16). (Item 8) The fusion protein described in item 7, wherein the peptide linker comprises the amino acid sequence of DAASSHSHSSAR (SEQ ID NO: 14), DAASSHSHSSAA (SEQ ID NO: 15), or DAAGANANAGAR (SEQ ID NO: 16). (Item 9) The fusion protein according to any one of items 1 to 8, wherein the first peptide has an amino acid sequence that is at least about 95% identical to the entire amino acid sequence of SEQ ID NO: 2, the second peptide has an amino acid sequence that is at least about 95% identical to the entire amino acid sequence of SEQ ID NO: 1, and the fusion protein has innate relaxin-2 activity. (Item 10) The fusion protein described in item 9, wherein the amino acid sequence of the first peptide is selected from the group consisting of SEQ ID NOs: 2, 7, 8, 9, and 10, and the amino acid sequence of the second peptide is selected from the group consisting of SEQ ID NOs: 1 and 6. (Item 11) The fusion protein described in item 9, wherein the first peptide comprises a substitution selected from the group consisting of M4K, M25K, W28A, and combinations thereof. (Item 12) The fusion protein described in item 11, wherein the first peptide comprises substituted M4K, M25K, and W28A. (Item 13) A fusion protein comprising a first peptide, a peptide linker, and a second peptide, wherein the amino acid sequence of the fusion protein is at least about 85% identical to the entire amino acid sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, and 55. (Item 14) A fusion protein comprising a first peptide, a peptide linker, and a second peptide, wherein the amino acid sequence of the fusion protein is at least about 85% identical to the entire amino acid sequence shown in SEQ ID NO: 55. (Item 15) The fusion protein described in item 14, wherein the amino acid sequence of the fusion protein is shown in Sequence ID No. 55. (Item 16) A fusion protein according to any one of items 1 to 15, further comprising a first detectable label. (Item 17) The fusion protein according to item 16, wherein the first detectable label is operably ligated to the N-terminus of the first peptide or the C-terminus of the second peptide. (Item 18) The fusion protein according to item 16 or 17, wherein the first detectable label is an immunoglobulin G (IgG)Fc peptide having an amino acid sequence that is at least about 85% identical to the entire amino acid sequence of SEQ ID NO: 20. (Item 19) The fusion protein according to item 18, wherein the first detectable label has the amino acid sequence of SEQ ID NO: 20 or 21. (Item 20) The fusion protein according to item 18 or 19, wherein the first detectable label is operably ligated to the N-terminus of the first peptide. (Item 21) The fusion protein according to item 20, further comprising a second linker, the second linker being operably ligated to the C-terminus and N-terminus of the first detectable label. (Item 22) The fusion protein described in item 21, wherein the second linker is selected from the group consisting of Gly-Gly-Ser, Ala-Ala-Ala, Pro-Pro-Pro, Gly-Ser-Gly, (Gly-Ser-Gly)2 (SEQ ID NO: 57), and (Gly-Gly-Ser)4 (SEQ ID NO: 17). (Item 23) A fusion protein described in any one of items 18 to 22, having an in vivo circulating half-life longer than approximately 10 hours. (Item 24) A fusion protein as described in item 23, having an in vivo circulating half-life of approximately 130 hours. (Item 25) The fusion protein according to item 16 or 17, wherein the first detectable label is a polyhistidine tag having an amino acid sequence that is at least about 85% identical to the entire amino acid sequence selected from the group consisting of SEQ ID NOs. 18 and 19. (Item 26) The fusion protein according to item 18, wherein the first detectable active substance comprises the amino acid sequence of SEQ ID NO: 18 or 19. (Item 27) A fusion protein according to any one of items 16 to 26, further comprising a second detectable label. (Item 28) The fusion protein according to item 27, wherein the first detectable label is operably ligated to the N-terminus of the first peptide, and the second detectable label is operably ligated to the C-terminus of the second peptide. (Item 29) The fusion protein according to item 27, wherein the first detectable label and the second detectable label are operably ligated to the N-terminus of the first peptide. (Item 30) The fusion protein according to any one of items 27 to 29, wherein the first detectable label and the second detectable label are different peptides. (Item 31) A fusion protein according to any one of items 1 to 30, further comprising a cleavable linker. (Item 32) The fusion protein according to item 31, wherein the cleavable linker is a peptide that undergoes specific digestion by a protease. (Item 33) The fusion protein described in item 32, wherein the protease is HRV 3C protease or thrombin. (Item 34) The fusion protein according to item 33, wherein the cleavable linker is a peptide or a variant thereof having the sequence of SEQ ID NO: 23. (Item 35) The fusion protein according to any one of items 1 to 34, further comprising a signal peptide at the N-terminus of the aforementioned fusion protein. (Item 36) A fusion protein comprising a detectable label, a second linker, a first peptide, a peptide linker, and a second peptide, wherein the amino acid sequence of the fusion protein is at least about 85% identical to the entire amino acid sequence shown in SEQ ID NO: 41, SEQ ID NO: 60, or SEQ ID NO: 61. (Item 37) The fusion protein described in item 36, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO: 41. (Item 38) The fusion protein according to item 36, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO: 60 or SEQ ID NO: 61. (Item 39) A peptide linker containing an amino acid sequence that has at least approximately 85% amino acid identity with the entire amino acid sequence of an amino acid selected from the group consisting of DAASSHSHSSAR (SEQ ID NO: 14) and DAAGANANAGAR (SEQ ID NO: 16). (Item 40) From the N-terminus to the C-terminus, The first peptide containing the relaxin B amino acid sequence; Peptide linker; and A second peptide containing the relaxin A amino acid sequence. A fusion protein containing, The aforementioned fusion protein has the activity of the natural relaxin-2 protein, and The aforementioned fusion protein is (i) Activating the relaxin-2 receptor RXFP1 on the cell surface at an EC50 of approximately 4.2 nM or less; (ii) A melting temperature of at least about 57°C; (iii) a circulating half-life of at least approximately 77.5 hours; and (iv) Any combination of them A fusion protein having properties selected from the group consisting of the following. (Item 41) From the N-terminus to the C-terminus, A first peptide containing an amino acid sequence that is at least approximately 90% identical to the entire amino acid sequence of SEQ ID NO: 10; Peptide linker; and A second peptide containing the relaxin A amino acid sequence. A fusion protein containing [the specified ingredient]. (Item 42) The fusion protein described in item 41, wherein the amino acid in the first peptide corresponding to amino acid 4 of SEQ ID NO: 10 is K; the amino acid in the first peptide corresponding to amino acid 25 of SEQ ID NO: 10 is K; and the amino acid in the first peptide corresponding to amino acid 28 of SEQ ID NO: 10 is A. (Item 43) The fusion protein described in item 41 or 42, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 16. (Item 44) From the N-terminus to the C-terminus, The first peptide containing the relaxin B amino acid sequence; A peptide linker containing the amino acid sequence of SEQ ID NO: 16; and A second peptide containing the relaxin A amino acid sequence. A fusion protein containing [the specified ingredient]. (Item 45) The first peptide comprises the amino acid sequence of SEQ ID NO: 10; The amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 16; The second peptide contains an amino acid sequence that is at least approximately 85% identical to the entire amino acid sequence of SEQ ID NO: 1; The second peptide comprises the amino acid sequence of SEQ ID NO: 1; The first peptide comprises an amino acid sequence that is at least about 90% identical to the entire amino acid sequence of SEQ ID NO: 10; the peptide linker comprises the amino acid sequence of SEQ ID NO: 16; and the second peptide comprises an amino acid sequence that is at least about 85% identical to the entire amino acid sequence of SEQ ID NO: 1. Contains an acid sequence; or A fusion protein according to any one of items 1 to 44, wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 10; the peptide linker comprises the amino acid sequence of SEQ ID NO: 16; and the second peptide comprises the amino acid sequence of SEQ ID NO: 1. (Item 46) A polypeptide containing an amino acid sequence that is at least approximately 90% identical to the entire amino acid sequence of SEQ ID NO: 10. (Item 47) The polypeptide described in item 46, wherein the amino acid corresponding to amino acid 4 in SEQ ID NO: 10 is K; the amino acid corresponding to amino acid 25 in SEQ ID NO: 10 is K; and the amino acid corresponding to amino acid 28 in SEQ ID NO: 10 is A. (Item 48) Polypeptides as described in item 46 or 47, comprising the amino acid sequence containing SEQ ID NO: 10. (Item 49) The polypeptide according to any one of items 46 to 48, wherein the amino acid sequence of the polypeptide is sequence number 10. (Item 50) A polypeptide according to any one of items 46 to 49, further comprising an amino acid sequence that is at least approximately 85% identical to the entire amino acid sequence of SEQ ID NO: 16. (Item 51) A polypeptide according to any one of items 46 to 50, further comprising the amino acid sequence containing SEQ ID NO: 16. (Item 52) A polypeptide according to any one of items 46 to 51, further comprising an amino acid sequence that is at least approximately 85% identical to the entire amino acid sequence of SEQ ID NO: 1. (Item 53) A polypeptide according to any one of items 46 to 52, further comprising the amino acid sequence containing SEQ ID NO: 1. (Item 54) A polypeptide containing the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 16. (Item 55) A polypeptide comprising an amino acid sequence containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 16, wherein the amino acid sequence of SEQ ID NO: 16 is inserted between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 10. (Item 56) A polynucleotide comprising a nucleotide sequence encoding a fusion protein as described in any one of items 1 through 45 or a polypeptide as described in any one of items 46 through 55. (Item 57) A polynucleotide, which is an RNA molecule, as described in item 56. (Item 58) An expression vector containing the polynucleotides described in item 56. (Item 59) A plasmid, an expression vector as described in item 58. (Item 60) A viral vector, an expression vector as described in item 58. (Item 61) Recombinant cells containing a polynucleotide as described in item 56 or 57, or an expression vector as described in any one of items 58 to 60. (Item 62) Recombinant cells, as described in item 61, that are prokaryotic or eukaryotic cells. (Item 63) Recombinant cells as described in item 62, which are prokaryotic cells selected from the group consisting of E. coli cells and Bacillus cells. (Item 64) Recombinant cells as described in item 62, which are eukaryotic cells selected from the group consisting of yeast cells, insect cells, and mammalian cells. (Item 65) Recombinant cells as described in item 64, which are mammalian cells selected from the group consisting of CHO cells, HeLa cells, and 293 cells. (Item 66) Recombinant cells, specifically Expi293 cells, as described in item 65. (Item 67) A method for producing a fusion protein as described in any one of items 1 to 45 or a polypeptide as described in any one of items 46 to 55, comprising the steps of culturing recombinant cells as described in any one of items 61 to 66, and purifying the fusion protein. (Item 68) A pharmaceutical composition comprising an effective amount of a fusion protein according to any one of items 1 to 45, a polypeptide according to any one of items 46 to 55, a polynucleotide according to item 56 or 57, or an expression vector according to any one of items 58 to 60. (Item 69) A method for enhancing relaxin-2-related activity in cells, comprising the step of contacting the cells with a fusion protein according to any one of items 1 to 45 or a polypeptide according to any one of items 46 to 55, thereby enhancing relaxin-2-related activity in the cells. (Item 70) The method according to item 69, wherein the fusion protein activates the relaxin-2 receptor RXFP1 on the cell surface. (Item 71) The method according to item 69 or 70, which increases cAMP levels in the cells to induce vasodilation, induce the expression of angiogenic factors, induce the expression of MMPs, and induce collagen degradation. (Item 72) The method according to any one of items 69 to 71, wherein the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts. (Item 73) The method according to any one of items 69 to 71, wherein the cells are within the scope. (Item 74) The method according to item 73, wherein the subject has relaxin-2 related disorder. (Item 75) The method according to item 74, wherein the relaxin-2 related disorder is selected from the group consisting of renal disease, fibrous disease, and cardiovascular disease. (Item 76) The method according to item 75, wherein the disorder is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, hepatorenal syndrome, scleroderma, idiopathic pulmonary fibrosis, renal fibrosis, cardiac fibrosis, NASH, dilated cardiomyopathy, diastolic heart failure, pulmonary arterial hypertension, chronic heart failure, acute heart failure, congestive heart failure, coronary artery disease, hypertension, and pre-eclampsia. (Item 77) A method for treating relaxin-related disorder in a subject requiring treatment for the disorder, comprising the step of administering to the subject an effective amount of a fusion protein according to any one of items 1 to 45, a polypeptide according to any one of items 46 to 55, a polynucleotide according to item 56 or 57, an expression vector according to any one of items 58 to 60, or a pharmaceutical composition according to item 68, thereby treating the relaxin-related disorder. (Item 78) The method according to item 77, wherein the relaxin-2 related disorder is selected from the group consisting of renal disease, fibrous disease, and cardiovascular disease. (Item 79) The method according to item 78, wherein the aforementioned disorder is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, hepatorenal syndrome, scleroderma, idiopathic pulmonary fibrosis, renal fibrosis, cardiac fibrosis, NASH, dilated cardiomyopathy, diastolic heart failure, pulmonary arterial hypertension, chronic heart failure, acute heart failure, congestive heart failure, coronary artery disease, hypertension, and pre-eclampsia. (Item 80) The method described in any one of items 77 to 79, which reduces arterial pressure, increases renal artery blood flow, increases diastolic cardiac filling, resolves established fibrosis, or inhibits the development of new fibrosis. (Item 81) An effective amount of any one of items 1 to 45, a polypeptide as described in any one of items 46 to 55, a polynucleotide as described in item 56 or 57, an expression vector as described in any one of items 58 to 60, or a pharmaceutical composition as described in item 68, and Instructions for use A kit that includes this.

Claims

1. A fusion protein comprising a first peptide, a peptide linker, and a second peptide from the N-terminus to the C-terminus, The peptide linker comprises SEQ ID NO: 14 or 16, The fusion protein wherein the amino acid sequence of the fusion protein consists of one of the amino acid sequences of SEQ ID NOs. 25, 28, 29, 31-46, 60, and 61.

2. (a) A cleavable linker wherein the cleavable linker is a peptide that is specifically digested by a protease, and / or (b) A signal peptide at the N-terminus of the fusion protein The fusion protein according to claim 1, further comprising:

3. A polynucleotide comprising a nucleotide sequence encoding the fusion protein according to claim 1 or 2.

4. An expression vector comprising the polynucleotide described in claim 3, (a) The expression vector is a plasmid, or (b) An expression vector wherein the expression vector is a viral vector.

5. Recombinant cells comprising the polynucleotide described in claim 3 or the expression vector described in claim 4: (a) The cells are prokaryotic cells selected from the group consisting of E. coli cells and Bacillus cells. (b) The cells are eukaryotic cells selected from the group consisting of yeast cells, insect cells and mammalian cells, or (c) Recombinant cells, wherein the cells are mammalian cells selected from the group consisting of CHO cells, HeLa cells, and 293 cells.

6. A method for producing the fusion protein described in claim 1 or 2, comprising the steps of culturing the recombinant cells described in claim 5, and purifying the fusion protein.

7. A pharmaceutical composition comprising an effective amount of the fusion protein according to claim 1 or 2, the polynucleotide according to claim 3, or the expression vector according to claim 4.

8. An ex vivo method for enhancing relaxin-2-related activity in cells, comprising the step of contacting the cells with the fusion protein described in claim 1 or 2: (a) The relaxin-2 related activity includes activating the relaxin-2 receptor RXFP1 on the cell surface, and / or (b) The relaxin-2 related activity includes increasing cAMP levels in the cells, thereby inducing vasodilation, inducing the expression of angiogenic factors, inducing the expression of MMPs, and inducing collagen degradation. A method in which the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, cardiomyocytes, and fibroblasts.

9. A composition for use in a method of treating relaxin-2-related disorders in subjects requiring treatment for relaxin-2-related disorders, comprising the fusion protein described in claim 1 or 2, wherein the relaxin-2-related disorder is selected from the group consisting of renal disease, fibrous disease and cardiovascular disease, and / or the relaxin-2-related disorder is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, hepatorenal syndrome, scleroderma, idiopathic pulmonary fibrosis, renal fibrosis, cardiofibrosis, NASH, dilated cardiomyopathy, diastolic heart failure, pulmonary arterial hypertension, chronic heart failure, acute heart failure, congestive heart failure, coronary artery disease, hypertension and preeclampsia: (a) The method comprises enhancing relaxin-2 related activity in cells within the subject and activating the relaxin-2 receptor RXFP1 on the cell surface; and / or (b) The method comprises enhancing relaxin-2 related activity in cells within the subject, increasing cAMP levels in the cells, inducing vasodilation, inducing the expression of angiogenic factors, inducing the expression of MMPs, and inducing collagen degradation. The composition wherein the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, cardiomyocytes, and fibroblasts.

10. A composition for use in a method of treating relaxin-2-related disorder in subjects requiring treatment for relaxin-2-related disorder, comprising a fusion protein according to claim 1 or 2, a polynucleotide according to claim 3, an expression vector according to claim 4, or a pharmaceutical composition according to claim 7: A composition in which the relaxin-2 related disorder is selected from the group consisting of kidney disease, fibrous disease and cardiovascular disease, and / or the relaxin-2 related disorder is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, hepatorenal syndrome, scleroderma, idiopathic pulmonary fibrosis, renal fibrosis, cardiac fibrosis, NASH, dilated cardiomyopathy, diastolic heart failure, pulmonary arterial hypertension, chronic heart failure, acute heart failure, congestive heart failure, coronary artery disease, hypertension and pre-eclampsia.

11. A kit comprising an effective amount of the fusion protein according to claim 1 or 2, the polynucleotide according to claim 3, the expression vector according to claim 4, or the pharmaceutical composition according to claim 7, and instructions for use.

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