TW201315739A - Relaxin fusion polypeptides and uses thereof - Google Patents

Abstract

The present invention provides Relaxin fusion polypeptides A-L-B with a non-wild type array of the Relaxin A-chain and Relaxin B-chain, wherein the A and B-chains are connected by a linker peptide. The invention further provides Relaxin fusion polypeptides with extended half-life. Furthermore, the invention provides nucleic acid sequences encoding the foregoing fusion polypeptides, vectors containing the same, pharmaceutical compositions and medical use of such fusion polypeptide.

Description

Relaxin (RELAXIN) fusion polypeptides and uses thereof

The present invention provides a relaxin fusion polypeptide A-L-B having a relaxin A chain and a relaxin B chain non-wild type array, wherein the A chain and the B chain are linked by a linker peptide. The invention further provides relaxin fusion polypeptides having an extended half-life. Furthermore, the present invention provides a nucleic acid sequence encoding the above fusion polypeptide, a vector comprising the same, a pharmaceutical composition of the fusion polypeptide, and a medical use.

Background of the invention

Relaxin 2 (H2 relaxin, RLN2), a member of the insulin superfamily, is a 2-chain peptide exhibiting a typical B-C-A chain pro-hormone structure arranged from the N to the C-terminus at the gene level. The other members of this superfamily are encoded by seven human genes, the relaxin genes RLN 1, RLN3, and the insulin-like peptide genes INSL3, INSL4, INSL5, and INSL6. The overall sequence homology between members of this family is low; however, phylogenetic analysis revealed that these gene lines evolved from the RLN3 ancestral gene [Hsu, S.Y. (2003); Wilkinson, T.N. et al. (2005)]. The mature protein has a molecular weight of approximately 6000 Da, which is a pre-hormone enzyme cleavage product of prohormone-converting enzyme 1 (PC1) and 2 (PC2) [Hudson P. et al. (1983)]. The resulting A and B chains are linked by two intermolecular cysteine bridges; the A chain has an additional intramolecular disulfide bond.

Relaxin initiates pleiotropic effects on various cell types via multiple pathways; it is called G protein called LGR7 via a class I (retinoid) Coupling receptor (G-protein coupled receptor 7 rich in leucine), also known as RXFP1 (relaxin family peptide 1 receptor), confers activity and significantly reduces LRG8/RXFP2 (relaxin) Affinity of the Family Peptide 2 Receptor) [Kong RC et al. (2010) Mol Cell Endocrinol. 320: 1-15]. In the relaxin molecule, the amino acid motif in the B chain (Arg-XXX-Arg-XX-Ile/Val-X) [Schwabe and Büllesbach (2007) Adv Exp Med Biol. 612: 14-25 It is stored in all relaxin peptides with Büllesbach and Schwabe J Biol Chem. 2000 Nov 10; 275(45): 35276-80 and is the key to the interaction of these peptides with the corresponding receptors. Binding of relaxin to LGR7/RXFP1 results in activation of adenosine cyclase and an increase in the second messenger molecule cAMP. Through this mechanism, for example, relaxin 2 mediates the release of rat cardiac atrial diuretic peptide [Toth, M. et al. (1996)]. The positive effect of relaxin 2 on rat atrial myocytes has also been demonstrated [Piedras-Renteria, E.S. et al. (1997)]. Other signaling molecules activated by the relaxin/LGR7 complex are phosphoinositide-3 kinase, tyrosine kinase, and phosphodiesterase [Bartsch, O. et al. (2001), Bartsch, O. et al. (2004)]. Additional signaling pathways activated by this system include one of the increased nitric oxide (NO) pathways in the rat and guinea pig heart resulting in an increase in circulating GMP [Bani-Sacchi, T. et al. (1995)].

Relaxin exhibits the action of pleiotropic hormones [Dschietzig T. et al. (2006)] with biological activity on, for example, the lungs, kidneys, brain, and heart organs. Relaxin in various animal disease patterns and clinical studies The strong anti-fibrotic and vasodilator activity is the most obvious cause of the positive effect of using this peptide [McGuane J.T. et al. (2005)]. In pathological conditions, RLN2 has a variety of beneficial effects in the cardiovascular system; during the physiological processes of various diseases, the tissue itself is maintained stable and protects the injured myocardium; it exhibits significant vasodilating effects, for example, affecting rodent coronary arteries [Nistri, S. et al. (2003)] and flow and vasodilation in other organ vascular beds. In spontaneously hypertensive rats, RLN2 lowers blood pressure, a role that is passed through increased NO production.

The cardioprotective activity of relaxin 2 has been assessed in different animal models such as guinea pigs, rats and pigs [Perna A. M. et al. (2005), Bani, D. et al. (1998)]. RLN2 ameliorate myocardial injury, inflammatory cell infiltration, and subsequent fibrosis, thereby alleviating severe ventricular dysfunction [Zhang J. et al. (2005)].

Relaxin 2 exhibits strong anti-fibrotic activity. In injured tissues, activation and proliferation of fibroblasts lead to increased collagen production and interstitial fibrosis. Increased fibrosis of the heart due to biomechanical loading, affecting ventricular dysfunction, remodeling, and arrhythmia. In the animal model, sustained perfusion of relaxin 2 inhibits or even reverses cardiac dysfunction caused by cardiomyopathy, hypertension, isoproterenol-induced cardiotoxin, diabetic cardiomyopathy, and myocardial infarction. This inhibition of fibrosis or reversal of established fibrosis reduces ventricular sclerosis and improves diastolic function. It is worth noting that although relaxin 2 reduces the accumulation of abnormal collagen, it does not affect the basic collagen content of healthy tissues. Amount that highlights its safety for therapeutic use.

Relaxin 2 has been tested in several clinical studies to provide a promising pleiotropic vasodilator for the treatment of patients with acute heart failure. In their studies, relaxin 2 was associated with beneficial relief of dyspnea and other clinical outcomes [Teerlink J.R. et al. (2009), Metra M. et al. (2010)].

Due to the limited half-life of relaxin, the patient must be treated every 14 to 21 days, so compound administration must be continuously perfused for at least 48 hours.

Furthermore, relaxin 2 can also be used to treat, for example, pancreatitis, inflammation-related diseases such as rheumatoid arthritis, and cancer [Cosen-Binker LI et al. (2006) Santora K. Et al. (2007)], or Scleroderma, lung, kidney, and liver fibrosis [Bennett RG. (2009)] and other diseases. Relaxin 2 reduces the growth of xenograft tumors in human MDA-MB-231 breast cancer cells [Radestock Y, Hoang-Vu C, Hombach-Klonisch S. (2008) Breast Cancer Res. 10: R71].

It is difficult to synthesize relaxin 2 by chemical methods; due to the low solubility of the B chain and the laborious need to introduce a cysteine bridge between the A and B chains, the yield of the active peptide obtained by these methods is extremely low [( Barlos KK et al. (2010)]. Alternatively, recombinant expression of relaxin 2 can be performed. To allow for the cleavage and maturation of the prepro-peptide and the secretion of the biologically active peptide during post-translational modification, Host cells are routinely co-transfected with expression constructs encoding pre-hormone-convertase 1 and/or 2 [(Park JI et al. (2008)]. However, pre-existing cells are pre-existing The efficiency of endoproteolytic treatment of peptides often significantly limits the production of bioactive molecules [Shaw J. A. et al. (2002)].

Thus, it would be advantageous to generate an irrelevant endoproteolytic treatment that is mediated by a particular protease that exhibits complete biological activity and that can be produced in a significant yield using a heterologous expression system.

In the case of human insulin, a single-chain variant in which the insulin B chain and the insulin A chain are linked by a polypeptide that is not cleaved has been produced [Rajpal G. et al. (2009)]. Endoproteolytic treatment is not necessary for these variants.

Surprisingly, Applicants et al. identified a relaxin variant in which the two active strands designated A and B were aligned, and the cleavable C chain was replaced by a linker peptide. As shown in Figure 1, the orientation of the modified molecular chain is: A chain-linker peptide-B chain, rather than the orientation of the gene-determined relaxin single chain, ie, the B chain-C chain-A chain. The resulting molecule exhibits complete biological activity regardless of any endoproteolytic treatment. This novel single-chain relaxin variant provided by the present invention thus addresses the problem of low performance yield or the need to co-transfect proteases.

In humans, the half-life of relaxin 2 administered intravenously is less than 10 minutes [Dschietzig T. et al. (2009)]; thus, in clinical trials, relaxin 2 must be administered continuously for more than 48 hours. Therefore, it may be advantageous to increase the biological half-life of relaxin.

Increasing the biological half-life can be carried out by chemical modification, such as pegylation or hydroxyethyl amylation of the polypeptide of interest, introduction of additional, non-native N-glycosylation sites; The gene fusion method is used to bind the polypeptide to other molecules such as antibody immunoglobulin Fc fragments, transferrin, albumin, in vivo, to other binding modules that mediate longer half-life molecules, or other proteins. The present invention provides single-chain relaxin variants having a half-life enhancing fusion to the Fc portion of an antibody. Surprisingly, their variants show biological activity in the range of wild-type relaxin.

Summary of invention

The present invention relates to fusion polypeptides, hereinafter also referred to as single-stranded relaxin (scRelaxin).

The current standard for the production of relaxin is the chemical synthesis of this molecule, a complex and expensive step. Due to the fact that relaxin is post-translationally modified, in particular by cleavage of the pre-protease of prehormone converting enzyme 1 and prohormone converting enzyme 2, selection of an appropriate expression system is necessary for recombinant performance. Endoproteolytic treatment of proteins belonging to the insulin superfamily often limits the production of bioactive molecules from heterologous cells. In order to avoid endoproteolytic treatment of relaxin, the fusion polypeptide of the present invention is a molecule in which the orientation of the gene encoding the two active chains of the relaxin of the A chain and the B chain is reversed, wherein the A chain and the B chain are linked. The sub-peptide is linked. In particular, in the relaxin variant provided by the present invention, the orientation of the DNA fragment is: A-chain-peptide linker-B chain, and the gene-specific orientation of the individual DNA fragments encoding the relaxin functional region, ie, the B-chain -C chain-A chain; thus producing a single-stranded relaxin in which the carboxy terminus of the relaxin A chain is fused to the amino terminus of the linker polypeptide L, and the carboxy terminus of the linker polypeptide L is fused to the amine end of the relaxin B chain , named ALB (see Figure 1 for description); the resulting molecular exhibition It is similar to wild-type relaxin, but its performance is not related to endoproteolytic treatment.

A specific example of the invention is a fusion polypeptide comprising A-L-B, wherein A comprises a relaxin A chain polypeptide or a functional variant thereof, B comprises a relaxin B chain polypeptide or a functional variant thereof, and L is a linker polypeptide.

In a further embodiment, the relaxin A chain polypeptide of A-L-B comprises a relaxin 2A chain polypeptide or a functional variant thereof, and the relaxin B chain polypeptide comprises a relaxin 2B chain polypeptide or a functional variant thereof.

In a preferred embodiment, the relaxin A chain polypeptide of ALB comprises a human minimal relaxin 2A chain polypeptide (SEQ ID NO: 118) or a functional variant thereof, or comprises a human relaxin 2A chain polypeptide (SEQ ID NO: 117) ) or a functional variant thereof.

In a preferred embodiment, the relaxin B chain polypeptide of A-L-B comprises a human relaxin 2 B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof.

In a more preferred embodiment, the relaxin A chain polypeptide of ALB comprises a human minimal relaxin 2A chain polypeptide (SEQ ID NO: 118) or a functional variant thereof, or comprises a human relaxin 2A chain polypeptide (SEQ ID NO: 117) Or a functional variant thereof and a relaxin B chain polypeptide comprising a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof.

In still a further preferred embodiment, the relaxin A chain polypeptide of ALB is a human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof and a relaxin B chain polypeptide is a human relaxin 2 B chain polypeptide (SEQ. ID NO: 119) or a functional variant thereof.

In a specific embodiment, the linker polypeptide L of the above fusion polypeptide ALB is composed of a polypeptide having a length of 6 to 14 amino acid residues; further preferably a polypeptide linker L having a length of 7 to 13 amino acid residues Further preferred is a polypeptide linker L having a length of 8 to 12 amino acid residues; more preferably a polypeptide linker L having a length of 7 to 11 or 9 to 11 amino acid residues; more preferably a polypeptide linker L having a length of 9 amino acid residues; in a further preferred embodiment, the integer length of the polypeptide linker L is selected from the group consisting of integers 6, 7, 8, 9, 10, 11, 12, Groups of 13 and 14.

The linker peptide L may be composed of any amino acid; in a preferred embodiment, the linker polypeptide L comprises at least one Gly, Ser, Arg, Leu, Cys, Ala, Leu and/or Lys residue; In the example, the linker polypeptide L comprises Gly and Ser residues; further preferred embodiments are linker L comprising Gly and Ser residues and having a Gly to Ser ratio of at least 3 to 1.

In a further embodiment, the linker L comprises at least one linker for covalent coupling of a half-life extending group. In a specific embodiment of the invention, the above-mentioned linking site is a Lys or Cys residue.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, and the B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and L-system 6-14, 7-13, 8-12, 7-11, 9-11, or 9 amino acid residues Long linker polypeptide. The linker peptide L can be composed of any amino acid. In a preferred embodiment, the linker polypeptide L comprises at least one Gly, Ser, Arg, Leu, Cys, Ala, Leu and/or Lys residue. In a more preferred embodiment, the linker polypeptide L comprises Gly and Ser residues. Further preferred embodiments are linker L comprising a Gly to Ser residue and a Gly to Ser ratio of at least 3 to 1. In a further embodiment, the linker L comprises at least one linker for covalent coupling of a non-proteinaceous polymer half-life extending group. In a specific embodiment of the invention, the above-mentioned linking site is a Lys or Cys residue.

A preferred embodiment of the invention is a fusion polypeptide A-L-B further comprising a half-life extending group.

In a further embodiment, the fusion polypeptide described above has relaxin activity. In a further preferred embodiment, the relaxin activity is activation of the relaxin receptor LGR7. In still further preferred embodiments, the activation of the relaxin receptor LGR7 is determined using the methods disclosed in the experimental methods.

In another aspect, the invention provides a polynucleotide encoding the fusion polypeptide described above. Such polynucleotides may further comprise a coding sequence for a message peptide that permits secretion of the fusion polypeptide; also a vector comprising a polynucleotide to obtain such a fusion polypeptide; a suitable vector is, for example, an expression vector. A further embodiment of the invention is a host cell comprising a polynucleotide, vector, or expression vector encoding the fusion polypeptide described above. The host cell of the invention may be a eukaryotic cell or a prokaryotic cell. The eukaryotic cell can be a mammalian cell or a yeast or insect cell, preferably a mammalian cell. original The nuclear cell can be, for example, an E. coli cell.

In another embodiment, the invention provides a pharmaceutical composition comprising the fusion polypeptide described above; the composition can be formulated for intravenous, intraperitoneal or subcutaneous administration.

Another embodiment of the invention provides a pharmaceutical composition or fusion polypeptide for use as a medicament. A further specific example is the use of a pharmaceutical composition or fusion polypeptide for the treatment of cardiovascular disease, pancreatitis, inflammation, cancer, scleroderma, lung, kidney, and liver fibrosis.

Detailed description of the invention definition:

The term "amino acid residue" is intended to be encompassed by alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E). , phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine ( Leu or L), methionine (Met or M), aspartame (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), proline (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues Amino acid residues in the constituent groups.

The terms "relaxin activity" or "relaxin activity" are defined as the ability of relaxin or its variant to activate a stimulatory G protein Gs, thereby producing a second messenger ring AMP, and/or stimulating PI3 kinase. Relaxin or a variant thereof binds to LGR7, resulting in intracellular activation of the stimulatory G protein Gs, with the result that a second messenger loop AMP (cAMP) is subsequently produced. however, cAMP produces a time-dependent biphasic response. After the initial transient Gs-adenonucleotide cyclase-mediated cAMP response, the receptor signal is converted to an inhibitory G protein activation and thus a PI3 kinase-mediated response [Halls ML, Bathgate RA, Summers RJ (2005) Signal Switching after Stimulation of LGR7 Receptors by Human Relaxin 2, Ann. NY Acad. Sci. 1041: 288-291].

The term "half-life extending group" refers to a pharmaceutically acceptable group, functional region, or "vehicle" covalently linked ("conjugated") to a relaxin fusion polypeptide, either directly or via a linker. Compared with the unconjugated binding type relaxin fusion polypeptide, the proteolytic degradation or other chemical modification of the relaxin fusion polypeptide can be prevented or reduced, and the half-life or other pharmacokinetic properties of the relaxin fusion polypeptide can be increased, for example. However, it is not limited to increasing absorption rate, reducing toxicity, increasing solubility, increasing biological activity and/or target selectivity, increasing the manufacturability of relaxin fusion polypeptides, and/or reducing immunogenicity. The term "half-life extending group" includes non-proteinaceous half-life extending groups such as PEG or HES; and proteinaceous half-life extending groups such as serum albumin, transferrin or Fc functional regions.

"Polypeptide", "peptide" and "protein" are used interchangeably herein and include molecular chains of two or more amino acids joined via a peptide bond. They do not refer to products of a particular length; they include post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like. In addition, protein fragments, analogs, mutant or variant proteins, and fusion proteins are all included within the meaning of the polypeptide. Their terms also include those that are synthesizable, or Molecules of one or more amino acid analogs or non-standard or non-natural amino acids are represented using known protein engineering techniques. Furthermore, the fusion proteins of the invention can be derivatized as described herein using well-known organic chemistry techniques.

The term "functional variant" refers to a variant polypeptide that retains at least some of its natural biological activity. In the case of a variant of relaxin 2 according to the invention, the functional variant system exhibits at least a number of variants of its natural activity, such as activation of the relaxin receptor LGR7. Activation of the relaxin receptor LGR7 can be determined by the methods disclosed in the experimental methods.

The terms "fragment," "variant," "derivative," and "analog" when referring to a polypeptide of the invention include any polypeptide that retains at least several receptor binding properties of the corresponding wild-type relaxin polypeptide. Polypeptide fragments of the invention include proteolytic fragments, deletion fragments, and polypeptides having altered amino acid sequences due to substitutions, deletions, or insertions of amino acids. Variants may occur naturally or non-naturally. Non-naturally occurring variants can be produced using mutation techniques known in the art. Variant polypeptides may comprise substitutions, deletions, or additions of conservative or non-conservative amino acids. A variant polypeptide is also referred to herein as a "polypeptide analog." A "derivative" of a polypeptide as used herein refers to a subject polypeptide having one or more residues that are chemically derivatized via a reactive side group reaction. "Derivatives" also include polypeptides containing one or more of the naturally occurring one or more amino acid derivatives of twenty standard amino acids. For example, 4-hydroxyproline can replace valine, 5-hydroxy-amino acid can replace lysine, 3-methylhistamine can replace histidine, and homoserine can replace serine. And ornithine can be substituted for lysine.

The term "fusion protein" refers to a protein comprising a polypeptide component derived from more than one parent protein or polypeptide, and/or a protein function derived from one or more parent proteins or polypeptides that are not in their wild-type orientation. Fusion protein of the region. In general, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a protein polypeptide sequence is affixed to a framework of nucleotide sequences encoding different protein polypeptide sequences, and optionally separated by a linker or an extension. The fusion gene can then be expressed as a single protein using a recombinant host cell.

The terms "nucleotide sequence" or "polynucleotide" mean the continuous extension of two or more nucleotide molecules. The nucleotide sequence can be obtained from a gene, cDNA, RNA, semi-synthetic, synthetic source, or any combination thereof.

"EC _50" (half maximal effective concentration) shall mean a therapeutic compound after exposure to a certain specific time, half of the effective concentration inducing a reaction between the baseline and maximum.

The term "immunogenicity" as used in relation to a particular substance means the ability of the substance to elicit a response from the immune system. The immune response can be a response of a cell or antibody (for further definition of immunogenicity, see, for example, Roitt: Essential Immunology (8th Edition, Black-well)]. Generally, antibody reactivity is reduced to a sign of reduced immunogenicity. Reduced immunogenicity can be determined using any suitable method (e.g., in vivo or in vitro) known in the art.

The terms "polymerase chain reaction" or "PCR" generally refer to a method of amplifying a desired nucleotide sequence in vitro, for example, as described in US Pat. No. 4,683,195 and US Pat. No. 4,683,195. Usually, PCR methods involve the use of The primer extension of the oligonucleotide primer which can preferentially hybridize to the template nucleic acid is synthesized and repeated.

The term "vector" refers to a plastid or other nucleotide sequence that can be replicated in a host cell or embedded in a host cell's genome, and thus can be used in conjunction with a compatible host cell (vector-host system) to perform different functions: Promoting the transfer of a nucleotide sequence, that is, generating a usable amount of sequence; directing the expression of the gene product encoded by the sequence and embedding the nucleotide sequence into the host cell genome. The carrier contains different components depending on the function to be performed.

"Cell", "host cell", "cell strain" and "cell culture" are used interchangeably herein, and all such words are to be understood to include the generation of progeny from cell growth or culture.

The term "functional in vivo half-life" is used in its normal sense, that is, 50% of the polypeptide biological activity is present in the body/target organ, or the polypeptide activity is 50% of the initial value.

An alternative method for determining functional in vivo half-life can be to determine the "serum half-life", which is the time in which 50% of the polypeptide is recycled in the plasma or bloodstream before being cleared. Determination of serum half-life is often simpler than measuring functional in vivo half-life, and serum half-life is usually a good indicator of functional in vivo half-life. Alternatives to serum half-life include "plasma half-life", "circulatory half-life", "serum clearance", "plasma clearance", and "clearance half-life." Polypeptide clearance utilizes one or more reticuloendothelial systems (RES), kidney, spleen, or liver function, using tissue factor, SEC receptor or other receptors that are excluded by mediation, or by specific or non-specific protein hydrolysis. Usually, clearance rate depends on size (relative to glomerulus) Filtered Responsive), charge, additional carbohydrate chains, and the presence of protein receptors. The functionality to be retained is typically selected from receptor binding or receptor activation. The functional in vivo half-life and serum half-life can be determined by any suitable method known in the art, for example, generally involving the step of appropriately administering to a mammal a suitable dose of a therapeutic amino acid sequence or compound; periodically collected from the mammal. a blood sample or other sample; determining the content or concentration of the amino acid sequence or compound of the present invention in the blood sample; and calculating the initial concentration at the time of administration from the data thus obtained (map), the present invention The time or concentration of the amino acid sequence or compound is reduced by 50%. Reference is now made to the reference standard manuals, such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinete analysis: A Practical Approach (1996); reference may also be made to "Pharmacokinetics" by Marcel Dekker, M Gibaldi & D Perron, 2nd Rev.edition (1982).

"Glycosylation" is a chemical modification in which a sugar group is added to a specific portion of a polypeptide. The glycosylation of a polypeptide is generally an N-linked or O-linked. N-linked refers to the attachment of a carbohydrate group to the side chain of an aspartate residue. Tripeptide sequence, Asn-X-Ser and Asn-X-Thr ("NXS/T"), where X is any amino acid other than lysine, which is used to attach a carbohydrate group to aspartate The identification sequence of the side chain. Thus, the presence of any of the other tripeptide sequences (or characteristic structures) in the polypeptide results in a potential N-linked glycosylation site. O-linkage refers to the attachment of a carbohydrate group to the hydroxyl oxygen of serine and threonine.

"Isolated" fusion polypeptides are those that have been identified and separated from the cellular components that express them. The contaminating component of the cell will interfere with the diagnostic or therapeutic use of the fusion polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the fusion polypeptide is purified to (1), for example, by the Lowry method, ultraviolet-visible spectroscopy, or by SDS-capillary gel electrophoresis (eg, in a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer device). Above) greater than 95% by weight of the fusion polypeptide, and in further preferred embodiments greater than 99% by weight; (2) to a degree sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues; (3) Uniformity is achieved via SDS-PAGE using Coomassie blue or preferably silver staining under reducing or non-reducing conditions. Typically, however, the isolated fusion polypeptide will be prepared by at least one purification step.

Review

The present application provides an ALB fusion polypeptide, and the term also used herein is a single-chain relaxin abbreviated as scRelaxin or scR, wherein the "A" relaxin A chain, the "B" relaxin B chain and the "L" are Linker polypeptide. The present application describes an enhanced relaxin molecule in which the C-terminus of the A chain is linked to the N-terminus of the B chain via a polypeptide linker such that the fusion polypeptide is expressed as a functional scRelaxin. Part of the present application relates to the surprising discovery that A-L-B fusion polypeptides may not be required to be endoproteolytically treated as known for wild-type relaxin, but may be functionally expressed.

Single-stranded form of relaxin Relaxin A and B functional areas:

A specific example of the invention is a fusion polypeptide comprising A-L-B, wherein A comprises a relaxin A chain polypeptide or a functional variant thereof, B comprises a relaxin B chain polypeptide or a functional variant thereof and L is a linker polypeptide.

A further embodiment of the invention is a fusion polypeptide comprising ALB, wherein A comprises a relaxin A chain polypeptide or a functional variant thereof, B comprises a relaxin B chain polypeptide or a functional variant thereof, and L is a linker polypeptide; The prime system is selected from the group consisting of relaxin 1, relaxin 2, relaxin 3, INSL3, INSL4, INSL5, and INSL6. In a further preferred embodiment, the relaxin is relaxin 2 or relaxin 3. In a further embodiment, the relaxin is human relaxin.

In a further embodiment, the relaxin A chain polypeptide of A-L-B comprises a relaxin 2A chain polypeptide or a functional variant thereof. In a further embodiment, the relaxin B chain polypeptide of A-L-B comprises a relaxin 2B chain polypeptide or a functional variant thereof.

In a further embodiment, the relaxin A chain polypeptide of A-L-B comprises a relaxin 2A chain polypeptide or a functional variant thereof and a relaxin B chain polypeptide comprising a relaxin 2B chain polypeptide or a functional variant thereof.

In a preferred embodiment, the relaxin A chain polypeptide of ALB comprises a human minimal relaxin 2A chain polypeptide (SEQ ID NO: 118) or a functional variant thereof, or comprises a human relaxin 2A chain polypeptide (SEQ ID NO: 117) ) or a functional variant thereof. In a preferred embodiment, the relaxin B chain polypeptide of A-L-B comprises a human relaxin 2 B chain polypeptide (SEQ ID NO: 119) Or a functional variant thereof.

In a more preferred embodiment, the relaxin A chain polypeptide of ALB comprises a human minimal relaxin 2A chain polypeptide (SEQ ID NO: 118) or a functional variant thereof, or comprises a human relaxin 2A chain polypeptide (SEQ ID NO: 117) Or a functional variant thereof, and a relaxin B chain polypeptide comprising a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof.

In a further embodiment, the relaxin A chain polypeptide of A-L-B comprises a relaxin 3A chain polypeptide or a functional variant thereof. In a further embodiment, the relaxin B chain polypeptide of A-L-B comprises a relaxin 3B chain polypeptide or a functional variant thereof.

In a further embodiment, the relaxin A chain polypeptide of A-L-B comprises a human relaxin 3A chain polypeptide (SEQ ID NO: 124) or a functional variant thereof. In a further embodiment, the relaxin B chain polypeptide of A-L-B comprises a human relaxin 3B chain polypeptide (SEQ ID NO: 125) or a functional variant thereof. In a preferred embodiment, the relaxin A chain polypeptide of ALB comprises a human relaxin 3A chain polypeptide (SEQ ID NO: 124) or a functional variant thereof and a relaxin B chain polypeptide comprising a human relaxin 3B chain polypeptide (SEQ ID NO: 125) or a functional variant thereof.

In a preferred embodiment of the above fusion polypeptide ALB, the functional variants of relaxin A or B chain have 1, 2, 3, 4, 5, 6, 7 respectively compared to the wild type relaxin A and B chains. Replacement, insertion, and/or deletion of 8, 9, or 10 amino acids. It is further preferred that the relaxin 2B variant further comprises a conserved characteristic structure Arg-X-X-X-Arg-X-X-Ile/Val-X.

Relaxin A and B chain variants are known in the art. The well-characterized relaxin binding site geometry is well understood to provide guidance to those skilled in the art of designing relaxin A and B chain variants, for example, regarding changes in the relaxin B chain, see Büllesbach and Schwabe J Biol Chem. 2000 Nov 10; (45): 35276-80; for changes in the relaxin A chain and the "minimum" relaxin A chain, see Hossain et al. J Biol Chem. 2008 Jun 20; 283(25): 17287-97. For example, in the case of the conserved relaxin 2B characteristic structure (Arg-XXX-Arg-XX-Ile/Val-X), X represents an example of the formation of a helical structure to select an appropriate amino acid X in a conserved characteristic structure. As the amino acid of the amino acid defined by the triangular contact region on the surface of the relaxin B chain [Büllesbach and Schwabe J Biol Chem. 2000 Nov 10; 275 (45)].

In still a further preferred embodiment, the relaxin A chain polypeptide of ALB is a human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof and a relaxin B chain polypeptide is a human relaxin 2 B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof. In yet a further preferred embodiment, the functional variant of the human relaxin 2A chain polypeptide (SEQ ID NO: 117) has 1, 2, 3, 4, 5, 6, compared to SEQ ID NO:117. A functional variant of 7, 8, 9, or 10 amino acid substitutions, deletions, and/or insertions. Further preferred is a functional variant of the human relaxin 2B chain polypeptide (SEQ ID NO: 119), wherein the functional variant has 1, 2, 3, 4, 5 compared to SEQ ID NO: 6, 6, 8, 9, or 10 amino acid substitutions, deletions, and/or insertions. Still further preferably further comprising a conserved feature structure Arg-X-X-X-Arg-X-X- The above human relaxin 2B variant of Ile/Val-X.

In still a further preferred embodiment, the relaxin A chain polypeptide of ALB is a human relaxin 2A chain polypeptide (SEQ ID NO: 117) or, when compared to SEQ ID NO: 117, has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid exchange functional variants, and relaxin B chain polypeptide is a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or SEQ ID NO: When 119 is compared, it has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid exchanges and contains a conserved characteristic structure Arg-XXX-Arg-XX-Ile/Val-X Functional variants.

Those skilled in the art will know how to obtain functional variants; examples of functional variants are disclosed in Hossain et al J Biol Chem. 2008 Jun 20; 283(25): 17287-97, or US patents on the relaxin A chain. Bulletin No. US 2011/0130332; and Schwabe and Büllesbach (2007) Adv Exp Med Biol. 612: 14-25 on the relaxin B chain and Büllesbach and Schwabe J Biol Chem. 2000 Nov 10; 275(45): 35276- 80.

Linker L:

In a specific embodiment, the linker polypeptide L of the above fusion polypeptide ALB is composed of a polypeptide having a length of 6 to 14 amino acid residues; further preferably a polypeptide linker L having a length of 7 to 13 amino acid residues Further preferred is a polypeptide linker L having a length of 8 to 12 amino acid residues; more preferably a polypeptide linker L having a length of 7 to 11 or 9 to 11 amino acid residues; more preferably a polypeptide linker L having a length of 9 amino acid residues; in a further preferred embodiment, the integer length of the polypeptide linker L is selected from the group consisting of an integer of 6, Groups of 7, 8, 9, 10, 11, 12, 13 and 14.

The amino acid composition of the linker can vary, but a linker that exhibits a low immunogenic score is preferred. Examples of linkers are well known to those skilled in the art and include sequences such as (GGGS)n, (GGSG)n, where n is an integer. The linker peptide L can be composed of any amino acid. In a preferred embodiment, the linker polypeptide L comprises at least one Gly, Ser, Arg, Cys, Leu and/or Lys residue. In a more preferred embodiment, the linker polypeptide L comprises Gly and Ser residues. In a further preferred embodiment, the linker peptide L is a glycine-rich linker, for example, comprising a peptide of the [GGGGS] _n sequence as disclosed in U.S. Patent No. 7,271,149. In other embodiments, a serine-rich linker as described in U.S. Patent No. 5,525,491 is used.

Further preferred embodiments are linker L comprising a Gly to Ser residue and a Gly to Ser ratio of at least 3 to 1.

In a further embodiment, the linker L comprises at least one linker for covalent coupling of a non-proteinaceous polymer half-life extending group. In a specific embodiment of the invention, the above-mentioned linking site is a Lys or Cys residue.

Examples of such a linker are [GlyGlyGlySerGlyGly] (SEQ ID NO: 137), [GlyGlyGlySerGlyGlyGly] (SEQ ID NO: 138), [GlyGlyGlySerGlyGlyGlySerGly] (SEQ ID NO: 139), [GlyGlyGlySlyGlyGlyGlySerGlyGlyGlySer] (SEQ ID NO: 140) , [GlyGlyGlySerGlyCysGlyGlySerGly] (SEQ ID NO: 141), [GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGly] (SEQ ID NO: 143), [LysArgSerLeuSerArgLysLysArg] (SEQ ID NO: 144), [GlyGlyGlySerGlyLysGlyGlySerGly] (SEQ ID NO: 142), [GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly] (SEQ ID NO: 145), and [GlyGlyGlySerGlyGlyGlySerGlyGlyGly] (SEQ ID NO: 146).

In general, the length of the optimal linker and the amino acid composition can be determined using routine methods known in the art.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117), or 1, 2, 3, 4, compared to SEQ ID NO: a functional variant of 5, 6, 7, 8, 9, or 10 amino acid substitutions, a B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119), or a comparison thereof to SEQ ID NO: 119 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and comprising a functional variant of the conserved signature Arg-XXX-Arg-XX-Ile/Val-X, and L-linker polypeptides of 6-14, 7-13, 8-12, 7-11, 9-11, or 9 amino acid residues are long.

In a preferred embodiment, the linker polypeptide L of the above fusion polypeptide A-L-B is 7-11, or 9-11 amino acid residues are long. More preferably, it is a polypeptide linker L of 9 amino acid residues. In a further preferred embodiment, the integer of the length of the polypeptide linker L is selected from the group consisting of integers 6, 7, Groups of 8, 9, 10, 11, 12, 13 and 14. The linker polypeptide L can be composed of any amino acid. In a preferred embodiment, the linker polypeptide L is a bendable linker.

In a preferred embodiment, linker polypeptide L comprises at least one Gly, Ser, Arg, Leu, Cys, and/or Lys residue. In a further preferred embodiment, the linker polypeptide L consists of an amino acid residue selected from the group consisting of Gly, Ser, Arg, Leu, Cys, and the amino acid group of the Lys residue.

In a more preferred embodiment, the linker polypeptide L comprises Gly and Ser residues. In a further preferred embodiment, the linker peptide L is a glycine-rich linker, for example, comprising a peptide of the [GGGGS] _n sequence as disclosed in U.S. Patent No. 7,271,149. In other embodiments, a serine-rich linker peptide L as described in U.S. Patent No. 5,525,491 is used.

Further preferred embodiments are linker polypeptides L comprising Gly and Ser residues and having a Gly to Ser ratio of at least 2 to 1.

Further preferred embodiments are linker polypeptides L comprising Gly and Ser residues and having a Gly to Ser ratio of at least 3 to 1.

A further preferred embodiment is a linker polypeptide L comprising a Gly to Ser residue and a Gly to Ser ratio of at least 1 to 2.

Further preferred embodiments are linker polypeptides L comprising Gly and Ser residues and having a Gly to Ser ratio of at least 1 to 3.

Further preferred embodiments are the linker polypeptides L having the preferred length described above, wherein the amino acid residues of the linker L are all but four. The moiety consists of Gly and/or Ser residues, and the remaining four amino acid residues are selected from the group of natural amino acids.

Further preferred embodiments are the linker polypeptides L having the preferred length described above, wherein the amino acid residues of the linker L are all composed of Gly and/or Ser residues except for three, and the remaining three amino acids are The residue is selected from the group of natural amino acids.

Further preferred embodiments are the linker polypeptides L having the preferred length described above, wherein the amino acid residues of the linker L are all composed of Gly and/or Ser residues except for two, and the remaining two amino acids are The residue is selected from the group of natural amino acids.

Further preferred embodiments are the linker polypeptides L having the above preferred length, wherein the amino acid residues of the linker L are all composed of Gly and/or Ser residues except for one, and the remaining amino acid residues are It is selected from the group of natural amino acids.

In a further preferred embodiment, the natural amino acid group described above does not include a phthalic acid amino acid.

Further preferred embodiments are the linker polypeptides L having the above preferred length, wherein the amino acid residues of the linker L are all composed of Gly and/or Ser except for one, and the remaining amino acid residues are It is selected from the group of Cys and Lys.

In a further preferred embodiment, the linker polypeptide L is comprised of an amino acid residue selected from the group consisting of amino acid residues comprising Gly and Ser residues.

In a further preferred embodiment, the linker L is selected from the group consisting of Gly is composed of an amino acid residue of the amino acid group of the Ser residue, wherein the ratio of Gly to Ser is at least 2 to 1.

In a further preferred embodiment, linker L is comprised of an amino acid residue selected from the group consisting of amino acid groups comprising Gly and Ser residues, wherein the ratio of Gly to Ser is at least 3 to 1.

In a further preferred embodiment, linker L is comprised of an amino acid residue selected from the group consisting of amino acid groups comprising Gly and Ser residues, wherein the ratio of Gly to Ser is at least 1 to 2.

In a further preferred embodiment, linker L is comprised of an amino acid residue selected from the group consisting of amino acid groups comprising Gly and Ser residues, wherein the ratio of Gly to Ser is at least 1 to 3.

In a further embodiment, the linker L comprises at least one linker for covalent coupling of a non-proteinaceous polymer half-life extending group. In a specific embodiment of the invention, the above-mentioned linking site is a Lys or Cys residue.

Preferably, the linker polypeptide L is selected from the group consisting of a linker polypeptide comprising: [GlyGlyGlySerGlyGly] (SEQ ID NO: 137), [GlyGlyGlySerGlyGlyGly] (SEQ ID NO: 138), [GlyGlyGlySerGlyGlyGlySerGly] (SEQ ID NO: 139), [GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer] (SEQ ID NO: 140), [GlyGlyGlySlyGlyCysGlyGlySerGly] (SEQ ID NO: 141), [GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGly] (SEQ ID NO: 143), [LysArgSerLeuSerArgLysLysArg] (SEQ ID NO: 144), [GlyGlyGlySerGlyLysGlyGlySerGly] (SEQ ID NO: 142), [GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly] (SEQ ID NO: 145), and [GlyGlyGlySlyGlyGlyGlySerGlyGlyGly] (SEQ ID NO: 146).

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, and the B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and a linker polypeptide having an L-system of 7, 8, 9 or 10 amino acids.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or its SEQ ID NO: 117 has 1, 2, 3, 4, 5 a functional variant of 6, 7, 8, 9, or 10 amino acid substitutions, B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119) or 1 compared to SEQ ID NO: 119 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and functional variants containing the conserved feature Arg-XXX-Arg-XX-Ile/Val-X, and the L system 7, 8, 9 or 10 amino acid long linker polypeptides.

A preferred embodiment of the invention is a fusion polypeptide comprising A-L-B, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117), B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119), and a L-linked 7, 8, 9 or 10 amino acid long linker polypeptide.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, and the B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and L-linked 9 amino acid long linker polypeptides.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or its SEQ ID NO: 117 has 1, 2, 3, 4, 5 a functional variant of 6, 7, 8, 9, or 10 amino acid substitutions, B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119) or 1 compared to SEQ ID NO: 119 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and functional variants containing the conserved feature Arg-XXX-Arg-XX-Ile/Val-X, and the L system 9 amino acid long linker polypeptides.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117), the B is a human relaxin 2 B chain polypeptide (SEQ ID NO: 119), and the L system 9 amino acid long linker polypeptides.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, and the B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and L-linked 7, 8, 9 or 10 amino acid long linker polypeptides comprising a glycine acid and a serine residue in a ratio of at least 3 to 1.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or its SEQ ID NO: 117 has 1, 2, 3, 4, 5 a functional variant of 6, 7, 8, 9, or 10 amino acid substitutions, B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119) or 1 compared to SEQ ID NO: 119 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and functional variants containing the conserved feature Arg-XXX-Arg-XX-Ile/Val-X, and the L system 7, 8, 9 or 10 amino acid long linker polypeptides comprising a glycine acid and a serine residue in a ratio of at least 3 to 1.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117), the B is a human relaxin 2 B chain polypeptide (SEQ ID NO: 119), and the L system 7, 8, 9 or 10 amino acid long linker polypeptides, including ratio Examples are at least 3 to 1 glycine and serine residues.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, and the B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and L-linked 9 amino acid long linker polypeptide comprising a glycine acid and a serine residue in a ratio of at least 3 to 1.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or its SEQ ID NO: 117 has 1, 2, 3, 4, 5 a functional variant of 6, 7, 8, 9, or 10 amino acid substitutions, B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119) or 1 compared to SEQ ID NO: 119 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and functional variants containing the conserved feature Arg-XXX-Arg-XX-Ile/Val-X, and the L system A 9 amino acid long linker polypeptide comprising a glycine acid and a serine residue in a ratio of at least 3 to 1.

A preferred embodiment of the invention is a fusion polypeptide comprising A-L-B, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117), the B is a human relaxin 2 B chain polypeptide (SEQ ID NO: 119), and L is a 9 amino acid long linker polypeptide comprising a glycine acid and a serine residue in a ratio of at least 3 to 1.

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, and the B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and a linker polypeptide having the sequence of GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139).

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or its SEQ ID NO: 117 has 1, 2, 3, 4, 5 a functional variant of 6, 7, 8, 9, or 10 amino acid substitutions, B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119) or 1 compared to SEQ ID NO: 119 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and functional variants containing the conserved feature Arg-XXX-Arg-XX-Ile/Val-X, and the L system A linker polypeptide having the sequence of GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139).

A preferred embodiment of the invention is a fusion polypeptide comprising A-L-B, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117), B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119), and L is a linker polypeptide having a GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139) sequence.

A more preferred embodiment of the invention is a fusion polypeptide comprising the sequence of scR4 (SEQ ID NO: 4).

A more preferred embodiment of the invention is a fusion polypeptide comprising a sequence of a markerless scR4 (SEQ ID NO: 45).

A preferred embodiment of the invention is a fusion polypeptide comprising ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or its SEQ ID NO: 117 has 1, 2, 3, 4, 5 a functional variant of 6, 7, 8, 9, or 10 amino acid substitutions, B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119) or 1 compared to SEQ ID NO: 119 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and functional variants containing the conserved feature Arg-XXX-Arg-XX-Ile/Val-X, and the L system A linker polypeptide selected from the group consisting of a linker peptide having a linker of the amino acid sequence of SEQ ID NO: 137-146.

The linker can be between 6 and 14 amino acids in length, while the longer linker peptide itself can convey additional functions as understandable.

In a further embodiment, the fusion polypeptide ALB has relaxin activity. In a further preferred embodiment, the relaxin activity is activation of the relaxin receptor LGR7. Methods for determining relaxin activity are known in the art or are provided herein. In still further preferred embodiments, the activation of the relaxin receptor LGR7 is determined using the methods disclosed in the experimental methods herein. In yet further preferred embodiment, the relaxin receptors LGR7 activation of the measurement system ₅₀ measurement values EC. In another more preferred embodiment, the above-relaxin activity compared to the corresponding wild type induced an effective concentration of the half maximum active relaxin, which reduces less than ¹⁰⁵ times, ¹⁰⁴ times, ¹⁰³ times, 100 times, 75 times, 50 Times, 25 times or 10 times. For example, in the case of a fusion polypeptide of human relaxin 2, the corresponding wild-type relaxin of ALB is a human relaxin 2 protein.

Enhancement of biological half-life of single-chain relaxin variants

The increase in the half-life of the fusion polypeptides of the invention can be achieved by the addition of a half-life extending group.

In a particular embodiment of the invention, the fusion polypeptide A-L-B further comprises at least one half-life extending group. In one embodiment, the half-life extending group is a proteinaceous or non-proteinaceous polymer.

The half-life extension of the extended half-life group via a non-proteinaceous polymer:

Promoting the biological half-life of the fusion polypeptide ALB, which can be covalently coupled to a non-proteinaceous polymer half-life extending group comprising an extended polypeptide comprising a non-proteinaceous polymer half-life extending group attachment site fused to the ALB N and/or C-terminus Achieving; methods of attaching such groups are known in the art.

The non-proteinaceous polymer half-life extending group can be covalently coupled to the junction of the fusion polypeptide A-L-B. The linking site may be within A, L or B, or a polypeptide comprising such a joining site fused to the N-terminus and/or C-terminus of the above-described fusion polypeptide A-L-B. Preferred by connection The sub-peptide L, or a fusion of the N and/or C-terminus of the fusion polypeptide A-L-B comprising an extension of the junction. The linking site can be a linking amino acid, such as Cys or Lys, or a sugar moiety of a carbohydrate.

The non-proteinaceous polymeric molecule to be coupled to the variant polypeptide can be any suitable polymeric molecule, such as a natural or synthetic homopolymer or heterogeneous polymer, typically having a molecular weight in the range of from about 300 to 100,000 Da, for example It is preferably in the range of about 500 to 20,000 Da; more preferably in the range of about 500 to 15,000 Da; more preferably in the range of about 2 to 12 kDa, for example, in the range of about 3 to 10 kDa. The use of the term "about" in reference to a particular molecular weight herein refers to an approximate average molecular weight and reflects the fact that a particular molecular weight distribution is typically present in the preparation of a given polymer. Examples of homopolymers include polyols (i.e., multiple OH), polyamines (i.e., multiple NH2), and polycarboxylic acids (i.e., poly-COOH). A heterogeneous polymer is a polymer comprising different coupling groups, such as a hydroxyl group and an amine group.

Examples of suitable polymer molecules include those selected from polyalkylene oxides (PAO) including polyalkylene glycols (PAG) such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched chain PEG, hydroxyalkyl starch (HAS). For example, hydroxyethyl starch (HES), polysialic acid (PSA), polyvinyl alcohol (PVA), polycarboxylate, poly(vinylpyrrolidone), ethylene-maleic anhydride copolymer, styrene - maleic anhydride copolymer, glucan including carboxymethyl-glucan, or any other biopolymer suitable for reducing immunogenicity and/or increasing functional in vivo half-life and/or serum half-life Group of polymer molecules. Another example of a polymer molecule is human albumin or another high plasma protein. Usually, the polyalkylene glycol-derived polymer is Biocompatible, non-toxic, non-antigenic, non-immunogenic, with a variety of water solubility, and easy to excrete from the organism.

PEG is a preferred polymer molecule because it has only a few crosslinkable reactive groups compared to, for example, polysaccharides such as dextran. In particular, monofunctional PEGs, such as methoxypolyethylene glycol (mPEG), are of interest due to their relatively simple coupling chemistry (only one reactive group can be used to conjugate to a linking group on a polypeptide). As a result, since the risk of cross-linking is excluded, the resulting conjugated fusion polypeptide of the present invention is more homogenous, and thus the reaction of the polymer molecule with the variant polypeptide is more easily controlled.

In order to covalently link a polymer molecule to a fusion polypeptide of the present invention, the hydroxyl end group of the polymer molecule must be in an activated form, that is, a reactive functional group [examples thereof include a primary amine group, hydrazine (HZ), sulfur Alcohol, succinate (SUC), amber succinimide succinate (SS), amber succinimide succinimide (SSA), amber succinyl propionate (SPA), amber succinimide Root (SBA), amber quinone iminocarboxymethyl (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenyl carbonate (NPC), and Provided by trifluoroethanesulfonate (TRES). Suitable activated polymer molecules are commercially available, for example, from Shearwater Polymers, Inc., Huntsville, AL, USA, or from PolyMASC Pharmaceuticals plc, UK.

Alternatively, the polymer molecules can be activated using conventional methods known in the art (e.g., as disclosed in WO 90/13540). Specific examples of activated linear or branched polymer molecules useful in the present invention are described in Shearwater Polymers, Inc. 1997 and 2000. (Functionalized Biocompatible Polymers for Research and Pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEG: NHS-PEG (eg, SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM- PEG), NOR-PEG, BTC-PEG, EPOXPEG, NCO-PEG, NPC-PEG, CD1-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched-chain PEG For example, PEG2-NHS and US 5, 932, 462 and US 5, 643, 575, both incorporated herein by reference. Furthermore, the following publications disclose useful polymer molecules and/or PEGylation chemistry: US 5,824,778, US 5,476,653, WO 97/32607, EP 229,108, EP 402,378, US 4,902,502, US 5,281,698, US 5,122,614, US 5,219,564 WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO 95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, US 5,736, 625, WO 98/05363, EP 809 996, US 5,629,384, WO 96/41813, WO 96/07670, US 5,473,034 US 5,516,673, EP 605 963, US 5,382,657, EP 510 356, EP 400 472, EP 183 503 and EP 154 316.

Specific examples of particularly preferred activated PEG polymers for coupling to cysteine residues include the following linear PEG: vinyl hydrazine-PEG (VS-PEG), preferably vinyl hydrazine-mPEG (VS-mPEG); Butyleneimine-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) and o-pyridyl-disulfide-PEG (OPSS-PEG), preferably O-pyridyl-disulfide-mPEG (OPSS-mPEG). In general, such PEG or mPEG polymers have a size of about 5 kDa, about 10 kD, about 12 kDa, or about 20 kDa.

The conjugated binding of the fusion polypeptides of the invention to the activated polymer molecules is carried out by using any of the conventional methods described, for example, in the following references, which also describe suitable methods for activating polymer molecules: Harris and Zalipsky, Eds., Poly(ethylene glycol) Chemistry and Biological Applications, AZC Washington; RF Taylor, (1991), "Protein immobilisation. Fundamental and applications", Marcel Dekker, NY; SSWong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; GTHermanson et al., (1993), "Immobilized Affinity Ligand Techniques", Academic Press, NY).

It is known to those skilled in the art that the activation method to be used and/or the linking group of the conjugated chemical fusion polypeptide, examples of which are described above, and the functional groups of the polymer (for example, amines, hydroxyl groups, carboxyl groups, aldehydes, sulfhydryl groups, Amber quinone imine, maleimide, vinyl hydrazine or haloacetate). PEGylation can directly target all available linking groups on the fusion polypeptide (ie, such a linking group that is exposed to the surface of the polypeptide) or may be directed to one or more specific linking groups, such as the N-terminal amine group described in US 5,985,265 or the conjugated binding to a cysteine residue. . Furthermore, conjugate binding can be achieved in one step or stepwise manner (for example as described in WO 99/55377).

For pegylation of cysteine residues (see above), prior to pegylation, the fusion polypeptide is typically treated with a reducing agent such as dithiothreitol (DDT); followed by any conventional method ( For example, desalting method) removes the reducing agent. Conjugation of PEG to cysteine residues is typically carried out in a suitable buffer at pH 6-9, at temperatures ranging from 4 ° C to 25 ° C, for up to 16 hours.

It will generally be understood that PEGylation is designed to produce the number of PEG molecules involved in the linkage, the size and shape of such molecules (eg, whether they are linear or branched), and the attachment sites in the fusion polypeptide. Optimum molecule. The molecular weight of the polymer to be used, for example, can be selected according to the desired efficacy to be achieved.

With respect to a single linking group (e.g., an N-terminal amine group) conjugated only to a protein, the polymer molecule which may be a linear or branched chain has a high molecular weight (preferably about 10-25 kDa, for example, about 15- 25 kDa, for example about 20 kDa) is advantageous.

Normally, the polymer is conjugated to the reaction for reacting a plurality of available polymer linking groups with the polymer molecules; this is achieved using a polymer with a suitable molar excess relative to the polypeptide. Typically, the molar ratio of activated polymer molecules to polypeptides is up to about 1000-1, for example Such as up to about 200-1, or up to about 100-1; however, in some cases, this ratio may be slightly lower, such as up to about 50-1, 10-1, 5-1, 2-1, or 1-1. Get the best response.

The attachment of a polymer molecule to a polypeptide via a linker is also contemplated in accordance with the invention. Suitable linkers are well known to those skilled in the art; a preferred example is cyanuric chloride [Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; US 4,179,337; Shafer et al., ( 1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378])].

After conjugation, the remaining activated polymer molecules are blocked according to methods known in the art, for example, by adding a primary amine to the reaction mixture, and the resulting inactivated polymer molecules are removed in a suitable manner.

It will generally be understood that depending on the circumstances, such as the amino acid sequence of the fusion polypeptide, the nature of the activated PEG compound used, and the specific PEGylation conditions (including the molar ratio of PEG to the polypeptide), different degrees of PEGylation can be obtained. Higher PEG-to-fusion polypeptide ratios typically result in higher degrees of pegylation. However, the PEGylated fusion polypeptide produced by any given pegylation procedure typically comprises a randomly distributed conjugated fusion polypeptide having a slightly different degree of PEGylation.

For the biological half-life of the relaxin or the fusion polypeptide of the present invention, chemical modification methods such as pegylation or hydroxyethylamylation are applicable.

HAS and HES non-proteinaceous polymers, as well as methods for producing HAS or HES conjugates are disclosed, for example, in WO 02/080979, WO 03/070772, WO057092391 and WO057092390.

Polysialylation is another technique that uses natural polymer polysialic acid (PSA) to extend half-life and enhance the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar); when used for protein and therapeutic peptide delivery, polysialic acid provides a protective niche for conjugate binding; this increases the active life of the therapeutic protein in the circulation, preventing It is recognized by the immune system. PSA polymers are naturally found in humans; they are absorbed by specific bacteria that have evolved over millions of years to cover the surface of their cell walls; then their natural polysialytic bacteria can block the body's defenses by virtue of molecular mimicry. system. PSA, the ultimate stealth technology of nature, can be easily mass produced from such bacteria and has predetermined physical properties. Since the bacterial PSA is chemically identical to the PSA in the human body, it is completely non-immunogenic even when it is linked to a protein.

The half-life of the extended group is extended by the half-life of the protein:

Further possibilities for enhancing the half-life of the fusion polypeptide ALB, commonly used as a prolonged group of protein properties, such as an immunoglobulin Fc fragment of an antibody, a transferrin, a transferrin receptor or at least a transferrin binding portion thereof, serum white A fusion of a protein, or a variant thereof, or a binding module, such as a serum albumin binding protein, that binds to a longer half-life molecule in vivo.

The above sc relaxin polypeptide can be fused to the Fc portion of the immunoglobulin directly or via a peptide linker. An "immunoglobulin" is a molecule comprising a polypeptide chain joined together by disulfide bonds, typically having two light chains and two heavy chains. In each chain, a functional region (V) has a molecular antibody-specific The variable amino acid sequence; the other functional regions (C) have a fairly constant sequence shared by the same class of molecules.

The "Fc" portion of an immunoglobulin as used herein has the meaning given to the term commonly used in the field of immunology. Specifically, the term refers to an antibody fragment obtained by removing two antigen-binding regions (Fab fragments) from an antibody. One method of removing Fab fragments is to hydrolyze immunoglobulins with papain. Thus, the Fc portion is formed by fragments of approximately the same size from the two heavy chain constant regions joined together via non-covalent interactions and disulfide bonds. The Fc portion can include a hinge region and extends to the C-terminus of the antibody via the CH2 and CH3 functional regions. Representative hinge regions for human and mouse immunoglobulins can be found in Antibody Engineering, A Practical Guide, Borrebaeck, C.A.K., ed., W.H. Freeman and Co., 1992.

There are five human immunoglobulin Fc regions with different effector and pharmacokinetic properties: IgG, IgA, IgM, IgD, and IgE. IgG is the largest amount of immunoglobulin in serum; it also has the longest half-life (23 days) in the serum of any immunoglobulin. Unlike other immunoglobulins, IgG is efficiently recirculated after binding to Fc receptors. The IgG subpopulations are G1, G2, G3, and G4, each with different effector functions. These effector functions are usually mediated via interaction with the Fc receptor (FcγR) or by binding to C1q and fixed complement. Binding to FcγR results in antibody-dependent cell-mediated cell lysis, whereas binding to complement factors results in the breakdown of cells that complement-mediated. In designing heterologous Fc fusion proteins in which the Fc portion is utilized only for its half-life extending ability, it is important to minimize any effector function. All IgG subpopulations can bind For Fc receptors (CD16, CD32, CD64), G1 and G3 are more potent than G2 and G4. The Fc receptor binding region of IgG is formed by residues located in the hinge region of the CH2 functional region as well as the carboxy terminal region.

Depending on the desired in vivo effect, a heterologous fusion protein of the invention may comprise any of the above isotypes or may comprise a mutated Fc region in which complement and/or Fc receptor binding functions have been altered. Thus, a heterologous fusion protein of the invention may comprise an entire immunoglobulin Fc portion, a fragment of an immunoglobulin Fc portion, or an analog thereof fused to a sc relaxin compound.

Regardless of the final structure of the fusion protein, the Fc or Fc-like region must be useful for extending the in vivo plasma half-life of sc relaxin compounds fused to the C-terminus or the N-terminus. Preferably, the fused sc relaxin compound retains several biological activities. Biological activity can be determined using in vitro and in vivo methods known in the art.

Preferably, the Fc region for the heterologous fusion protein of the invention is derived from the IgGl or IgG2 Fc region.

In general, the Fc region for use in the heterologous fusion proteins of the invention can be derived from any species, including, but not limited to, humans, rats, mice, and pigs. Preferably, the Fc region for use in the present invention is derived from human or rat; however, the best is the human Fc region and fragments and variants thereof to reduce the risk of immunogenicity of the fusion protein in humans. The "primary sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from the original sequence Fc region by alteration of at least one amino acid. Preferably, the variant Fc region has at least one amino acid compared to the native sequence Fc region or the parent polypeptide Fc region. Alternatively, for example, from about one to about ten amino acid substitutions, preferably from about one to about five amino acid substitutions, in the native sequence Fc region or the parent polypeptide Fc region. The variant Fc region herein preferably has at least about 80% sequence identity to the native sequence Fc region and/or to the parent polypeptide Fc region, preferably at least about 90% sequence identity, more preferably at least about 95%. Sequence identity.

The above sc relaxin compound can be fused to albumin or an analog, fragment or derivative thereof directly or via a peptide extender. Typically, the albumin protein that constitutes a fusion protein of the invention can be derived from albumin that is transfected from any species. However, human albumin and its fragments and analogs are preferred to reduce the risk of immunogenicity of the fusion protein in humans. Human serum albumin (HSA) consists of a single non-glycosylated polypeptide chain of 585 amino acids with a molecular weight of 66,500. The amino acid sequence of HAS (SEQ ID NO: 123) has been described, for example, in Meloun, et al. (1975); Behrens, et al. (1975); Lawn, et al. (1981) and Minghetti, et al. (1986). ) in the narrative. Various polymorphic variants of albumin, as well as analogs and fragments, have been described [see Weitkamp, et al. (1973)]. For example, EP 0 322 094 and EP 0 399 666 disclose various human serum albumin fragments. It is generally understood that the heterologous fusion protein of the present invention comprises a sc relaxin compound linked to any albumin protein comprising a fragment, an analog, and a derivative, wherein the fusion protein is biologically active and has more activity than the sc relaxin compound alone. Long plasma half-life. Therefore, the albumin portion of the fusion protein does not necessarily need to have a plasma half-life equal to that of the native human albumin. These fragments, analogs, and derivatives are known or can produce longer half-lives or have a native human albumin and sense The half-life between the compounds of interest sc relaxin. Such techniques are well known in the art, see, for example, WO 93/15199, WO 93/15200, WO 01/77137 and EP 0413622.

In a particular embodiment of the invention, the protein nature half-life extending group is immunogenic and is human or anthropomorphic. In a preferred embodiment, the proteinaceous half-life extending group is a human, such as human transferrin (SEQ ID NO: 122), human serum albumin (SEQ ID NO: 123), or human IgG1 Fc (SEQ ID NO: 120).

Additionally, other proteins, protein functional regions or peptides that enhance biological half-life can also be used as fusion partners.

The half-life extension via fusion to human serum albumin is disclosed, for example, in WO 93/15199; albumin binding for general strategies for enhancing protein pharmacokinetics is described, for example, in Dennis et al., The Journal of Biological Chemistry, Vol. 277, No 38. , Issue of September 20, pp. 35035-35043; extended half-life via fusion to human serum albumin binding proteins is disclosed, for example, in US20100104588; extended half-life via fusion to human serum albumin or IgG-Fc binding proteins is disclosed, for example, in WO01/ Further examples of half-life extension by fusion to human serum albumin binding peptides are disclosed in WO2010/054699.

The half-life extension via fusion to the Fc functional region is disclosed, for example, in WO2001/058957.

Biological activity determines the preferred orientation of the protein of interest to its fusion partner; the C-terminus and N-terminal orientation of the fusion partner are included. In addition, In order to enhance biological half-life or other functions, phosphorylation, sulfation, propylene deuteration, glycosylation, deglycosylation, methylation, farnesylation, acetamylation, guanidation, etc. may be utilized. Integration partner.

The proteinaceous half-life extending group is recombinantly fused to the N-terminus and/or C-terminus of the above-described fusion polypeptide A-L-B; the fusion may or may not have an additional stretcher polypeptide. Examples of protein nature half-life extending groups are transferrin, transferrin receptor or at least its transferrin binding moiety, serum albumin, serum albumin binding protein, immunoglobulin, and immunoglobulin Fc functional regions. Preferred are human protein property half-life extending groups, such as human transferrin, human transferrin receptor or at least its transferrin binding moiety, human serum albumin, human immunoglobulin or human Fc functional region. The fusion partner is linked directly or by an extended amino acid (also known as an extender). A fusion junction is defined as the position between the first protein or the last C-terminal amino acid of the peptide and the first protein or the first N-terminal amino acid of the peptide. Thus, the fusion junction or extension comprises any amino acid between the last amino acid of the N-terminal fusion partner and the first amino acid of the C-terminal fusion partner.

Extended subunit:

Such extenders are known in the art and are from 1 to about 100 amino acids long, from 1 to about 50 amino acids long, from 1 to about 25 amino acids long, from 1 to about 15 The length of the amino acid is from 1 to 10 amino acids, from 4 to 25 amino acids, from 4 to 20 amino acids, from 4 to 15 amino acids, or from 4 to The 10 amino acids are long.

The amino acid composition of the stretcher sequence can be altered to demonstrate low immunity The extension of the original score is preferred. In a particular embodiment of the invention, the stretcher polypeptide linking the fusion polypeptide ALB to the proteinaceous half-life extending group can be comprised of any amino acid; for example, the stretcher polypeptide used in scR-Fc1 is derived from a charged and bulky amino acid (eg, The Glu, Arg or Asp) composition, while the stretcher polypeptide in scR-Fc2 consists of an uncharged amino acid (eg Gly and Ser).

In a preferred embodiment, the stretcher polypeptide comprises at least one Gly, Ser, Ile, Glu, Arg, Met, and/or Asp residue. In a more preferred embodiment, the stretcher polypeptide comprises Gly and Ser residues. In a further preferred embodiment, the stretcher peptide is a glycine-rich linker, for example, comprising a peptide of the [GGGGS] _n sequence as disclosed in U.S. Patent No. 7,271,149. In other embodiments, a serine-rich stretcher polypeptide as described in U.S. Patent No. 5,525,491 is used. Further preferred embodiments are extender polypeptides comprising a Gly to Ser residue and a Gly to Ser ratio of at least 3 to 1. Further preferred are extender polypeptides having a proline residue at the C- and/or N-terminus.

Preferred extender peptides are [GlyGlySerPro] (SEQ ID NO: 148), [GlyGlySerGlyGlySerPro] (SEQ ID NO: 149), and [GlyGlySlyGlyGlySerGlyGlySerPro] (SEQ ID NO: 150).

Such fusion polypeptides having a enhanced half-life may comprise a fusion polypeptide representation of a (Rl) _m- (S1) _n- ALB-(S2) _o- (R2) _p sequence.

A further embodiment of the invention is a fusion polypeptide comprising (R1) _m -(S1) _n -ALB-(S2) _o -(R2) _p , wherein A, L and B have the definitions as disclosed above, R1 And the R2 line protein nature half-life extending group, S1 and S2 are as defined above as the extender peptide, and wherein m, n, o, and p independently have an integer of 0 or 1, except m, n, o, and At least one of p is 1. For example, (S1)n=0 means that no linker S1 is present in the fusion polypeptide.

In a further specific example, if m is an integer of 1, n is an integer of one. In a further specific example, if p is an integer of 1, then o is an integer of one.

In a preferred embodiment, n and m are 0 and o and p are 1. In a further preferred embodiment, n and m are 1 and o and p are 0.

A further embodiment of the invention is a fusion polypeptide comprising (R1) _{m = 1} - (S1) _{n = 0 -} ALB - (S2) _{o = 0} - (R2) _{p = 0} .

A further embodiment of the invention is a fusion polypeptide comprising (R1) _{m = 0} - (S1) _{n = 0 -} ALB - (S2) _{o = 0} - (R2) _{p = 1} .

In a preferred embodiment, the proteinaceous half-life extending group is selected from the group consisting of serum albumin, transferrin, Fc functional region, IgGl Fc functional region, and serum albumin binding protein.

In a further embodiment, the fusion polypeptide further comprises at least one half-life extending group having an extended half-life compared to the corresponding wild-type relaxin, wherein the half-life is extended by at least 5, 10, 20, 50, 100 or 500-fold. Preferably, the half-life is determined by serum half-life, meaning that the fusion protein is detected in serum or whole blood, for example using commercially available quantitative ELISA assays (eg R&D Systems, Human Relaxin-2) Quantikine ELISA kit, catalog number DRL200). The half-life is preferably the human blood half-life. Preferably, the half-life is a functional in vivo half-life assay, meaning the activity of the fusion polypeptide in a serum or blood sample is determined. Assays for determining the activity of the fusion polypeptide A-L-B of the present invention are known in the art and are described herein.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1) _m -(S1) _n -ALB-(S2) _o -(R2) _p , wherein A is a human relaxin 2A chain polypeptide (SEQ ID NO :117) or a functional variant thereof, B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, L-line 9 amino acid long linker polypeptides, R1 and R2 line half-life An extended group, preferably a protein-life half-life extending group, S1 and S2 are as defined above as an extender peptide, and wherein m, n, o, and p independently have an integer of 0 or 1, except m, n And o, and at least one of p is 1, preferably at least m or p is 1, more preferably m and n are 0 and o and p are 1, and preferably m and n are 1 and o and p are 0.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1) _m -(S1) _n -ALB-(S2) _o -(R2) _p , wherein A is a human relaxin 2A chain polypeptide (SEQ ID NO : 117), B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119), L is a 9 amino acid long linker polypeptide, R1 and R2 are half-life extending groups, preferably a protein nature half-life extending group. a group, S1 and S2 are an extender peptide as defined above, and wherein m, n, o, and p independently have an integer of 0 or 1, except that m, n, o, and p are at least one, Preferably, at least m or p is 1, more preferably m and n are 0 and o and p are 1, and most preferably m and n are 1 and o and p are 0.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1) _m -(S1) _n -ALB-(S2) _o -(R2) _p , wherein A is a human relaxin 2A chain polypeptide (SEQ ID NO :117) or a functional variant thereof, B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and L is a linker polypeptide having a sequence of GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139), R1 And a R2 system half-life extending group, preferably a protein nature half-life extending group, S1 and S2 are as defined above as an extender peptide, and wherein m, n, o, and p independently have an integer of 0 or 1, Preferably, at least one of m, n, o, and p is 1, preferably at least m or p is 1, more preferably m and n are 0 and o and p are 1, and preferably m and n are 1 and o and p are 0.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1) _m -(S1) _n -ALB-(S2) _o -(R2) _p , wherein A is a human relaxin 2A chain polypeptide (SEQ ID NO :117), a B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119), a L-linker polypeptide having a GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139) sequence, and a R1 and R2 system half-life extending group, preferably a protein a half-life extending group of properties, S1 and S2 are as described above as an extender peptide, and wherein m, n, o, and p independently have an integer of 0 or 1, but at least one of m, n, o, and p Preferably, it is at least m or p is 1, more preferably m and n are 0 and o and p are 1, and most preferably m and n are 1 and o and p are 0.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, B-lineage Human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, L line is a linker polypeptide having the sequence of GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139), R1 is a half-life extending group, preferably a half-life of protein properties An extended group, and the S1 is an extender peptide as defined above.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-A-L-B, wherein A line human relaxin 2A chain polypeptide (SEQ ID NO: 117), B line human relaxin 2 B chain polypeptide (SEQ ID NO: 119), L line linker polypeptide having the sequence GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139), R1 A half-life extending group, preferably a proteinaceous half-life extending group, and the S1 line is an extender peptide as defined above.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, B-lineage Human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, L line has a GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139) sequence linker polypeptide, R1 is a protein nature half-life extending group, S1 line 4 to The 10 amino acid long stretcher peptides are preferably selected from the group consisting of GlyGlySerPro (SEQ ID NO: 148), GlyGlySerGlyGlySerPro (SEQ ID NO: 149), and GlyGlySlyGlyGlySerGlyGlySerPro (SEQ ID NO: 150).

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-A-L-B, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117), B line human relaxin 2B chain polypeptide (SEQ ID NO: 119), L line linker polypeptide having the sequence of GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139), R1 line protein nature half-life extending group, S1 line 4 to 10 amine groups The acid long stretcher peptide is preferably selected from the group consisting of GlyGlySerPro (SEQ ID NO: 148), GlyGlySerGlyGlySerPro (SEQ ID NO: 149), and GlyGlySlyGlyGlySerGlyGlySerPro (SEQ ID NO: 150).

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, B-lineage Human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, L line has a linker polypeptide of GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139) sequence, R1 is a protein-life half-life extending group, and S1 is 10 Amino acid long extender peptide.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117), and the B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119), L line has GlyGlyGlySerGlyGlyGlySerGly (SEQ ID NO: 139) a linker polypeptide of the sequence, R1 is a protein half-life extending group, and S1 is a 10 amino acid long extender peptide.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, B-lineage Human relaxin 2B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, L line has a GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139) sequence linker polypeptide, R1 is a protein nature half-life extending group, and S1 is a GlyGlySerGlyGlySerGlyGlySerPro (SEQ ID NO: 150) an extender peptide consisting of.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117), and the B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119), L is a linker polypeptide having a sequence of GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139), an R1 line protein nature half-life extending group, and an S1 line is an extension of GlyGlySerGlyGlySerGlyGlySerPro (SEQ ID NO: 150). Peptide.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-ALB, wherein the A-line human relaxin 2A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof, the B-line human relaxin 2B chain polypeptide (SEQ ID NO: 119) or A functional variant thereof, L is a linker polypeptide having a sequence of GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139), an Fc domain of an R1 antibody, preferably a human IgG1 or IgG2 Fc domain, and S1 is a GlyGlySerGlyGlySerGlyGlySerPro (SEQ ID NO) : 150) The stretcher peptide consisting of.

A preferred embodiment of the invention is a fusion polypeptide comprising (R1)-(S1)-ALB, wherein the A is a human relaxin 2A chain polypeptide (SEQ ID NO: 117), and the B is a human relaxin 2B chain polypeptide (SEQ ID NO: 119), L is a linker polypeptide having a sequence of GlyGlyGlySlyGlyGlyGlySerGly (SEQ ID NO: 139), an Fc domain of an R1 antibody, preferably a human IgG1 or IgG2 Fc domain, and S1 is a GlyGlySerGlyGlySerGlyGlySerPro (SEQ) ID NO: 150) The stretcher peptide consisting of.

A further preferred embodiment of the invention is a fusion polypeptide comprising a polypeptide as set forth in Table 3.

Further preferred embodiments of the present invention are as set forth in Table 3 Polypeptide.

In a further embodiment, the fusion polypeptide ALB further comprises a half-life extending group having relaxin activity. In a further preferred embodiment, the relaxin activity is activation of the relaxin receptor LGR7. Methods for determining relaxin activity are known in the art or are provided herein. In still further preferred embodiments, the activation of the relaxin receptor LGR7 is determined using the methods disclosed in the experimental methods herein. In yet further preferred embodiment, the relaxin receptors LGR7 activation of the measurement system ₅₀ measurement values EC. And more preferably in the particular example, compared to the corresponding wild type activity Relaxin, relaxin activity is reduced less than above ¹⁰⁵ times, ¹⁰⁴ times, ¹⁰³ times, 100 times, 75 times, 50 times, 25 times, or 10 times . For example, in the case of the fusion polypeptide ALB of human relaxin 2, the corresponding wild-type relaxin is a human relaxin 2 protein.

Transfer, vector system, performance, host, and purification

The invention also provides vectors comprising an isolated nucleic acid molecule encoding a fusion polypeptide of the invention; the vector system is operably linked to a sequence of expression that directs its expression in a host cell.

Suitable host cells may be selected from the group consisting of bacterial cells (e.g., E. coli), yeast cells (e.g., Saccharomyces cerevisiae ), fungal cells, plant cells, insect cells, and animal cells. Animal cells include, but are not limited to, HEK293 cells, CHO cells, COS cells, BHK cells, HeLa cells, and various primary mammalian cells; derivatives of mammalian cells, such as HEK293T cells, can also be used.

DNA molecule of the invention

The invention also relates to DNA molecules encoding the fusion proteins of the invention; such sequences include, but are not limited to, the DNA molecules set forth in Table 4.

The DNA molecules of the present invention are not limited to the sequences disclosed herein, but include variants thereof. The DNA variants of the present invention can be described with reference to their physical properties at the time of hybridization. Familiar workers will recognize that DNA hybridization techniques can be used to identify complements and, because DNA is a double strand, its equivalent or homologue; it will also be recognized that less than 100% complementarity can occur heterozygous, however Hybridization techniques can be used to identify differences between DNA sequences based on their structural relationships with specific probes under specific appropriate selection conditions. For guidance on such conditions, see Sambrook et al., 1989 (supra) and Ausubel et al., 1995 [Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA , & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons].

The structural similarity between the two polynucleotide sequences can be expressed as a function of the "stringency" of the conditions in which the two sequences are heterozygous. The term "stringency" as used herein refers to the degree of unfavorable heterozygous conditions. Stringent conditions are extremely detrimental to heterozygosity, under which only the most structurally related molecules are heterozygous. Conversely, imprecise conditions facilitate the display of heterogeneity of molecules of lesser structural relevance. Therefore, the degree of heterozygosity is directly related to the structural relationship of the two nucleic acid sequences. Heterozygous for the following relation is associated with each correlation (where the melting temperature T _m of a double-stranded nucleic acid molecule):

a. T _m =69.3+0.41(G+C)%

b. Each 1% increase in the number of mismatch base pairs, DNA double-stranded molecule of the T _m decreased 1 ℃.

c. (T _m ) _μ2 - (T _m ) _μ1 = 18.5 log ₁₀ μ2 / μ1 where μ1 and μ2 are the ionic strength of the two solutions.

Hybrid rigor is a function of many factors, including overall DNA concentration, ionic strength, temperature, probe size, and the presence of destructive hydrogen bonding agents. Factors that promote hybridization include high DNA concentration, high ionic strength, low temperature, longer probe size, and the absence of destructive hydrogen bonding agents. Hybridization is generally carried out in two phases: the "combination" phase and the "washing" phase.

First, in the binding phase, the probe is bound to the target under conditions of favorable hybridization. This stage uses the temperature control to control the rigor. For high stringency, temperatures are typically between 65 °C and 70 °C unless a short oligonucleotide probe (<20 nucleotides) is used. A representative hybrid solution comprises 6X SSC, 0.5% SDS, 5X Denhardt solution and 100 micrograms of non-specific carrier DNA. See Ausubel et al., section 2.9, supplement 27 (1994). Of course, many different, but equally functional, buffering conditions are known. Lower temperatures can be selected when the correlation is low. Low stringency combined temperature between about 25 ° C and 40 ° C; medium stringency between at least about 40 ° C to less than about 65 ° C; Be careful at least about 65 ° C.

Second, excess probe is removed by washing. At this stage, more stringent conditions are usually applied. Therefore, this "washing" phase is most important in determining the correlation via heterozygosity. The wash solution typically contains a lower salt concentration. An example of a medium-stringency solution containing 2X SSC and 0.1% SDS. The high stringency wash solution contains less than about 0.2X SSC equivalent (on ionic strength), preferably a stringent solution containing about 0.1X SSC. The temperature associated with various stringency is the same as discussed above for "combination." The washing solution is usually also replaced many times during the washing. For example, generally high stringency wash conditions include washing twice at 55 ° C for 30 minutes and washing at 60 ° C for 15 minutes three times.

A specific example of the invention is an isolated nucleic acid sequence encoding a fusion polypeptide of the invention.

Recombinant DNA constructs and performance

The invention further provides recombinant DNA constructs comprising one or more nucleotide sequences of the invention. The recombinant DNA construct of the present invention is for use in a ligation vector, such as a plastid, phage, phage or viral vector, into which a DNA molecule encoding a fusion polypeptide of the present invention is inserted.

A fusion polypeptide as provided herein can be prepared by recombinantly expressing in a host cell a nucleic acid sequence encoding a fusion polypeptide. To recombine the expression fusion polypeptide, the host cell can be transfected with a recombinant expression vector carrying a DNA fragment encoding the fusion polypeptide, and the fusion polypeptide can be expressed in a host cell. For example, see Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989); Ausubel, FM et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and the standard of U.S. Patent No. 4,816,39 to Boss et al. Recombinant DNA methods, preparing and/or obtaining nucleic acids encoding fusion polypeptides, incorporating such nucleic acids into recombinant expression vectors and introducing such vectors into host cells.

To express a fusion polypeptide, standard recombinant DNA expression methods can be used [see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)]. For example, a DNA encoding a desired polypeptide can be inserted into an expression vector and then transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells; examples of prokaryotic host cells are, for example, bacteria, and examples of eukaryotic host cells are yeast, insect or mammalian cells. It is generally understood that the design of the expression vector, including the choice of regulatory sequences, is influenced by factors such as the choice of host cell, the amount of expression of the desired protein, and the constitutive or inducible nature of the expression.

Bacterial performance

The construction of an efficient expression vector for bacteria utilizes an operable reading phase with a functional promoter, inserting a structural DNA sequence encoding the desired protein, along with appropriate translation initiation and termination signals. The vector comprises one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, amplification in the host. Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis , Salmonella typhimurium and Pseudomonas , Streptomyces , and Staphylococcus . Many varieties within the genus.

The bacterial vector can be, for example, a phage, plastid or phage system. These vectors may contain a selection marker derived from a commercially available plastid that normally contains the well-known transcription vector pBR322 (ATCC 37017) and a bacterial origin of replication. After transformation of the appropriate host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-inhibited/induced by an appropriate method (e.g., temperature shift or chemical induction) to allow the cells to be cultured for a further period of time. The cells are typically collected by centrifugation, destroyed by physical or chemical means, and the resulting crude extract is retained for further purification.

In bacterial systems, depending on the intended use of the protein, some expression vectors can be advantageously selected. For example, when a large number of such proteins are to be produced, it may be desirable to direct a large number of vectors that exhibit a fusion polypeptide product that is readily purified. The fusion polypeptide of the present invention includes a purified product, a product of a chemical synthesis procedure, and a recombinant host from a prokaryotic host (including, for example, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Many species within the genus Staphylococcus, preferably produced from E. coli cells.

Eukaryotic cell expression

Eukaryotic cells can be used to express the polypeptides of the invention; systems for expressing proteins are known in the art. Such systems include, for example, eukaryotic cells, growth media, and corresponding expression vectors. Common eukaryotic cells for expression are, for example, mammalian cells, yeast cells, plant cells, or insect cells.

Mammalian performance and purification

Preferred regulatory sequences for mammalian host cell expression include viral elements that direct expression of a large number of proteins in mammalian cells, such as derived from cellular giant virus (CMV) (eg, CMV mover/enhancer), simian virus type 40 (SV40) (eg, SV40 promoter/enhancer), adenovirus [eg, adenovirus major late promoter (AdMLP)] and polyomavirus promoter and/or enhancer. For further description of the viral regulatory elements and their sequences, see, for example, U.S. Patent No. 5,168,062 to Stinski, U.S. 4,510,245 to Bell et al., and U.S. Patent No. 4,968,615 to Schaffner et al. Recombinant expression vectors can also include an origin of replication and a selectable marker (see, for example, U.S. 4,399,216, 4,634,665 and U.S. 5,179,017 to Axel et al.). Suitable selectable markers include genes that confer resistance to an agent (e.g., G418, hygromycin or methotrexate) to a host cell into which the vector has been introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to amidoxime and the anti-neomycin gene confers resistance to G418.

Transfection of the expression vector into the host cell can be performed using standard techniques, such as electroporation, calcium phosphate precipitation, and DEAE-polyglucose, lipofection or multivalent cation transfection.

Suitable mammalian host cells for use in the expression of the fusion polypeptides provided herein include Chinese hamster ovaries (CHO cells) [including DHFR as described in, for example, RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621. The use of selection markers together is described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. dhfr-CHO cells in USA 77:4216-4220], NSO bone marrow cancer cells, COS cells and SP2 cells. In a number of specific examples, the expression vector is designed to secrete the expressed protein into the culture medium in which the host cells are grown. Transient transfection/expression of antibodies can be achieved, for example, according to the experimental protocol of Durocher et al (2002) Nucl. Acids Res. Vol 30 e9; stable transfection/expression of antibodies can be based, for example, on the UCOE system [T. Benton et al. (2002). ) The experimental process of Cytotechnology 38:43-46] was reached.

The fusion polypeptide can be recovered from the culture medium using standard protein purification methods.

The fusion polypeptide of the present invention can be subjected to well-known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A chromatography, protein G chromatography, anion or cation exchange chromatography, and phosphocellulose chromatography. Method, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, recovery and purification from recombinant cell culture. High performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), for example, Chapters 1, 4, 6, 8, 9, and 10, each All contents are incorporated herein by reference.

Fusion polypeptides of the invention include purified or isolated products, products of chemical synthesis procedures, and recombinant eukaryotic hosts using recombinant techniques {including, for example, yeast [eg, Pichia ], higher plants, insects and Mammalian cells, preferably produced from mammalian cells. Depending on the host used in the recombinant production procedure, the fusion polypeptides of the invention may be glycosylated or non-glycosylated, with glycosylation being preferred. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, vol. 17.37-17.42; Ausubel, supra, chapters 10, 12, 13, 16, 18 and 20.

Use for treatment

A specific example of the present invention is the use of the pharmaceutical composition or fusion polypeptide of the present invention for the treatment of cardiovascular diseases, kidney diseases, pancreatitis, inflammation, cancer, scleroderma, lung, kidney, and liver fibrosis.

Cardiovascular diseases

Cardiovascular diseases, or cardiovascular disorders, in the present specification means, for example, the following conditions: hypertension, peripheral and cardiac vascular disorders, coronary heart disease, stable and unstable angina pectoris, myocardial insufficiency, persistent ischemic dysfunction ("Sleeping myocardium"), transient ischemic dysfunction ("salvatory myocardium"), heart failure, abnormal peripheral blood flow, acute coronary syndrome, heart failure, and myocardial infarction.

In the present specification, the term heart failure includes both acute and chronic manifestations of heart failure, as well as more specific or related types of diseases, such as acute non-compensatory heart failure, right heart failure, left heart failure, overall failure, ischemia. Myocardial lesions, dilated cardiomyopathy, congenital heart defects, heart valve defects, heart failure associated with heart valve defects, mitral stenosis, mitral insufficiency, aortic stenosis, aortic insufficiency, three Mitral stenosis, tricuspid atresia, pulmonary stenosis, pulmonary valve atresia, complex heart valve defects, myocardial inflammation (myocarditis), chronic myocarditis, acute myocarditis, viral myocarditis, diabetic heart failure, alcoholic cardiomyopathy Heart accumulation disease, and Shu Zhang type and contractile heart failure and acute stage worsen heart failure.

The compounds according to the invention are further suitable for reducing the range of myocardium affected by infarction and for preventing secondary infarction.

Furthermore, the compounds according to the invention are useful for the prevention and/or treatment of thrombotic embolism disorders, post-ischemic reperfusion injury, microvascular and macrovascular disease (vasculitis), arterial and venous thrombosis, edema, ischemia such as myocardium Infarction, stroke and transient ischemic attack; suitable for coronary artery bypass surgery (CABG), primary percutaneous coronary angioplasty (PTCA), PTCA after thrombolysis, rescue PTCA, heart transplantation and happy Surgical-related cardioprotection; and for organ protection associated with transplantation, bypass surgery, cardiac catheterization, and other surgical procedures.

Other indications are, for example, prevention and/or treatment of respiratory disorders, for example, chronic obstructive pulmonary disease (chronic bronchitis, COPD), asthma, emphysema, bronchiectasis, cystic fibers. Symptoms (pancreatic ductal fibrosis) and pulmonary hypertension, especially pulmonary arterial hypertension.

Kidney disease

The present invention relates to the use of the fusion polypeptide of the present invention as an agent for preventing and/or treating kidney diseases, particularly acute and chronic kidney diseases and acute and chronic renal insufficiency, and acute and chronic renal failure, including the need And acute and chronic stage renal failure without dialysis, and potential or related kidney diseases such as insufficient renal perfusion, dialysis-induced hypotension, non-inflammatory glomerular lesions, glomerular and renal tubular proteinuria, kidney Edema, hematuria, primary, secondary, and Acute and chronic glomerulonephritis, membranous and membrane proliferative glomerulonephritis, Alport syndrome, glomerular sclerosis, tubulointerstitial lesions, nephropathy (eg primary and natural kidney disease) Kidney inflammation, immune nephropathy such as renal transplant rejection, immune complex-induced nephropathy, and poisoning-induced nephropathy, diabetic and non-diabetic nephropathy, pyelonephritis, cystic kidney, renal sclerosis, hypertensive nephrosclerosis, Renal syndrome; characteristics of their diseases and abnormal diagnosis and creatinine clearance and / or water excretion, abnormal increase in blood urea, nitrogen, potassium and / or creatinine concentrations, such as glutamine synthase kidney Enzyme activity, changes in urine osmolality and urine volume, increased microalbuminuria, massive albuminuria, glomerular and arteriolar lesions, tubular dilatation, hyperphosphatemia, and/or dialysis requirements.

In addition, the fusion polypeptide of the present invention can be used for preventing and/or treating kidney cancer after incomplete renal resection, dehydration after excessive use of diuretics, uncontrollable blood pressure elevation with malignant hypertension, urinary tract obstruction and infection, starch-like Degenerative diseases, and systemic diseases associated with glomerular injury (eg, lupus erythematosus, rheumatoid immune system disease), renal artery stenosis, renal artery thrombosis, renal venous thrombosis, analgesic-induced nephropathy For use with agents for renal tubular acidosis.

Further, the fusion polypeptide of the present invention can be used as a medicament for preventing and/or treating a contrast medium-induced and drug-induced acute and chronic interstitial kidney disease, metabolic syndrome, and abnormal dyslipidemia.

Furthermore, the invention encompasses the use of the fusion polypeptides of the invention as agents for the prevention and/or treatment of acute and/or chronic kidney diseases such as pulmonary edema, Use of heart failure, uremia, anemia, electrolyte abnormalities (eg, hyperkalemia, hyponatremia), and side effects associated with bone metabolism and carbohydrate metabolism.

Lung disease

Furthermore, the fusion polypeptides according to the invention are also suitable for the treatment and/or prevention of pulmonary diseases, in particular asthmatic disorders, pulmonary arterial hypertension (PAH) and other forms of pulmonary hypertension (PH) including left heart disease, HIV, sickle cell anemia, thromboembolism (CTEPH), sarcoma-like, pulmonary hypertension associated with pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), Acute lung injury (ALI), alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, emphysema (eg, cigarette smoke-induced emphysema), and cystic fibrosis (CF).

Fibrotic disease

The fusion polypeptides according to the invention are furthermore suitable for the treatment and/or prevention of fibrotic disorders of internal organs (for example, the lungs, heart, kidneys, bone marrow and especially the liver), as well as skin fibrosis and fibrotic eye disorders. In the present specification, the term fibrotic disease specifically includes the following words: liver fibrosis, cirrhosis, pulmonary fibrosis, endomyocardial fibrosis, nephropathy, glomerulonephritis, renal interstitial fibers. Fibrosis injury caused by diabetes, bone marrow fibrosis and similar fibrotic diseases, scleroderma, morphea, scar growth, thick phlegm (also after surgery), sputum, diabetic retinopathy, proliferation Vitreoretinopathy and connective tissue disorders (eg sarcoma-like).

cancer

Cancer is a disease in which a group of cells exhibit uncontrolled growth. Cancer is usually classified as a malignant tumor derived from epithelial cancer (this group covers many of the most common cancers, including breast cancer, prostate cancer, lung cancer and colon cancer); sarcoma derived from connective tissue or mesenchymal cells; Lymphoma and leukemia from hematopoietic cells; germ cell tumors derived from pluripotent; and blastomas derived from immature "precursor" or embryonic tissue.

The invention further provides for the use of a fusion polypeptide of the invention for the preparation of a medicament for the treatment and/or prophylaxis of disorders, in particular the aforementioned disorders.

The invention further provides a method of treating and/or preventing a condition (especially the above mentioned conditions) using an effective amount of at least one fusion polypeptide of the invention.

The present invention further provides a fusion polypeptide of the present invention for use in the treatment and/or prevention of coronary heart disease, acute coronary syndrome, heart failure, and myocardial infarction.

Pharmaceutical composition and investment

The invention also provides a pharmaceutical composition comprising a single-chain relaxin fusion protein in a pharmaceutically acceptable vehicle; the single-chain relaxin fusion protein can be administered systemically or locally; Any suitable manner of administration is known, including, but not limited to, intravenous, intraperitoneal, intraarterial, intranasal, by inhalation, orally, subcutaneously, by topical injection or in the form of a surgical implant.

The invention also relates to a pharmaceutical composition which may comprise at least one other preparation, alone or in combination [for example, in any sterile, biocompatible medicine A fusion polypeptide of the invention for administration to a drug carrier (including, but not limited to, salt solution, buffered saline, glucose, and water). Any of these molecules can be administered to a patient in a pharmaceutical composition in admixture with the excipient(s) or pharmaceutically acceptable carrier, alone or in combination with other agents, drugs or hormones. In one embodiment of the invention, the pharmaceutically acceptable carrier is inert to the drug.

The invention also relates to the administration of pharmaceutical compositions; such administration is achieved either orally or parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. In addition to the active ingredients, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers which comprise the excipients and auxiliaries which are pharmaceutically acceptable for use in the preparation of the active compound. Further details regarding formulation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulated for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical compositions to be formulated into lozenges, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like for ingestion by a patient.

Pharmaceutical formulations for parenteral administration comprise an aqueous solution of the active compound. For administration, the pharmaceutical composition of the present invention can be formulated in an aqueous solution [preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological buffered saline). Aqueous injection suspension The agent may contain a substance which increases the viscosity of the suspending agent, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, the active compound suspension may be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. If desired, the suspending agent may also contain suitable stabilizers or increase the solubility of the compound to prepare a highly concentrated solution.

The fusion polypeptides according to the invention may be used alone or, if desired, in combination with other active compounds. The invention furthermore provides an agent comprising at least one fusion polypeptide according to the invention and one or more further active ingredients, in particular for the treatment and/or prevention of the abovementioned conditions.

Suitable active ingredients for combination, for example and preferred use: active ingredients for regulating lipid metabolism, anti-diabetic agents, hypotensive agents, perfusion-reinforcing and/or anti-thrombotic agents, antioxidants, chemokine receptor antagonists, P38-kinase inhibitors, NPY agonists, appetite agonists, anorectic agents, PAF-AH inhibitors, anti-inflammatory agents (COX inhibitors, LTB ₄ -receptor antagonists), analgesics (eg aspirin) Ling), antidepressants and other psychotic drugs.

The invention relates in particular to a composition according to at least one fusion polypeptide according to the invention and at least one active ingredient which alters lipid metabolism, an anti-diabetic agent, a blood pressure lowering active ingredient and/or an antithrombotic preparation.

The fusion polypeptide according to the invention is preferably combined with one or more of the following active ingredients: 活性 an active ingredient that modulates lipid metabolism, for example, and preferentially derived from an HMG-CoA reductase inhibitor, an HMG-CoA reductase expression inhibitor , squalene synthesis inhibitor, ACAT inhibitor, LDL receptor sensor, cholesterol absorption inhibitor, polymeric bile acid adsorbent, bile acid reuptake inhibitor, MTP inhibitor, lipase inhibitor, LpL activator, fiber Acids, niacin, CETP inhibitors, PPAR-α, PPAR-γ and/or PPAR-δ agonists, RXR modulators, FXR modulators, LXR modulators, thyroid hormones and/or thyroid mimics, ATP lemons Acid lyase inhibitors, Lp(a) antagonists, cannabinoid receptor 1 antagonists, leptin receptor agonists, bombesin receptor agonists, histamine receptor agonists and antioxidants/ a group of free radical scavengers; anti-diabetic agents as described in Chapter 12 of Rote Liste 2004/II, and, for example, and prioritized from sulfonylureas, biguanides, meglitinide derivatives Glucosidase inhibitor, dipeptidyl peptidase I V inhibitor (DPP-IV inhibitor), Diazolidinone, thiazolidinedione, GLP 1 receptor agonist, glycosidic antagonist, insulin sensitizer, CCK 1 receptor agonist, leptin receptor agonist, involved in stimulating sugar a group of hepatic enzyme inhibitors, glucose uptake regulators, and potassium channel openers (for example, as disclosed in WO 97/26265 and WO 99/03861) of the nascent and/or hepatic glycolysis; Blood pressure active ingredients, for example, and preferentially derived from calcium antagonists, angiotensin AII antagonists, ACE inhibitors, renin inhibitors, beta-blockers, alpha-blockers, aldosterone antagonists , mineral cortisol receptor antagonists, ECE inhibitors, ACE/NEP inhibitors, and vasopeptidase inhibitors; and/or sputum antithrombotic agents, for example, and preferentially derived from platelet aggregation inhibitors or antibodies Coagulant group; loop diuretics; vasopressin receptor antagonist; ̇ organic nitrates and NO donors; compounds with positive effects on myocardial contractile activity; ̇ inhibition of cyclic guanosine monophosphate (cGMP) And/or a compound that degrades adenosine monophosphate (cAMP), for example, phosphoric acid Diesterase (PDE) 1, 2, 3, 4 and/or 5 inhibitors, particularly PDE 5 inhibitors, such as sildenafil, vardenafil and tadalafil, and PDE 3 inhibitors, such as milrinone; vasopressin, for example, "atrial diuretic peptide" [ANP, anaritide], "B-diuretic peptide" or "brain diuretic peptide""[BNP, nesiritide], "C-type diuretic peptide" (CNP), and urodilatin; ̇prostacyclin receptor (IP receptor) agonist, for example, For example, iloprost, beraprost, cicaprost; ̇I _f channel (Trolltech channel) inhibitors, for example, ivabradine Calcium sensitizer, for example, and prioritized, levosimendan; strontium potassium supplement; ̇ unrelated to NO, but dependent on heme guanylate cyclase stimulator, for example, special Is described in WO 00/06568, WO 00/06569, WO 02/42301 and WO 03/095451; ̇ unrelated to NO and heme guanylate cyclase activators, for example, in particular, see Said in WO 01/19 355, WO 01/19776, WO 01/19778, WO 01/19780, WO 02/070462 and WO 02/070510; ̇ human neutrophil elastase (HNE) inhibitors, for example, Sivelestat and DX-890 (Reltran); a compound that inhibits the signaling cascade, for example, tyrosine kinase inhibitors, especially sorafenib, imatinib (imatinib), gefitinib and erlotinib; and/or ̇ compounds that modulate cardiac energy metabolism, for example, etomoxir, dichloroacetate, Renault (ranolazine) and Qumei (trimetazidine).

An active ingredient for modifying lipid metabolism is understood to mean, preferably, obtained from an HMG-CoA reductase inhibitor, a squalene synthesis inhibitor, an ACAT inhibitor, a cholesterol absorption inhibitor, an MTP inhibitor, a lipase inhibition. Agent, thyroid hormone and/or thyroid mimetic, nicotinic acid receptor agonist, CETP inhibitor, PPAR-α agonist, PPAR-γ agonist, PPAR-δ agonist, polymeric bile acid sorbent Bile acid reuptake inhibition a compound, an antioxidant/radical scavenger, and a compound of the cannabinoid receptor 1 antagonist group.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with an HMG-CoA reductase inhibitor derived from a statin, such as, for example, and prioritized, lovastatin, simvastatin Statvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin or pitavastatin.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a squalene synthesis inhibitor, such as, by way of example and priority, BMS-188494 or TAK-475.

In a preferred embodiment of the invention, the fusion polypeptides according to the invention are administered in combination with an ACAT inhibitor, such as, for example, and prioritized, avasimibe, melinamide, patil. Pactimibe, eflucimibe or SMP-797.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a cholesterol absorption inhibitor, such as, for example, and prioritized, ezetimibe, tiqueside or pama Glycosides (pamaqueside).

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with an MTP inhibitor, such as, for example, and prioritized, implitapide, BMS-201038, R-103757 or JTT-130. .

In a preferred embodiment of the invention, the fusion polypeptide according to the invention It is administered in combination with a lipase inhibitor, such as, for example, and prioritized, orlistat.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a thyroid hormone and/or a thyroid mimetic, such as, for example, and prioritized, D-thyroxine or 3,5,3'-three Iodine thymidine (T3).

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a nicotinic acid receptor agonist, such as, for example, prioritized, niacin, acipimox, a Acifran or radecol.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a CETP inhibitor, such as, for example, and prioritized, dalcetrapib, BAY 60-5521, Ansetripe ( Anacetrapib) or CETP vaccine (CETi-1).

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with, for example, a PPAR-gamma agonist derived from a thiazolidinedione, such as, by way of example and preference, pioglitazone or lo格iglintazone.

In a preferred embodiment of the invention, the fusion polypeptides according to the invention are administered in combination with a PPAR-delta agonist, such as, by way of example and priority, GW-501516 or BAY 68-5042.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a polymeric bile acid sorbent, such as, for example, and prioritized, cholestyramine, colestipol, Colesolvam, CholestaGel or colestimide.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a bile acid reuptake inhibitor, such as, for example and prioritized, an ASBT (=IBAT) inhibitor, for example, AZD- 7806, S-8921, AK-105, BARI-1741, SC-435 or SC-635.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with an antioxidant/radical scavenger, such as, for example, and prioritized, probucol, AGI-1067, BO-653 Or AEOL-10150.

In a preferred embodiment of the invention, the fusion polypeptides according to the invention are administered in combination with a cannabinoid receptor 1 antagonist, such as, by way of example and priority, rimonabant or SR-147778.

An anti-diabetic agent is to be understood as meaning, preferably, an insulin and an insulin derivative, and an orally effective hypoglycemic active ingredient. Herein, insulin and insulin derivatives include insulin of animal, human or biotechnological origin, and mixtures thereof. The orally effective hypoglycemic active ingredient preferably comprises a sulfonylurea, a biguanide, a mergentin derivative, a glucosidase inhibitor, and a PPAR-gamma agonist.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with insulin.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a sulfonylurea, such as, by way of example and preference, toluene Sulfonamide, glibenclamide, glimepiride, glipizide or gliclazide.

In a preferred embodiment of the invention, the fusion polypeptides according to the invention are administered in combination with a biguanide, such as, by way of example and priority, metformin.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a meglitin derivative, such as, for example, and prioritized, repaglinide or nateglinide .

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a glucosidase inhibitor, such as, by way of example and preference, miglitol or acarbose.

In a preferred embodiment of the invention, the fusion polypeptides according to the invention are administered in combination with a DPP-IV inhibitor, such as, for example and prioritized, sitagliptin and vildagliptin.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with, for example, a PPAR-gamma agonist derived from a thiazolidinedione, such as, by way of example and preference, pioglitazone or rosiglitazone. .

A hypotensive agent is preferably understood to mean a compound derived from a calcium antagonist, an angiotensin AII antagonist, an ACE inhibitor, a beta-blocker, an alpha-blocker, and a diuretic group. .

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a calcium antagonist, such as, for example, and prioritized, nifedipine, amlodipine, verapamil. (verapamil) or diltiazem.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with an angiotensin AII antagonist, such as, for example, and prioritized, losartan, valsartan, kan Desartantan, embusartan, olmesartan or telmisartan.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with an ACE inhibitor, such as, for example, and prioritized, enalapril, captopril, lisino Lisinopril, ramirril, deLapril, fosinopril, quinopril, perindopril or trandolapril (trandopril).

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a beta-blocker, such as, for example, and prioritized, propranolol, atenolol ), timolol, pindolol, aprenolol, oxprenolol, penbutolol, bupranolol, Metipranolol, nadolol, mepindolol, carazalol, sotalol, metoprolol, beta Betaxolol, celiprolol, bisoprolol, carteolol, esmolol, labetalol, carvedilol ( Carvedilol), adalolol, landiolol, nebivolol, Epanolol or bucindolol.

In a preferred embodiment of the invention, the fusion polypeptides according to the invention are administered in combination with an alpha-blocker, such as, by way of example and preference, prazosin.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a diuretic, such as, for example, and prioritized, furosemide, bumetanide, tonoxime (torsemide), benzyl fluorothiazide (bendroflumethiazide), chlorothiazide (chlorothiazide), hydrochlorothiazide (hydrochlorothiazide), hydrofluorothiazide (hydroflumethiazide), methylchlorothiophene (methyclothiazide), pelolithia (polythiazide), trichlorothiazide (trichloromethiazide), chlorothalidone, indapamide, metolazone, quinethazone, acetazolamide, dichlorophenamide , methazolamide, glycerol, isosorbide, mannitol, amiloride or triamteren.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with an aldosterone or a mineral cortisol receptor antagonist, such as, for example, and preferably, spironolactone or eplerenone.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a vasopressin receptor antagonist, such as, for example, and prioritized, conivaptan, tolvaptan ( Tolvaptan), lixivaptan or SR-121463.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention And organic nitrate or NO donors, such as, for example, and prioritized, sodium nitroprusside, nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, molsidomin or SIN- 1, or in combination with inhaled NO.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a compound which positively affects myocardial contraction, such as, for example, prioritized, cardiac glycoside [digoxin], β- Adrenalin and dopaminergic agonists such as isoproterenol, adrenaline, norepinephrine, dopamine or dobutamine.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is associated with antisympathotonics such as reserpine, clonidine or alpha-methyldopa, Or with potassium channel agonists such as minoxidil, dinitrogen (diazoxide), biguanide (dihydralazine) or grievance (hydralazine), or in combination with a substance that releases nitrogen oxides [eg, glycerol nitrate or sodium nitroprusside].

An antithrombotic agent is to be understood as meaning, preferably, a compound derived from a platelet aggregation inhibitor or an anticoagulant group.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a platelet aggregation inhibitor, such as, for example, and prioritized, aspirin, clopidogrel, ticlopidine ( Ticlopidine) or dipyridamol.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention In combination with thrombin inhibitors, for example, and prioritized, ximelagatran, melagatran, dabigatran, bivalirudin or gram Clement (clexane).

In a preferred embodiment of the invention, the fusion polypeptides according to the invention are administered in combination with a GPIIb/IIIa antagonist, such as, by way of example and preference, tirofiban or abciximab.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with a factor Xa inhibitor, such as, for example, and prioritized, rivaroxaban (BAY 59-7939), DU-176b , apixaban, otamixaban, fidexaban, razaxaban, fondaparinux, idraparinux, PMD -3112, YM-150, KFA-1982, EMD-503982, MCM-17, MLN-1021, DX 9065a, DPC 906, JTV 803, SSR-126512 or SSR-128428.

In a preferred embodiment of the invention, the fusion polypeptide according to the invention is administered in combination with heparin or a low molecular weight (LMW) heparin derivative.

In a preferred embodiment of the invention, the fusion polypeptides according to the invention are administered in combination with a vitamin K antagonist, such as, by way of example and preference, coumarin.

In the present specification, it is particularly preferred to use a fusion polypeptide comprising at least one of the invention according to the invention and one or more selected from the group consisting of HMG-CoA reductase inhibitors (statins), diuretics, beta-blockers, organic Nitrate and NO donors, ACE inhibitors, vasopressin Composition of AII antagonist, aldosterone and mineral cortisol receptor antagonist, vasopressin receptor antagonist, platelet aggregation inhibitor and further active ingredient of anticoagulant group, and its treatment and/or prevention The use of the above disorders.

Further, the present invention provides an agent comprising at least one fusion polypeptide according to the present invention, usually together with one or more inert, non-toxic pharmaceutically suitable adjuvants, and the use thereof for the above purposes.

Effective therapeutic dose

Pharmaceutical compositions suitable for use in the present invention include compositions in which an effective amount of the active ingredient is included to achieve the intended purpose (e.g., heart failure). The determination of the effective dose is well within the skill of the artisan.

For any compound, the effective therapeutic dose can be initially in an in vitro assay (eg, LGR7 receptor activation), in vivo in an isolated perfused rat heart, or in an animal model (usually a mouse, rabbit, dog, or Evaluation in pigs). Animal models are also used to achieve the desired concentration range and route of administration. This information can then be used to determine the useful dose and route of administration in the human body.

An effective therapeutic dose refers to an amount of a fusion polypeptide that improves symptoms or a state of health. Therapeutic efficacy and toxicity of such compounds can be determined in vitro or in experimental animals using standard pharmaceutical procedures, for example, ED50 (50% therapeutically effective dose) and LD50 (50% ethnically lethal dose). The dose ratio between therapeutic and toxic effects is the therapeutic index and can be expressed as an ED50/LD50 ratio; a pharmaceutical composition exhibiting a large therapeutic index is preferred. Use data obtained from in vitro and animal studies to develop agents for human use The range of quantities. The dosage of such compounds is preferably within a range of circulating concentrations that contain little or no toxic ED50. The dosage in this range will vary depending on the dosage form employed, the sensitivity of the patient, and the route of administration.

Depending on the route and route, normal doses may vary from 0.1 to 100,000 mg total dose. Guidance on specific dosages and delivery methods is provided in the literature; see U.S. Patent No. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art will use different formulations of polynucleotides than proteins or their inhibitors. Likewise, delivery of a polynucleotide or polypeptide is specific to a particular cell, condition, location, and the like.

The invention is further described by the following examples. The embodiments provided are merely illustrative of the invention by reference to the detailed embodiments. The exemplifications of the present invention are not intended to limit the scope of the disclosed invention.

All of the examples are carried out using standard techniques well known and routine to those skilled in the art, unless otherwise specified. The exemplary molecular biology techniques of the following examples can be as described in standard laboratory manuals (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Said to proceed.

Further preferred specific examples are:

A fusion polypeptide having a relaxin activity comprising A-L-B, wherein B comprises a relaxin B chain polypeptide or a functional variant thereof, and A comprises a relaxin A chain polypeptide or a functional variant thereof, L-linker polypeptide.

2. The fusion polypeptide according to item 1, wherein the B-type relaxin B-chain polypeptide or a functional variant thereof, the A-type relaxin A-chain polypeptide or a functional variant thereof, and the L-based linker polypeptide.

3. The fusion polypeptide according to Item 1 or 2, wherein the relaxin B chain is a relaxin 2B or a relaxin 3B chain.

4. The fusion polypeptide according to any one of the preceding claims, wherein the relaxin A chain is a relaxin 2A or a relaxin 3A chain.

5. The fusion polypeptide of any of the preceding clauses, wherein the relaxin A chain is a relaxin 2A chain.

6. The fusion polypeptide of any of the preceding clauses, wherein the relaxin A chain is a relaxin 3A chain.

The fusion polypeptide according to any one of the preceding claims, wherein the relaxin A chain relaxin 2A chain and the relaxin B chain relaxin 2B chain.

The fusion polypeptide according to any one of the preceding claims, wherein the relaxin A and B chains are human relaxin A and B chain.

9. The fusion polypeptide of any of the preceding claims, wherein the fusion polypeptide further comprises at least one extended half-life group.

10. The fusion polypeptide according to item 9, wherein the half-life extending group is a non-proteinaceous or proteinaceous half-life extending group.

11. The fusion polypeptide according to Item 9 or 10, wherein the polypeptide has the formula (R1)m-(S1)n-A-L-B-(S2)o-(R2)p, Wherein the R1 and R2 protein proteins have half-life extending groups, the S1 and S2 lines extend the peptide, and wherein m, n, o and p are independently the number 0 or 1, but at least one of m, n, o and p is 1.

12. The fusion polypeptide according to item 11, wherein m and n are 0 and o and p are 1.

13. The fusion polypeptide according to item 11, wherein m and n are 1 and o and p are 0.

14. The fusion polypeptide according to item 11, wherein m is 1 and n, o and p are 0.

15. The fusion polypeptide according to item 11, wherein m, n and o are 0 and p is 1.

The fusion polypeptide according to any one of items 11 to 15, wherein the R1 and R2 lines are comprised of an extended half-life of a protein consisting of an immunoglobulin Fc functional region, serum albumin, transferrin and serum albumin binding protein. A half-life extending group of protein properties in a group of groups.

The fusion polypeptide of any one of clauses 10 to 16, wherein the protein nature half-life extending group is an IgG1 Fc domain.

The fusion polypeptide according to any one of the items 10 to 17, wherein the protein half-life extending group is a human.

19. The fusion polypeptide of clause 10, wherein the non-proteinaceous half-life extending group is PEG or HES.

The fusion polypeptide according to any one of items 11 to 19, wherein the extension The polypeptides S1 and S2 are 1 to 25 amino acids long.

The fusion polypeptide according to any one of items 11 to 20, wherein the extended polypeptides S1 and S2 are 4 to 10 amino acids long, preferably 10 amino acids long.

22. The fusion polypeptide according to item 21, wherein the extension polypeptides S1 and S2 are comprised in the extended polypeptide group consisting of the polypeptides disclosed by SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NOs: 150.

The fusion polypeptide according to any one of the preceding claims, wherein the linker polypeptide L is 6 to 14 amino acids long.

The fusion polypeptide according to any one of the preceding claims, wherein the linker polypeptide L is 7 to 11 amino acids long.

The fusion polypeptide according to any of the preceding claims, wherein the linker polypeptide L is 8, 9, or 10 amino acid lengths.

The fusion polypeptide according to any of the preceding claims, wherein the linker polypeptide L is 9 amino acids long.

The fusion polypeptide according to any one of the preceding claims, wherein the linker polypeptide L is comprised of a linker having 6, 7, 8, 9, 10, 11, 12, 13 and 14 amino acid lengths. Connect to a subgroup.

28. The fusion polypeptide according to any one of the preceding claims, wherein in the linker polypeptide L, the linker L is composed entirely of Gly and/or Ser residues except for the four amino acid residues, and the remaining four amines The acid residue is selected from the group of natural amino acids.

The fusion polypeptide according to any one of the preceding claims, wherein the linker polypeptide L comprises at least one Gly, Ser, Arg, Cys, Leu and/or Lys Residues.

The fusion polypeptide according to any of the preceding claims, wherein the linker polypeptide L comprises Gly and Ser residues.

The fusion polypeptide according to any one of the preceding claims, wherein the linker polypeptide L consists of a Gly and a Ser residue.

The fusion polypeptide according to any one of the preceding claims, wherein the linker polypeptide L comprises Gly and Ser residues, and the ratio of Gly to Ser is at least 3 to 1.

The fusion polypeptide according to any one of the preceding claims, wherein the linker polypeptide L is comprised by SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO 141, a ligated polypeptide group consisting of the polypeptides disclosed by SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 145 and SEQ ID NO: 146.

The fusion polypeptide according to any one of the preceding claims, wherein the relaxin A chain is a human relaxin 2A chain (SEQ ID NO: 117), and the relaxin B chain is a human relaxin 2 B chain (SEQ ID NO: 119) ).

The fusion polypeptide according to any one of the preceding claims, wherein the A is a human relaxin 2A chain (SEQ ID NO: 117), and the B is a human relaxin 2B chain (SEQ ID NO: 119).

36. The fusion polypeptide of any of the preceding clauses comprising a polypeptide as described in Table 3.

The fusion polypeptide according to any one of the preceding claims, wherein the A-L-B is selected from the group consisting of scR3, scR4, scR5, unlabeled scR3, unlabeled scR4, The A-L-B polypeptide group of the marker-free scR5, scR-Fc5, scR-Fc6 and scR-Fc7.

38. A fusion polypeptide as described in Table 3.

39. A fusion polypeptide selected from the group consisting of scR3, scR4, scR5, unlabeled scR3, unlabeled scR4, unmarked scR5, scR-Fc5, scR-Fc6 and scR-Fc7.

40. A polynucleotide encoding a fusion polypeptide according to any of the preceding items.

41. A vector comprising the polynucleotide according to item 40.

42. A host cell comprising the vector according to item 41 or the polynucleotide according to item 40.

43. The host cell according to item 42, wherein the host cell is a eukaryotic or prokaryotic cell.

44. The host cell according to item 42 or 43, wherein the eukaryotic host cell is a mammalian, yeast, insect or plant cell.

The host cell according to item 44, wherein the mammalian host cell is a CHO cell.

The host cell according to item 43, wherein the prokaryotic host cell is a bacterial cell, preferably an Escherichia coli cell.

47. A method of producing a polypeptide according to any one of items 1 to 39, which comprises culturing a host cell according to items 42 to 46 and the step of isolating the polypeptide.

A pharmaceutical composition comprising the fusion polypeptide according to any one of items 1 to 39.

49. The pharmaceutical composition according to item 48, or the fusion polypeptide according to any one of items 1 to 39, for use as a medicament.

The pharmaceutical composition according to any one of items 1 to 39, which is a medicament for treating a cardiovascular disease, a pulmonary disease, a fibrotic disorder or a kidney disease.

51. A method of treating a cardiovascular disease, a pulmonary disease, a fibrotic condition or a kidney disease, the method comprising administering a therapeutically effective dose of a pharmaceutical composition according to items 48 and 49 or according to items 1 to 39 A fusion polypeptide.

52. The method of treatment according to clauses 50 and 51, wherein the cardiovascular disease is coronary heart disease, acute coronary syndrome, heart failure, and myocardial infarction.

Instance experiment process Construction of relaxin variants:

The cDNA sequence of the relaxin variant was generated by chemical gene synthesis; the synthetic gene was sub-transferred into the mammalian expression vector pCEP4 (Invitrogen, Cat. No. V044-50). The leader sequence for the message used to properly secrete the resulting protein is the leader sequence using LDL receptor-associated protein (LRP, amino acid composition MLTPPLLLLLPLLSALVAA) or CD33 (amino acid composition MPLLLLLPLLWAGALA). The secondary transformation of the synthetic construct was carried out using the restriction enzymes HindIII and BamH1 according to the manufacturer's instructions.

The performance of relaxin variants:

For small scale performance (up to 2 ml culture volume), HEK293 (ATCC, Cat. No. CRL-1573) cell line was transiently transfected according to the manufacturer's instructions using Lipofectamine 2000 Transfection Reagent (Invitrogen, Cat. No. 11668-019). Cells were plated at D-Mem F12 (Gibco, #31330), 1% penicillin-streptomycin (Gibco, #15140) and 10% fetal bovine serum (FCS, Gibco) at 37 ° C in a 5% carbon dioxide humidified incubator. , #11058) was cultured.

After 3 to 5 days of transfection, the activity of the conditioned medium of the transfected cells was determined using a stably transfected CHO-CRE-GR7 cell line.

For large-scale performance (10 ml culture volume and more), the constructs were transiently expressed in mammalian cells as described in Tom et al., 2007. Briefly, plastids were transfected into HEK293-6E cells and cultured in Fernbach-Flasks or Wave-Bags. The performance was carried out in F17 medium (Invitrogen) at 37 ° C for 5 to 6 days. After transfection, 5 g/L of tryptone TN1 (Organotechnie), 1% Ultra-Low IgG FCS (Invitrogen) and 0.5 mM valproic acid (Sigma) were supplemented.

Purification of relaxin variants:

The relaxin Fc-fusion construct was purified from mammalian cell culture supernatants. First, centrifugation was used to obtain a clear supernatant from the cell debris. The protein was purified using Protein A [MabSelect Sure (GE Healthcare)] affinity chromatography followed by size exclusion chromatography (SEC); therefore, the supernatant was applied to PBS previously at pH 7.4 ( Sigma /Aldrich) In a balanced Protein A column, the contaminants were removed in 10 column volumes of pH 7.4 PBS + 500 mM NaCl. The relaxin Fc fusion construct was eluted with 50 mM sodium acetate (pH 3.5) + 500 mM NaCl, and further purified by SEC in Superdex 200 column in PBS (pH 7.4).

For purification of the c-Myc marker protein or polypeptide, a c-Myc marker protein mild purified gel (Biozol Diagnostic, Protein Mild Purification Gel, Cat. No. 3306) was used according to the manufacturer's instructions.

For the purification of the His-tagged protein or polypeptide, a Ni-NTA centrifugal chromatography column (Qiagen, Ni-NTA Spin Kit, catalog number 31314) was used according to the manufacturer's instructions.

Quantification of the expression of relaxin variants:

For quantification of secreted and purified recombinant relaxin variants, a commercially available quantitative ELISA (R&D Systems, Human Relaxin-2 Quantikine ELISA Kit, catalog number DRL200) was used according to the manufacturer's instructions.

In addition, several constructor proteins were quantified using FC-ELISA. For the Fc ELISA, a 96-well microtiter plate (Nunc, Maxi Sorp black, Cat. No. 460918) was coated with anti-Fc antibody (Sigma Aldrich, Cat. No. A2136) at 4 ° C, 5 μg per ml. . The plate was washed once and 50 microliters of buffer consisting of PBS and 0.05% Tween 20 (Sigma Aldrich, Cat. No. 63158) buffer was used per well. Add 30 μl of blocking buffer (Candor Bioscience, Catalog No. 113500), the titration tray was incubated at 37 ° C for 1 hour. The plate was washed 3 times with 50 microliters of PBS/0.05% Tween 20 buffer per well. A sample was added, and the titration plate was incubated at 37 ° C for 1 hour. If necessary, the sample should be diluted with the above blocking buffer. After incubation, the plate was washed 3 times with 50 microliters of PBS/0.05% Tween 20 buffer per well.

For the test, 30 μl of anti-h-Fc-POD (Sigma Aldrich, Cat. No. A0170) diluted 1:10000 in 10% blocking buffer was added and incubated at 37 ° C for 1 hour. After incubation, the plate was washed 3 times with 50 microliters of PBS/0.05% Tween 20 buffer per well. 30 μl of BM Blue Substrate POD (Roche Diagnostics, Cat. No. 11484281001) was added, and after 5 minutes of incubation, a 1 M sulfuric acid solution was added to terminate the reaction. Absorbance was measured using a Tecan Infinite 500 reader (absorbance mode, 450 nm extinction, 690 nm emission).

When the Myc marker protein concentration was determined, a human c-Myc ELISA kit (EIAab & USCNLIFE, Wuhan EIAab Science Co., Ltd, catalog number E1290h) was used according to the manufacturer's instructions.

When measuring the concentration of His-tagged protein, the His-labeled protein ELISA kit (BIOCAT GmbH, catalog number AKR-130) was used according to the manufacturer's instructions.

When measuring the concentration of HA (hemagglutinin)-tagged protein, a human hemagglutinin (HA) ELISA kit (Hölzel Diagnostika, catalog number CSB-E09360h) was used according to the manufacturer's instructions.

Activity test:

CHO K1 cells (ATCC, catalog number CCL-61) were stably transfected with a circular AMP response element (CRE) luciferase reporter construct (Biomyx Technology, pHTS-CRE, catalog number P2100) to generate CHO-CRE - Luciferase cell line.

This was followed by stable transfection of the long DNA fragment into the mammalian expression vector pcDNA3.1(-) (Invitrogen, catalogue number V79520) human LGR7/RXFP1 receptor (accession number NM_021634.2) by transposing 2271 bases. The cell line produces a CHO-CRE-LGR7 cell line. On D-Mem F12 (Gibco, #31330), 2 mM Glutamax (Gibco, #35050), 100 nM Pyruvat (Gibco, #11360-070), 20 mM Hepes (Gibco, #15630), 1% penicillin-streptococcus This cell strain was cultured in Gibco (#15140) and 10% fetal bovine serum (FCS, Gibco, #11058).

To stimulate, the medium was changed to OptiMem (Gibco, #11058) + 1% FCS containing various concentrations of recombinant expression of relaxin variant protein (usually starting at a concentration of 100 nM followed by a 1:2 dilution). The positive control group exhibited human relaxin 2 (Genbank Accession No. NP_604390.1) (R&D Systems, Cat. No. 6586-RN-025) using commercially available recombinants. Next, the cells were cultured for 6 hours at 37 ° C in a humidified incubator of 5% carbon dioxide. After 6 hours, the luciferase activity of the cells was determined using a luciferase assay system (Promega, #E1500) and using a Tecan Infinite 500 reader (luminescence mode, 1000 millisecond integration time, measurement time 30 seconds).

Application computer program Graph Pad Prism version 5, using relative hair The luminescence unit measures the EC50 value of different molecules.

To test for relaxin and the surviving activity of the fusion polypeptide of the present invention, a cell line having an endogenously expressed LGR7 receptor (for example, THP1, ATCC catalog number TIB-202) or a primary cell [eg, Celprogen Inc., Human Cardiomyocyte Cell Culture (for example) is used. Human cardiomyocyte cultures, catalog number 36044-15], cultured their cells according to the manufacturer's instructions.

Methods for detecting cAMP production induced by relaxin or relaxin variants are known in the art. For example, such measurements are made using a cAMP ELISA (eg, IBL International GmbH, cAMP ELISA, catalog number CM 581001) according to the manufacturer's instructions.

Methods for detecting the activation of PI3 kinase induced by relaxin or relaxin variants are known in the art. For example, such measurements are made according to the manufacturer's instructions using PI3-kinase HTRF analysis (eg, Millipore, PI3-Kinase HTRF Assay, Cat. No. 33-016).

PEGylation

For pegylation of cysteine residues, the fusion polypeptide is typically treated with a reducing agent such as dithiothreitol (DDT) prior to PEGylation; followed by any conventional method (eg, desalting) In addition to reducing agents. Conjugation of PEG to cysteine residues is typically carried out in a suitable buffer at pH 6-9, at temperatures ranging from 4 ° C to 25 ° C, for up to 16 hours.

It will generally be understood that PEGylation is designed to produce the number of PEG molecules involved in the linkage, the size and shape of such molecules (eg, whether they are linear or branched), and the attachment sites in the fusion polypeptide. Optimal molecule. The molecular weight of the polymer to be used, for example, can be selected according to the desired efficacy to be achieved.

Immunogenicity test

The immunogenicity test was carried out using a computer program NetMHCIIpan (Center for Biological Sequence Analysis; Department of Systems Biology; Technical University of Denmark) which calculates the possible binding affinity of the protein or peptide to the MHC II complex. The higher the calculated binding affinity, the greater the risk of eliciting antibodies to the protein or polypeptide of interest.

The in vitro assay for T cell epitope mapping is based on the experimental protocol published by Reijonen and Kwok [Reijonen H., Kwok WW. (2003) Use of HLA class II tetramers in tracking antigen-specific T cells and mapping T-cell epitopes. Methods 29: 282-288] proceed.

Construction of single-stranded relaxin variants Determination of the optimal linker length for single-stranded relaxin variants

As described above, single-stranded relaxin variants linking the A and B chains with different linker lengths were generated. As described in the sequences, in the case of alternative assays for protein expression, the Myc marker (amino acid sequence EQKLISEEDL) was added to the construct with or without the hemagglutinin marker (amino acid sequence YPYDVPDYA) in several constructs. The N terminal end of the chain, and the addition of 6 histidine acid labels (amino acid sequence HHHHHH) to the C terminal end of the B chain.

Example 1: scR1

In scR1, the linker sequence connecting the A chain and the B chain of human relaxin 2 is composed of three amino acids and consists of a polypeptide having a GlyGlyGly sequence. In the alternative assay for protein performance, the Myc tag is added to the N-terminus of the A chain, and the hemagglutinin tag is added and the 6 histidine tag is labeled to the C-terminus of the B chain.

Example 2: scR2

In scR2, the linker sequence connecting the A chain and the B chain of human relaxin 2 is composed of five amino acids, and is composed of a polypeptide having a GlyGlyGlySerGly sequence. In the alternative assay for protein performance, the Myc tag is added to the N-terminus of the A chain, and the hemagglutinin tag is added and the 6 histidine tag is labeled to the C-terminus of the B chain.

Example 3: scR3

In scR3, the linker sequence connecting the A chain and the B chain of human relaxin 2 is composed of seven amino acids and consists of a polypeptide having a GlyGlyGlySerGlyGlyGly sequence. In the alternative assay for protein performance, the Myc tag is added to the N-terminus of the A chain, and the hemagglutinin tag is added and the 6 histidine tag is labeled to the C-terminus of the B chain.

Example 4: scR4

In scR4, the linker sequence connecting the A chain and the B chain of human relaxin 2 is composed of nine amino acids, and is composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGly sequence. For alternative assays for protein expression, add the Myc tag to the A chain. The N terminal, and the addition of a hemagglutinin label and 6 histidine labels to the C terminal of the B chain.

Example 5: scR5

In scR5, the linker sequence connecting the A chain and the B chain of human relaxin 2 is eleven amino acid long and is composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGlyGlyGly sequence. In the alternative assay for protein performance, the Myc tag is added to the N-terminus of the A chain, and the hemagglutinin tag is added and the 6 histidine tag is labeled to the C-terminus of the B chain.

Example 6: scR6

In scR6, the linker sequence connecting the A chain and the B chain of human relaxin 2 is composed of fifteen amino acids, and is composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGly sequence. In the alternative assay for protein performance, the Myc tag is added to the N-terminus of the A chain, and the hemagglutinin tag is added and the 6 histidine tag is labeled to the C-terminus of the B chain.

Example 7: scR7

In scR7, the linker sequence linking the A chain and the B chain of human relaxin 2 is composed of six amino acids and consists of a polypeptide having a GlyGlyGlySerGlyGly sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

Example 8: scR8

In scR8, the A chain and the B chain of human relaxin 2 are linked. The sequence of the linker is twelve amino acids long and consists of a polypeptide having the GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

Example 9: scR9

In scR9, the linker sequence linking the A and B chains of human relaxin 2 is composed of thirteen amino acids and consists of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

Example 10: scR10

In scR10, the linker sequence linking the A chain and the B chain of human relaxin 2 is composed of fourteen amino acids, and is composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGly sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

Example 11: scR11

In scR11, the linker sequence connecting the A chain and the B chain of human relaxin 2 is composed of ten amino acids, and is composed of a polypeptide having a GlyGlyGlySlyGlyCysGlyGlySerGly sequence. The activity test for unpegylated fusion polypeptides used unpurified proteins.

In order to increase the biological half-life of this construct, PEGylation of cysteine in the linker linking the A chain to the B chain was carried out following the experimental procedure described above. The activity of the PEGylated variant was determined according to the experimental procedure described above.

Example 12: scR12

In scR12, the linker sequence connecting the A chain and the B chain of human relaxin 2 is composed of ten amino acids, and is composed of a polypeptide having a GlyGlyGlySerGlyLysGlyGlySerGly sequence. The activity test for unpegylated fusion polypeptides used unpurified proteins.

In order to increase the biological half-life of this construct, it may be an option to PEGylate the deaminating acid in the linker linking the A chain to the B chain following the experimental procedure described above. The activity of the PEGylated variant was determined according to the experimental procedure described above.

Example 13: scR13

In scR13, the linker sequence of the A-chain C terminus and the B-chain N terminus of human relaxin 2 is composed of nine amino acids and consists of a polypeptide having a sequence of LysArgSerLeuSerArgLysLysArg. The activity test used an unpurified fusion polypeptide.

Example 14: scR14

In scR14, the linker sequence of the A-chain C terminus and the B-chain N terminus linked to human relaxin 3 (accession number NP_543140.1) is nine amino acids long and will be composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGly sequence. According to The above experimental procedure measures activity; the activity test uses an unpurified fusion polypeptide.

Example 15: scR15

In scR15, the linker sequence of the A-chain C terminus and the B-chain N terminus linked to human relaxin 3 (accession number NP_543140.1) is nine amino acids long and will be composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGly sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

Example 16: scR16

In scR16, the linker sequence connecting the C-terminus of the B-chain of human relaxin 2 to the N-terminus of the A-chain is composed of nine amino acids, and will be composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGly sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

Example 17: scR17

In scR17, the linker sequence of the B-chain N-terminus of the A-chain C-terminus of human relaxin 3 (accession number NP_543140.1) and human relaxin 2 (accession number NP_604390.1) is nine amino acids. Long, will consist of a polypeptide having the GlyGlyGlySlyGlyGlyGlySerGly sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

Example 18: scR18

In scR18, the linker sequence of the N-terminus of the A chain of the B-chain of human relaxin 2 (accession number NP_604390.1) and human relaxin 3 (accession number NP_543140.1) is nine amino acids. Long, will consist of a polypeptide having the GlyGlyGlySlyGlyGlyGlySerGly sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

Example 19: scR19

In scR19, the linker sequence of the B-terminus of the B-chain of the A-chain of the human relaxin 2 (accession number NP_604390.1) and human relaxin 3 (accession number NP_543140.1) is nine amino acids. Long, will consist of a polypeptide having the GlyGlyGlySlyGlyGlyGlySerGly sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

Example 20: scR20

In scR20, the linker sequence of the N-terminus of the A chain of the B-chain of human relaxin 3 (accession number NP_543140.1) and human relaxin 2 (accession number NP_604390.1) is nine amino acids. Long, will consist of a polypeptide having the GlyGlyGlySlyGlyGlyGlySerGly sequence. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain. The activity was determined according to the experimental procedure as described above.

A graphical representation of all single-stranded relaxin variants is shown in Figure 2.

Table 1 summarizes the results relating to the performance and biological activity of various scR constructs. Since the single-stranded relaxin variants with a linker of 3, 5, and 15 amino acids were not shown to have any detectable biological activity in the above analysis, unexpectedly, the length tested was 6,7, The linker of 9, 10, 11, 12, 13, and 14 amino acids results in the production of single-stranded variants that exhibit biological activity comparable to human relaxin 2.

The length of the linker connecting the C-terminus of the A chain and the N-terminus of the B-chain is quite important for the production of biologically active molecules, but the amino acid composition of the linker is variable; examples thereof are scR11 to scR13; thus, scR11 and scR12 are linked to the linker. In the case of an additional amino acid (C in the scR11 linker and K in the scR12 linker), or the construct scR13, there is a linker sequence which does not show any homology to the aforementioned linker sequence.

The production of the single-stranded relaxin variant is not limited to relaxin 2, and the constructs scR14 and 15 are single-chain variants of relaxin 3. Although the overall sequence homology between relaxin 2 and relaxin 3 is low, the genomes of the two genes of the insulin superfamily members are identical. Like relaxin 2, relaxin 3 consists of a typical B chain-C chain-A chain structure. Similarly to relaxin 2, the C chain is cleaved from the relaxin 3 pre-peptide using pre-hormone converting enzymes I and II, and the B and A chains are linked via a disulfide bridge to form an active molecule. The scR14 and scR15 constructs are single-stranded variants of relaxin 3, which have the same linker molecules as those shown in the previous example of the relaxin 2 and the construct scR4, which are linked to the C-terminus of the A-chain and the N-terminus of the B-chain. scR14 and scR15 exhibit detectable biological activity.

scR16, scR17, scR18, scR19, and scR20 are relaxin A chimera between the A chain of 3 and the B chain of relaxin 2 (or vice versa). Therefore, in order to activate the LGR7 receptor, it is necessary to forcibly place the B chain of relaxin 2 and relaxin 3 at the C-terminal portion of the relaxin 3/relaxin 2 chimera, respectively.

Dose-effect curve and comparison of hRelaxin2, scR3, scR4, and The corresponding EC50 values for scR5 activity are shown in Figure 4a; for hRelaxin2, scR7, scR8, scR9, and scR10 are shown in Figure 4b; for hRelaxin2, scR11 and scR12 are shown in Figure 4c; for hRelaxin2, hRelaxin3, scR14 and scR15 Figure 4d; and for hRelaxin3 and scR17 are shown in Figure 4e.

Conclusion: The results show that the length of the linker is greater than 5 amino acids and less than 15 amino acids are required for the biological activity of the single-stranded relaxin variant; the C-terminus of the A chain is linked to the B-chain via these linkers. The N end. Furthermore, the production of the single-chain relaxin of the present invention is not limited to relaxin 2.

The binding of relaxin 2 to its corresponding receptor LGR7 is a two-step process. In the first step, the A chain of human relaxin 2 binds to the N-terminal extracellular domain of the receptor. In the second step, the combined extracellular domain undergoes a conformational change, and the second interaction of the relaxin B chain with the LGR7 transmembrane functional interval mediates receptor signaling. This second step is most relevant for the activation of the ligand-receptor complex. Therefore, due to the fact that the variant scR17 contains human relaxin 3 instead of the A chain of human relaxin 2, a construct with reduced activity is produced. A further reduction in activity was observed in the variant BCR containing the B chain of human relaxin 3 rather than the B chain of human relaxin 2. Binding to the extracellular domain occurs via the A chain of human relaxin 2, but the activation of LGR7 is suboptimal via the B chain of relaxin 3. The corresponding receptor for relaxin 3 is LGR8. Thus, it is likely that when scR19 is used as a ligand and LGR8 is used as a corresponding receptor, the message intensity is much higher; this is also a method of modulating the activity of the fusion polypeptide of the present invention.

scR13 is not purified as an explanation for reduced activity because in the sample Possible impurities may result in incorrect determination of concentration or may have a negative impact on the accuracy of cell line luciferase assays.

In summary, the linker sequences shown to be useful are not limited to glycine/serine rich sequences, as other linker sequences (within the length of the invention) can also result in intact active single chain relaxin.

Construction of single-chain relaxin fusion protein with enhanced biological half-life

In order to enhance the biological half-life of the single-stranded relaxin variant, it is designed to add the Fc portion of the immunoglobulin molecule to the N-terminal or C-terminal construct of the single-stranded relaxin variant.

Thus, the single-stranded relaxin variant is directly fused to the Fc portion of the immunoglobulin, or linked by a polypeptide of different length and amino acid.

Another option to increase the biological half-life of the polypeptide is to fuse with a polypeptide such as transferrin (accession number P02787) or albumin (accession number P02768) [SR Schmid (2009)].

PEGylation is a commonly used method to increase the biological half-life of a polypeptide; in this way, a polyethylene glycol polymer chain is added to covalently attach to the polypeptide. Thereby, the reactive derivative of PEG is incubated with the polypeptide of interest. Preferred amino acids reactive with PEG are cysteine and lysine. Pasut and Veronese (2009))

Production of relaxin fusion protein-relaxin-Fc

In order to increase the biological half-life, the Fc portion of human IgG1 is bound to human relaxin 2 by chemical gene synthesis. The carboxy-terminal portion of human relaxin 2 (according to its genetic organization as follows: B-chain-C chain-A chain) is fused to the N-terminus of the human IgG1 Fc portion, thereby enabling fusion These two parts of the protein are linked by a 6 amino acid long linker sequence consisting of a polypeptide having an IleGluGlyArgMetAsp sequence encoding a cleavage site of the factor Xa. However, using the CHO-CRE-LGR7 cell strain assay, relaxin Fc showed no activity.

Example 16: scR-Fc 1

In scR-Fc 1, the sequence of the linker sequence connecting the C terminus of scR 4 to the N terminus of the Fc portion of human IgG1 is 6 amino acids long and consists of a polypeptide having an IleGluGlyArgMetAsp sequence encoding a cleavage site of factor Xa. . This polypeptide and the Fc portion replace the hemagglutinin label in scR 4 with 6 histidine labels. In the case of an alternative assay for protein performance, the Myc tag is added to the N terminus of the A chain.

Example 17: scR-Fc 2

In the scR-Fc 2, the C-terminus of the single-stranded relaxin scR4 and the N-terminal ligation sequence of the human IgG1 Fc portion are composed of four amino acids and consist of a polypeptide having a GlyGlySerPro sequence. In contrast to scR-Fc 1, the N-terminus of this construct A chain does not have the Myc tag.

Example 18: scR-Fc 3

In the scR-Fc 3, the C-terminus of the single-stranded relaxin scR4 and the N-terminal ligation sequence of the human IgG1 Fc portion are composed of seven amino acids and consist of a polypeptide having a GlyGlySerGlyGlySerPro sequence. In contrast to scR-Fc 1, the N-terminus of this construct A chain does not have the Myc tag.

Example 19: scR-Fc 4

In the scR-Fc 4, the C-terminus of the single-stranded relaxin scR4 and the N-terminal linker sequence of the human IgG1 Fc portion are composed of 10 amino acids, and are composed of a polypeptide having a GlyGlySerGlyGlySerGlyGlySerPro sequence. In contrast to scR-Fc 1, the N-terminus of this construct A chain does not have the Myc tag.

Example 20: scR-Fc 5

In scR-Fc 5, the linker sequence of the N-terminus of the single-stranded relaxin scR4 and the C-terminus of the human IgG1 Fc portion is composed of four amino acids and consists of a polypeptide having a GlyGlySerPro sequence. This Fc portion replaces the Myc tag of the A-chain N terminus. The C terminal of this construct does not have a hemagglutinin label and/or a 6 histidine label.

Example 21: scR-Fc 6

In scR-Fc 6, the sequence of the linker sequence connecting the N-terminus of the single-stranded relaxin scR4 to the C-terminus of the human IgG1 Fc portion is 7 amino acids long, and is composed of a polypeptide having a GlyGlySerGlyGlySerPro sequence. This Fc portion replaces the Myc tag of the A-chain N terminus. The C terminal of this construct does not have a hemagglutinin label and/or a 6 histidine label.

Example 22: scR-Fc 7

In scR-Fc 7, the sequence of the linker sequence connecting the N-terminus of the single-stranded relaxin scR4 to the C-terminus of the human IgG1 Fc portion is 10 amino acids long, and is composed of a polypeptide having a GlyGlySerGlyGlySerGlyGlySerPro sequence. This Fc portion replaces the Myc tag of the A-chain N terminus. The C terminal of this construct does not have a hemagglutinin marker and/or 6 groups. Amine acid labeling.

Example 23: scR-Fc 8

In the scR-Fc 8, the C-terminus of the single-stranded relaxin scR4 and the N-terminal ligation sequence of the Fc portion of the rat IgG2b are composed of four amino acids, and are composed of a polypeptide having a GlyGlySerPro sequence. In addition, six histidine labels were added to the C terminus of the Fc portion. In contrast to scR4, the N terminus of this construct A chain does not have the Myc tag. The rat IgG2b Fc portion replaces the hemagglutinin label and the 6 histidine label.

Example 24: scR-Fc 9

In scR-Fc 9, the C-terminus of the single-stranded relaxin scR4 and the N-terminal ligation sequence of the IgG2b Fc portion of the rat are composed of seven amino acids and consist of a polypeptide having a GlyGlySerGlyGlySerPro sequence. In addition, six histidine labels were added to the C terminus of the Fc portion. In contrast to scR4, the N terminus of this construct A chain does not have the Myc tag. The rat IgG2b Fc portion replaces the hemagglutinin label and the 6 histidine label.

Example 25: scR-Fc 10

In the scR-Fc 10, the C-terminus of the single-stranded relaxin scR4 and the N-terminal ligation sequence of the Fc portion of the rat IgG2b Fc are composed of 10 amino acids and consist of a polypeptide having a GlyGlySerGlyGlySerGlyGlySerPro sequence. In addition, six histidine labels were added to the C terminus of the Fc portion. In contrast to scR4, the N terminus of this construct A chain does not have the Myc tag. The rat IgG2b Fc portion replaces the hemagglutinin marker Record 6 histidine labels.

Example 26: scR-Fc 11

In scR-Fc 11, the sequence of the linker sequence connecting the N-terminus of the single-stranded relaxin scR4 to the C-terminus of the Fc portion of the rat IgG2b is composed of four amino acids, and is composed of a polypeptide having a GlyGlySerPro sequence. In addition, six histidine labels were added to the N terminus of the Fc portion. This rat IgG2b Fc portion replaces the Myc tag. At the same time, the C terminal of the construct does not have a hemagglutinin label and/or a 6 histidine label.

Example 27: scR-Fc 12

In the scR-Fc 11, the N-terminus of the single-stranded relaxin scR1 and the C-terminal ligation sequence of the Fc portion of the rat IgG2b are composed of seven amino acids and consist of a polypeptide having a GlyGlySerGlyGlySerPro sequence. In addition, six histidine labels were added to the N terminus of the Fc portion. This rat IgG2b Fc portion replaces the Myc tag. At the same time, the C terminal of the construct does not have a hemagglutinin label and/or a 6 histidine label.

Example 28: scR-Fc 13

In the scR-Fc 11, the N-terminus of the single-stranded relaxin scR4 and the C-terminal ligation sequence of the Fc portion of the rat IgG2b are composed of 10 amino acids, and are composed of a polypeptide having a GlyGlySerGlyGlySerGlyGlySerPro sequence. In addition, six histidine labels were added to the N terminus of the Fc portion. This rat IgG2b Fc portion replaces the Myc tag. At the same time, the C terminal of the construct does not have a hemagglutinin label and/or a 6 histidine label.

Example 29: scR-Fc 14

To analyze the effect of the linker sequence linking the single-stranded relaxin variant to the Fc portion, the C-terminus of the sequence scR4 was directly fused to the Fc portion of human IgG1 in scR-Fc 14. This Fc portion replaces the hemagglutinin marker in scR4 with six histidine markers. The N terminal of this construct A chain does not have the Myc flag.

Example 30: scR-Fc 15

In scR-Fc 15, the C-terminus of the single-stranded relaxin scR4 and the N-terminal ligation sequence of the human IgG1 Fc portion are composed of six amino acids and consist of a polypeptide having a GlySerGlySerGlySer sequence. The human IgG1 Fc portion replaces the hemagglutinin marker with six histidine markers. The N terminal of this construct A chain does not have the Myc flag.

Example 31: scR-Fc 16

The scR-Fc 16 line was designed to analyze the effect of the disulfide bridge in the Fc portion on protein expression and fusion protein activity; to this end, the cysteine at position 86 in the human IgG1 Fc portion of scR-Fc 15 was replaced with alanine. Residues.

Example 32: scR-Fc 17

In the scR-Fc 17, the sequence of the linker sequence connecting the C-terminus of the single-stranded relaxin scR4 to the N-terminus of the Fc portion of the rat IgG2b is 6 amino acids long, and is composed of a polypeptide having a GlySerGlySerGlySer sequence. The rat IgG2b Fc portion replaces the hemagglutinin label and the 6 histidine label. The N terminal of this construct A chain does not have the Myc flag.

Example 33: scR-Fc 18

In scR-Fc 18, the C terminus of the single-stranded relaxin scR4 is linked to The N-terminal linker sequence of the human IgG1 Fc portion is composed of 21 amino acids long and consists of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGlyThrLysValThrValSerSerGluSerLysTyrGly sequence. The human IgG1 Fc portion replaces the hemagglutinin marker and six histidine markers. The N terminal of this construct A chain does not have the Myc flag.

Example 34: scR-Var1

In scR-Var1, the linker sequence connecting the A chain and the B chain of human relaxin 2 is 9 amino acids long, and is composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGly sequence. In addition, 6 amino acids are long, and a polypeptide having a GlyGlySerGlyCysGly sequence is added to the C terminal of the B chain. The activity test for unpegylated fusion polypeptides used unpurified proteins.

In order to enhance the biological half-life of this construct, PEGylation of cysteine in the extended polypeptide fused to the B-chain C terminus was carried out following the experimental procedure described above. The activity of the PEGylated variant was determined according to the experimental procedure described above.

Example 35: scR-Var2

In scR-Var2, the linker sequence connecting the A chain and the B chain of human relaxin 2 is 9 amino acids long, and is composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGly sequence. In addition, 6 amino acids are long, and a polypeptide having a GlyCysGlySerGlyGly sequence is added to the N terminus of the A chain. The activity test for unpegylated fusion polypeptides used unpurified proteins.

In order to enhance the biological half-life of this construct, PEGylation of cysteine in the extended polypeptide fused to the N-terminus of the A chain was carried out following the experimental procedure described above. The activity of the PEGylated variant was determined according to the experimental procedure described above.

Example 36: scR-Var3

In scR-Var3, the linker sequence of the A-chain C terminus and the B-chain N terminus of human relaxin 2 is composed of 9 amino acids, and is composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGly sequence. At the N-terminus of the A chain, a polypeptide having an IleGluGlyArgMetAsp encoding a cleavage site of the factor Xa is ligated to the C terminus of the human transferrin (accession number NP_001054.1). The activity was determined according to the experimental procedure as described above.

Example 37: scR-Var4

Wild type relaxin 2 (gene body tissue) was fused to transferrin in scR-Var4. To this end, at the N-terminus of the B chain, a polypeptide having the IleGluGlyArgMetAsp encoding the cleavage factor Xa cleavage position of the C terminal of this variant and human transferrin (accession number NP_001054.1) was ligated. The activity was determined according to the experimental procedure as described above.

Example 38: scR-Var5

In scR-Var5, the linker sequence of the A-chain C terminus and the B-chain N terminus of human relaxin 2 is composed of 9 amino acids, and is composed of a polypeptide having a GlyGlyGlySerGlyGlyGlySerGly sequence. At the N-terminus of the A chain, a polypeptide having an IleGluGlyArgMetAsp encoding a cleavage site of the factor Xa is ligated to the human albumin C terminal of protein (accession number NP_000468.1). The activity was determined according to the experimental procedure as described above.

Example 39: scR-Var6

In scR-Var6, a polypeptide having an IleGluGlyArgMetAsp sequence encoding a cleavage site Xa cleavage site of the B-chain N terminus is ligated to the C-terminus of human albumin (accession number NP_000468.1). The activity was determined according to the experimental procedure as described above.

Example 40: scR-Var7

In scR-Var7, the linker sequence linking the A-chain C terminus of human relaxin 2 to the B-terminus N terminus of human relaxin 2 is 9 amino acids long and consists of a polypeptide having a sequence of LysArgSerLeuSerArgLysLysArg.

The linker sequence between the C terminus of the B chain and the N terminus of the human IgG1 Fc portion is 6 amino acids long and consists of a polypeptide having an IleGluGlyArgMetAsp sequence encoding the cleavage site of coagulation factor Xa.

Example 41: scR-Var8

In scR-Var8, the sequence of the linker sequence connecting the C terminus of the A chain to the N terminus of the B chain is 9 amino acids long, and is composed of a polypeptide having a sequence of LysArgSerLeuSerArgLysLysArg.

The linker sequence connecting the N terminus of the A chain to the C terminus of the human IgG1 Fc portion is 6 amino acids long and consists of a polypeptide having an IleGluGlyArgMetAsp sequence encoding the cleavage site of coagulation factor Xa.

A graphical representation of all single-stranded relaxin fusion proteins and variants designed for pegylation is shown in Figure 3.

Table 2 summarizes the performance and biological activity of various scR fusion protein constructs.

Human relaxin-2 can be used for all variants listed The performance was determined by the Quantikine ELISA kit and the activity was determined using the CHO-CRE-LGR7 cell line. Incidentally, dose-response curves of scR-Fc 1, scR-Fc 5 to scR-Fc 7, scR-Fc 11 to scR-Fc 13, and scR-Var3 to scR-Var6 are shown in Fig. 5 and Fig. 6, respectively. Figure 7, and Figure 8.

The human wild-type relaxin 2 molecule fused to the Fc portion of the human IgG molecule with its B chain-C chain-A chain did not exhibit any detectable activity. The possibility that this molecule is not active is explained by the incomplete treatment of the C chain. In contrast, all fusion constructs containing single-chain human relaxin 2 were able to detect significant activity. As indicated above, although not subjected to proteolytic treatment, the single-chain relaxin also exhibits activity comparable to human wild-type relaxin 2.

In the case of single-stranded relaxin 2 fusion constructs, the localization of the Fc portion appears to have a significant effect on the activity of their molecules. Constructs carrying the Fc portion (eg, scR-Fc 1 to scR-Fc 4 and scR-Fc 13 to scR-Fc 18) at the B-chain C terminus carry an Fc portion construct (eg, scR-) than the A-chain N-terminus Fc 5 to scR-Fc 6 and scR-Fc 11 to scR-Fc 12) exhibited slightly lower activity. As described above, after the A chain binds to the extracellular domain of the corresponding receptor LGR7, the conformational change in the receptor molecule causes the B chain to contact the extracellular loop of the transmembrane domain; this second step then leads to receptor activation. Thus, the Fc portion coupled to the B chain inhibits optimal binding of the B chain and thus inhibits complete activation of the receptor.

In vivo plasma stability analysis of Fc-single-chain relaxin

ScR-Fc 13 and hRelaxin2 at a concentration of 240 μg/kg Eight-week-old Wistar male rats were administered intravenously. Blood samples were taken at 0 hour, 1 hour, 3 days, 5 days, and 7 days after compound administration, using a commercially available quantitative ELISA (R&D Systems, Human Relaxin-2 Quantikine ELISA kit) , Catalog No. DRL200) Determine the concentration of Fc-single-chain relaxin and unmodified hRelaxin2.

As shown in Figure 9, unmodified hRelaxin2 was not detected after 3 days of administration, and even after 7 days of intravenous administration, significant concentrations of scR-Fc 13 were detected, even at concentrations higher than CHO-LGR7. The EC50 value obtained based on the activity test.

Determination of Fc-single-chain relaxin activity from plasma isolated

To determine whether scR-Fc 13 still exhibited activity after 3, 5, and 7 days of intravenous administration, plasma samples were tested using the CHO-CRE-LGR7 cell line. 10, for all three samples may be obtained by measuring the activity EC, and all activity values of the three samples using the purified 13 variants scR-Fc ₅₀ similar values.

Isolated perfused rat heart

Weiss male rats (200-250 g) were anesthetized with intraperitoneal injection of pentobarbital (Narcoren, 100 mg/kg). The heart was quickly removed and connected to a Langendorff perfusion system (FMI GmbH, Seeheim-Ober Beerbach, Germany). The heart was perfused with a Krebs-Henseleit bicarbonate buffer equilibrated with 95% O ₂ -5% CO ₂ at a fixed rate of 10 ml/min. The perfusion solution contained (mole/liter): NaCl 118; KCl 3 ; NaHCO ₃ 22; KH ₂ PO ₄ 1,2; MgSO ₄ 1.2; CaCl ₂ 1.8; glucose 10; sodium pyruvate 2. The perfusion pressure in the perfusion system is shown by a pressure transducer. Left ventricular pressure (LVP) was measured using a second pressure transducer connected to a water filled balloon inserted into the left atrium through the left ventricle. By adjusting the volume of the balloon, the end-diastolic pressure is initially set at 8 mm Hg; the heart naturally beats; the signal from the pressure transducer is amplified, displayed, and used to calculate the heart frequency and +dp/dt on a personal computer.

As shown in Figure 11, perfusion of human relaxin 2 (Figures 11a-d) and scR-Fc 13 (Figures 11e-h) resulted in a significant increase in heart rate and coronary blood flow, and left ventricular diastolic pressure and left ventricular pressure ( Reduction of +dp/dtmax). Thus, hRelaxin2 has a 10-fold more potency than scR-Fc 13, reflecting the difference in EC50 values between the sc relaxin fusion protein variant and relaxin 2 as measured by the CHO-CRE-LGR7 cell line.

Further references: Hsu, SY (2003). New insights into the evolution of the relaxin-LGR signaling system. Trends Endocrinol Metab 14:303-309; Wilkinson, TN, Speed, TP, Tregear, GW, Bathgate, RA ( 2005). Evolution of the relaxin-like peptide family. BMC Evol Biol 5:14; Hudson P, Haley J, John M, Cronk M, Crawford R, Haralambidis J, Tregear G, Shine J, Niall H. (1983) Structure Nature 301: 628-631; Toth, M., Taskinen, P., & Ruskoaho, H. (1996). Relaxin stimulates atrial natriuretic peptide secretion in perfused rat heart. J Endocrinol 150 : 487-495; Piedras-Renteria, ES, Sherwood, OD, and Best, PM (1997). Effects of relaxin on rat atrial myocytes: I. Inhibition of I(to) via PKA-dependent phosphorylation. Am J Physiol 272: H1791-H1797; Bartsch, O., Bartlick, B., and Ivell, R. (2001). Relaxin signaling links tyrosine phosphorylation to phosphodiesterase and adenylyl cyclase activity. Mol Hum Reprod 7:799-809; Bartsch, O., Bartlick, B., and Ivell, R. (2004). Phosphodiesterase 4 inhibition synergizes with relaxin Signaling to promote decidualization of human endometrial stromal cells. J Clin Endocrinol Metab 89:324-334; Bani-Sacchi, T., Bigazzi, M., Bani, D., Mannaioni, PF, and Masini, E. (1995) Relaxin -induced increased coronary flow through stimulation of nitric oxide production. Br J Pharmacol 116:1589-1594; Dschietzig T, Bartsch C, Baumann G, Stangl K. (2006) Relaxin - a pleiotropic hormone and its emerging role for experimental and clinical therapeutics Pharmacol. Ther. 112:38-56; McGuane JT, Parry LJ. (2005) Relaxin and the extracellularmatrix: Molecular mechanisms of actio Experts Rev. Mol.Med. 7:1-18; Nistri, S., Chiappini, L., Sassoli, C. and Bani, D. (2003) Relaxin inhibits lipopolysaccharide-induced adhesion of Neutrophils to coronary endothelial cells by a nitric Oxide-mediated mechanism. FASEB J. 17:2109-2111; Perna AM, Masini E, Nistri S, Briganti V, Chiappini L, Stefano P, Bigazzi M, Pieroni C, Bani Sacchi T, Bani D. (2005) Novel drug Development opportunity for relaxin in acute myocardial infarction: evidences from a swine model. FASEB J. 19:1525-1527; Bani, D., Masini, E., Bello, MG, Bigazzi, M. and Sacchi, TB (1998) Relaxin Amper J. Pathol. 152:1367-1376; Zhang J, Qi YF, Geng B, Pan CS, Zhao J, Chen L, Yang J, Chang JK, Tang CS. (2005) Effect of relaxin on myocardial ischemia injury induced by isoproterenol. Peptides 26:1632-1639; Teerlink JR, Metra M, Felker GM, Ponikowski P, Voors AA, Weatherley BD, Marmor A, Katz A, Grzybowski J, Unemori E, Teichman SL, Cotter G. (2009) Relaxin for the treatment of patients with acute heart failure (Pre-RELAX-AHF): a multicentre, randomised, placebo-controlled, parallel-group, dose-fi nding phase IIb study Lancet. 373:1429-39; Metra M, Teerlink JR, Felker GM, Greenberg BH, Filippatos G, Ponikowski P, Teichman SL, Unemori E, Voors AA, Weatherley BD, Cotter G. (2010) Dyspnoea and worsening heart failure in patients with Acute heart failure: results from the Pre-RELAX-AHF study. Eur J Heart Fail. 12:1130-1139; Cosen-Binker LI, Binker MG, Cosen R, Negri G, Tiscornia O. (2006) Relaxin prevents the development of severe acute pancreatitis. World J. Gastroenterol. 12:1558-1568; Santora K, Rasa C, Visco D, Steinetz BG, Bagnell CA. (2007) Antiarthritic effects of relaxin, in combination with estrogen, in rat adjuvant induced arthritis. J. Pharmacol. Exp. Ther. 322:887-893; Bennett RG. (2009) Relaxin and its role in the Development and treatment of fibrosis. Transl Res. 154:1-6; Barlos KK, Gatos D, Vasileiou Z, Barlos K. (2010) An optimized chemical synthesis of human relaxin-2. J Pept Sci. 16:200-211; Park JI, Semyonov J, Yi W, Chang CL, Hsu SY (2008) Regulation of receptor signaling by relaxin A chain motifs: derivation of pan-specific and LGR7-specific human relaxin analogs. J Biol Chem. 283:32099-32109; Shaw JA, Delday MI, Hart AW, Docherty HM, Maltin CA, Docherty K (2002) Secretion of bioactive human insulin following plasmid-mediated gene transfer to non-neuroendocrine cell lines, primary cul Tures and rat skeletal muscle in vivo. J Endocrinol 172:653-672; Rajpal G, Liu M, Zhang Y, Arvan P, (2009) Single-Chain Insulins as Receptor Agonists. Mol Endocrinol. 23:679-88; Dschietzig T , Teichmann S, Unemori E, Wood S, Boehmer J, Richter C, Baumann G, Stangl K (2009) Intravenous Recombinant Human Relaxin in Compensated Heart Failure: A Safety, Tolerability, and Pharmacodynamic Trial. J Cardiac Fail 5: 182-190; WO2006053299 A2, Site-directed modification of FVIII, Bayer Healthcare LLC; Harris JM, Martin NE, Modi M. (2001) Pegylation: a novel process for functional pharmacokinetics. Clin Pharmacokinet. 40:539-551; Schmid SR, (2009) Fusion-proteins as biopharmaceuticals--applications and challenges Curr Opin Drug Discov Devel. 12: 284-95; Pasut and Veronese (2009) PEGylation for improving the effectiveness of therapeutic biomolecules. Drugs Today 45: 687-695; WO 97/26265 WO 99/03861 WO 00/06568 WO 00 /06569 WO 02/42301 WO 03/095451 WO 01/19355 WO 01/19776 WO 01/19778 WO 01/19780 WO 02/070462 WO 02/070510.

Figure 1. Schematic diagram of the gene organization of wild-type relaxin and single-chain relaxin and its corresponding polypeptide functional regions.

Figure 2 is a schematic diagram of a single-stranded relaxin variant.

Figure 3 is a schematic diagram showing the functional region of a single-stranded relaxin fusion protein variant and a single-stranded relaxin variant of a PEGylated design.

Figures 4a-e use scR 3, scR 4, and scR 5 (Figure 4a), scR 7, scR 8, scR9, and scR10 (Figure 4b), scR11 and scE12 (Figure 4c), CHO-CRE-LGR7 cell line, Activity of human relaxin 3, scR14, and scR15 (Fig. 4d) and scR17 (Fig. 4e) in functional analysis. The control group used hRelaxin2 (R&D Systems, catalog number 6586-RN-025). Data are expressed as Relative Light Units, representing the activity of single-stranded relaxin variants and relaxin 2 induced luciferase expression. The symbol indicates the average value and the error bar indicates the SEM.

Figure 5 shows the activity of scR-Fc 1 of the CHO-CRE-LGR7 cell line in a functional assay. The control group used hRelaxin2 (R&D Systems, catalog number 6586-RN-025). Data are expressed as relative absorbance values, representing the activity of scR-Fc 1 and hRelaxin2 to induce luciferase expression. The symbol indicates the average value and the error bar indicates the SEM.

Figure 6 shows the activity of scR-Fc 5, scR-Fc 6, and scR-Fc 7 in a functional assay using the CHO-CRE-LGR7 cell line. The control group used hRelaxin2 (R&D Systems, catalog number 6586-RN-025). Data are expressed as relative absorbance values representing the activity of the scR-Fc variant and hRelaxin2 to induce luciferase expression. The symbol indicates the average value and the error bar indicates the SEM.

Figure 7 shows the activity of scR-Fc 11, scR-Fc 12, and scR-Fc 13 in a functional assay using the CHO-CRE-LGR7 cell line. The control group used hRelaxin2 (R&D Systems, catalog number 6586-RN-025). Data are expressed as relative absorbance values representing the activity of the scR-Fc variant and hRelaxin2 to induce luciferase expression. The symbol indicates the average value and the error bar indicates the SEM.

Figure 8 shows the activity of scR-Var 3, scR-Var 4, scR-Var 5, and scR-Var 6 in a functional assay using the CHO-CRE-LGR7 cell line. The control group used hRelaxin2 (R&D Systems, catalog number 6586-RN-025). Data are expressed as relative absorbance values representing the activity of the scR-Fc variant and hRelaxin2 to induce luciferase expression. The symbol indicates the average value and the error bar indicates the SEM.

Figure 9 In vivo half-life analysis of hRelaxin2 or scR-Fc 13 administered intravenously. Eight-week-old Wistar rat rats (3 animals per group) were administered a single dose of human relaxin 2 and scR-Fc 13 (0.24 mg/kg), respectively. Blood samples were collected at the indicated time points after administration, and the protein content in the serum was determined by quantitative ELISA.

Figure 10 shows the activity of relaxin 2 and relaxin variants in blood samples.

The relaxin activity in the blood samples obtained from the scR-Fc 13 treated rats was measured using a CHO-CRE-LGR7 cell strain. Blood samples collected 3, 5, and 7 days after intravenous administration of scR-Fc 13 were incubated in CHO-CRE-LGR7 cell line, and relative absorbance values were measured. The calibration curve was determined using hRelaxin2 (R&D Systems, catalog number 6586-RN-025) and purified scR-Fc 13. The EC50 within the dose response curve is labeled with X. Data are expressed as relative absorbance values representing the activity of the scR-Fc variant and hRelaxin2 to induce luciferase expression. The symbol indicates the average value and the error bar indicates S.E.M.

Figure 11: Effect of hRelaxin2 and scR-Fc 13 on heart rate, coronary blood flow and contractility in isolated perfused rat heart model.

At 1 nM, administration of hRelaxin2 resulted in increased heart rate and coronary blood flow, and exhibited negative inotropic activity (Fig. 11a-d); comparable efficacy was obtained with scR-Fc 13 but at 10 times higher concentration .

<110> Bayer Intellectual Property GmbH

<120> Relaxin fusion polypeptide and use thereof

<130> BHC 11 1 007

<160> 161

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<210> 48

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 48 50 55

<210> 49

<211> 64

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 49

<210> 50

<211> 65

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 50

<210> 51

<211> 66

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 51

<210> 52

<211> 298

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 52

<210> 53

<211> 291

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 53

<210> 54

<211> 294

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 54

<210> 55

<211> 297

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 55

<210> 56

<211> 292

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 56

<210> 57

<211> 295

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 57

<210> 58

<211> 298

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 58

<210> 59

<211> 240

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 59

<210> 60

<211> 246

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 60

<210> 61

<211> 252

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 61

<210> 62

<211> 258

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 62

<210> 63

<211> 264

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 63

<210> 64

<211> 276

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 64

<210> 65

<211> 204

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 65

<210> 66

<211> 222

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 66

<210> 67

<211> 225

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 67

<210> 68

<211> 228

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 68

<210> 69

<211> 186

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 69

<210> 70

<211> 186

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 70

<210> 71

<211> 183

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 71

<210> 72

<211> 180

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 72

<210> 73

<211> 210

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 73

<210> 74

<211> 924

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 74

<210> 75

<211> 876

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 75

<210> 76

<211> 885

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 76

<210> 77

<211> 894

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 77

<210> 78

<211> 876

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 78

<210> 79

<211> 885

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 79

<210> 80

<211> 894

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 80

<210> 81

<211> 891

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 81

<210> 82

<211> 900

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 82

<210> 83

<211> 909

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 83

<210> 84

<211> 894

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 84

<210> 85

<211> 903

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 85

<210> 86

<211> 912

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 86

<210> 87

<211> 864

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 87

<210> 88

<211> 882

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 88

<210> 89

<211> 882

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 89

<210> 90

<211> 882

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 90

<210> 91

<211> 933

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 91

<210> 92

<211> 201

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 92

<210> 93

<211> 201

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 93

<210> 94

<211> 2238

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 94

<210> 95

<211> 2538

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 95

<210> 96

<211> 1956

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 96

<210> 97

<211> 2256

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 97

<210> 98

<211> 894

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 98

<210> 99

<211> 894

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 99

<210> 100

<211> 165

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 100

<210> 101

<211> 171

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 101

<210> 102

<211> 177

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 102

<210> 103

<211> 183

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 103

<210> 104

<211> 189

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 104

<210> 105

<211> 201

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 105

<210> 106

<211> 174

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 106

<210> 107

<211> 192

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 107

<210> 108

<211> 195

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 108

<210> 109

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 109

<210> 110

<211> 894

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 110

<210> 111

<211> 873

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 111

<210> 112

<211> 882

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 112

<210> 113

<211> 891

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 113

<210> 114

<211> 876

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 114

<210> 115

<211> 885

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 115

<210> 116

<211> 894

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 116

<210> 117

<211> 24

<212> PRT

<213> Homo sapiens

<400> 117

<210> 118

<211> 16

<212> PRT

<213> Homo sapiens

<400> 118

<210> 119

<211> 28

<212> PRT

<213> Homo sapiens

<400> 119

<210> 120

<211> 231

<212> PRT

<213> Homo sapiens

<400> 120

<210> 121

<211> 226

<212> PRT

<213> Brown Rat (Rattus Norvegicus)

<400> 121

<210> 122

<211> 679

<212> PRT

<213> Homo sapiens

<400> 122

<210> 123

<211> 585

<212> PRT

<213> Homo sapiens

<400> 123

<210> 124

<211> 24

<212> PRT

<213> Homo sapiens

<400> 124

<210> 125

<211> 27

<212> PRT

<213> Homo sapiens

<400> 125

<210> 126

<211> 15

<212> PRT

<213> Homo sapiens

<400> 126

<210> 127

<211> 72

<212> DNA

<213> Homo sapiens

<400> 127

<210> 128

<211> 48

<212> DNA

<213> Homo sapiens

<400> 128

<210> 129

<211> 84

<212> DNA

<213> Homo sapiens

<400> 129

<210> 130

<211> 693

<212> DNA

<213> Homo sapiens

<400> 130

<210> 131

<211> 678

<212> DNA

<213> Brown Rat

<400> 131

<210> 132

<211> 2037

<212> DNA

<213> Homo sapiens

<400> 132

<210> 133

<211> 1755

<212> DNA

<213> Homo sapiens

<400> 133

<210> 134

<211> 72

<212> DNA

<213> Homo sapiens

<400> 134

<210> 135

<211> 81

<212> DNA

<213> Homo sapiens

<400> 135

<210> 136

<211> 45

<212> DNA

<213> Homo sapiens

<400> 136

<210> 137

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 137

<210> 138

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 138

<210> 139

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 139

<210> 140

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 140

<210> 141

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 141

<210> 142

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 142

<210> 143

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 143

<210> 144

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 144

<210> 145

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 145

<210> 146

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 146

<210> 147

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 147

<210> 148

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 148

Gly Gly Ser Pro

1

<210> 149

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 149

<210> 150

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 150

<210> 151

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 151

<210> 152

<211> 71

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 152

<210> 153

<211> 71

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 153

<210> 154

<211> 71

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 154

<210> 155

<211> 70

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 155

<210> 156

<211> 70

<212> PRT

<213> Artificial sequence

<220>

<223> Relaxin fusion polypeptide

<400> 156

<210> 157

<211> 213

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion

<400> 157

<210> 158

<211> 213

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion

<400> 158

<210> 159

<211> 213

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion

<400> 159

<210> 160

<211> 210

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion

<400> 160

<210> 161

<211> 210

<212> DNA

<213> Artificial sequence

<220>

<223> Relaxin fusion

<400> 161

Claims (16)

A fusion polypeptide having relaxin activity comprising A-L-B, wherein B comprises a relaxin B chain polypeptide or a functional variant thereof, and A comprises a relaxin A chain polypeptide or a functional variant thereof, and an L line linker polypeptide. The fusion polypeptide according to claim 1, wherein the fusion polypeptide further comprises at least one group having a half-life extension. A fusion polypeptide according to claim 2, wherein the half-life extending group is an IgG1 Fc domain, PEG or HES. The fusion polypeptide according to any one of the preceding claims, wherein the linker polypeptide L is 6 to 14 amino acids long. The fusion polypeptide according to any one of the preceding claims, wherein the relaxin A chain is a human relaxin 2A chain (SEQ ID NO: 117) and the relaxin B chain is a human relaxin 2B chain (SEQ ID NO: 119) . A fusion polypeptide comprising the polypeptide as described in Table 3. The fusion polypeptide according to any one of claims 1 to 6, wherein the ALB is selected from the group consisting of scR3, scR4, scR5, unlabeled scR3, unlabeled scR4, unlabeled scR5, scR-Fc5, scR-Fc6 and scR- A group of ALB polypeptides composed of Fc7. A polynucleotide encoding a fusion polypeptide according to any one of the preceding claims. A vector comprising the polynucleotide according to item 8 of the patent application. A host cell comprising the vector according to item 9 of the patent application or the polynucleotide according to item 8 of the patent application. A method of producing a polypeptide according to any one of claims 1 to 7 which comprises the step of culturing a host cell as claimed in claim 10 and the step of isolating the polypeptide. A pharmaceutical composition comprising the fusion polypeptide according to any one of claims 1 to 7. The pharmaceutical composition according to claim 12, or the fusion polypeptide according to any one of claims 1 to 7, which is used as a medicament. A pharmaceutical composition according to any one of claims 12 and 13 or a fusion polypeptide according to any one of claims 1 to 7 which is used as a medicament for the treatment of cardiovascular diseases, pulmonary diseases, fibrotic diseases or Kidney disease. A method for treating a cardiovascular disease, a pulmonary disease, a fibrotic disorder or a kidney disease, comprising administering a therapeutically effective dose of a pharmaceutical composition according to claims 12 and 13 or according to claims 1 to 7 A fusion polypeptide of any of the following. The method of treatment according to claims 14 and 15 wherein the cardiovascular disease is coronary heart disease, acute coronary syndrome, heart failure, and myocardial infarction.