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 polypeptides.

BACKGROUND OF THE INVENTION

Relaxin 2 (H2 relaxin, RLN2) as a member of the insulin superfamily is a 2-chain peptide exhibiting, on the genetic level, the typical B-C-A chain prohormone structure, arranged from N- to C-terminus. Other members of this superfamily, encoded by 7 genes in human, are 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; nevertheless, phylogenetic analysis indicates that these genes have 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 and is the product of an enzymatic cleavage of the prohormone catalyzed by the Prohormone-Convertase 1 (PC1) and 2 (PC2) (Hudson P. et al. (1983)). The resulting A- and B-chains are joined by two intermolecular cysteine bridges; the A-chain exhibits an additional intramolecular disulfide bond.

Relaxin initiates pleiotropic effects through multiple pathways on a variety of cell types. It confers its activity by binding to the class I (rhodopsin like) G-protein-coupled receptor termed LGR7 (leucine-rich G protein-coupled receptor 7) also named RXFP1 (relaxin family peptide 1 receptor), and with significantly lower affinity to LRG8/RXFP2 (relaxin family peptide 2 receptor) (Kong R C et al. (2010) Mol Cell Endocrinol. 320:1-15). Within the Relaxin molecule, an amino acid motif in the B-chain (Arg-X-X-X-Arg-X-X-Ile/Val-X) (Schwabe and BÃ¼llesbach (2007) Adv Exp Med Biol. 612:14-25 and BÃ¼llesbach and Schwabe J Biol Chem. 2000 Nov. 10; 275(45):35276-80) is conserved in all of the Relaxin peptides and is crucial for the interaction of these peptides with the corresponding receptor. Binding of Relaxin to LGR7/RXFP1 leads to activation of adenylate cyclase and to an increase of the second messenger molecule cAMP. Via this mechanism, Relaxin 2 for example mediates the release of atrial natriuretic peptide in rat hearts (Toth, M. et al. (1996)). A positive inotropic effect of Relaxin 2 on rat atrial myocytes has also been shown (Piedras-Renteria, E. S. et al. (1997)). Other signal transduction molecules which are activated by the Relaxin/LGR7 complex are the phosphoinositide-3 kinase, tyrosine kinases, and phosphodiesterases (Bartsch, O. et al. (2001), Bartsch, O. et al. (2004)). Additional signal transduction pathways activated by this system include the nitric oxide (NO) pathway leading to increased levels of cyclic GMP in rat and guinea-pig hearts (Bani-Sacchi, T. et al. (1995)).

Relaxin acts as a pleiotropic hormone (Dschietzig T. et al. (2006)) possessing biological activity on organs such as lung, kidney, brain, and heart. A strong antifibrotic and vasodilator activity of Relaxin is most notably responsible for the positive effects obtained with this peptide in various animal disease models as well as in clinical studies (McGuane J. T. et al. (2005)). RLN2 has multiple beneficial actions in the cardiovascular system under pathological conditions. It maintains tissue homeostasis and protects the injured myocardium during various pathophysiological processes. It exhibits prominent vasodilatory effects, e.g. affecting flow and vasodilation in rodent coronary arteries (Nistri, S. et al. (2003)) and in the vascular beds of other organs. In spontaneously hypertensive rats RLN2 lowered blood pressure, an effect mediated by increased NO production.

A cardioprotective activity of Relaxin 2 has been evaluated in different animal models such as guinea pig, rat and pig (Perna A. M. et al. (2005), Bani, D. et al. (1998)). RLN2 ameliorates myocardial injury, inflammatory cell infiltration and subsequent fibrosis, thereby alleviating severe ventricular dysfunction (Zhang J. et al. (2005)).

Relaxin 2 exhibits strong antifibrotic activity. In injured tissues, fibroblast activation and proliferation causes increased collagen production and interstitial fibrosis. Fibrosis in the heart is increased by biomechanical overload, and influences ventricular dysfunction, remodeling, and arrhythmogenesis. In animal models, continuous infusion of Relaxin 2 inhibits or even reverses cardiac dysfunction caused by cardiomyopathy, hypertension, isoprenaline-induced cardiac toxicity, diabetic cardiomyopathy and myocardial infarction. This inhibition of fibrogenesis or reversal of established fibrosis can reduce ventricular stiffening and improve diastolic function. Notably, although Relaxin 2 reduces aberrant collagen accumulation, it does not affect basal collagen content in healthy tissues, highlighting its safety for therapeutic use.

Relaxin 2 has been tested in several clinical studies as a pleiotropic vasodilator for the treatment of patients with acute heart failure with very promising outcome. In these studies, Relaxin 2 was associated with favourable relief of dyspnoea and other clinical outcomes (Teerlink J. R. et al. (2009), Metra M. et al. (2010))

Due to the limited in-vivo half life of Relaxin, treatment of patients has to be repeated every 14 to 21 days, whereby compound administration has to be performed as a continuous infusion for at least 48 hours.

Furthermore, Relaxin 2 may also be useful in the treatment of diseases such as pancreatitis, inflammation-related diseases like rheumatoid arthritis, and cancer (Cosen-Binker L. I. et al. (2006) Santora K. Et al. (2007)) or scleroderma, pulmonary, renal, and hepatic fibrosis (Bennett R G. (2009)). Relaxin 2 reduces xenograft tumour growth of human MDA-MB-231 breast cancer cells (Radestock Y, Hoang-Vu C, Hombach-Klonisch S. (2008) Breast Cancer Res. 10:R71).

The synthesis of Relaxin 2 by chemical methods is difficult. Due to the low solubility of the B-chain and the requirement for the laborious, specific introduction of cysteine bridges between A and B-chains, yields of active peptide obtained by these methods are extremely low (Barlos K. K. et al. (2010)). Alternatively, recombinant expression of Relaxin 2 can be performed. To allow efficient cleavage of the prepro-peptide during post-translational modifications and the secretion of mature and biological active peptides, expression host cells are routinely co-transfected with expression constructs encoding the Prohormone-Convertase 1 and/or 2 (Park J. I. et al. (2008)). Nevertheless, the endoproteolytic processing efficiency of prepro-peptides in heterologous cells often limits the production of bioactive molecules significantly (Shaw J. A. et al. (2002)).

Therefore, it would be of great advantage to generate a Relaxin molecule which independent of endoproteolytic processing mediated by specific proteases exhibits full biological activity and can be produced in significant yields using heterologous expression systems.

For human Insulin, single-chain variants have been generated in which an uncleavable polypeptide connects the insulin B-chain with the insulin A-chain (Rajpal G. et al. (2009)). For these variants, endoproteolytic processing is dispensable.

Surprisingly, we identified a Relaxin variant in which the orientation of the two active chains, designated as A chain and B chain, are exchanged and the cleavable C chain is substituted by linker peptide. As shown in FIG. 1, instead of the genetically determined orientation of the single chains encoding Relaxin, namely B chainâ€”C chainâ€”A chain, the orientation of the chains of the modified molecule is: A chainâ€”linker peptideâ€”B chain. The resulting molecule exhibits full biological activity, independent of any endoproteolytic processing. This new single-chain Relaxin variant provided by the invention thus solves the problem of low expression yields or the requirement of co-transfection with a processing protease.

The half-life of intravenously administrated Relaxin 2 in humans is less than 10 minutes (Dschietzig T. et al. (2009)). As a consequence, in clinical trials Relaxin 2 has to be administered continuously over 48 h. Therefore, the improvement of the biological half life of Relaxin could be of great advantage.

Improving biological half life can either be performed by chemical modification such as PEGylation or HESylation of the polypeptide of interest, introduction of additional, non-natural N-glycosylation sites or by genetically fusing this polypeptide with other molecules such as the immunoglobulin Fc fragment of antibodies, transferrin, albumin, binding modules that bind in-vivo to other molecules mediating longer half-life, or other proteins, respectively. This invention provides single-chain Relaxin variants fused to the Fc part of antibodies with improved half-life. Surprisingly, these variants show biological activity in the range of the wild-type Relaxin.

SUMMARY OF THE INVENTION

The invention concerns fusion polypeptides, hereafter also referred to as single chain Relaxin (scRelaxin).

Current standard of Relaxin 2 production is the chemical synthesis of this molecule, which is a complex and expensive procedure. Due to the fact that Relaxin undergoes posttranslational modifications, especially the cleavage of the prepro-protein by the Prohormon Convertase 1 and Prohormone Convertase 2, choice of an adequate expression system is mandatory for recombinant expression. Endoproteolytic processing of proteins belonging to the insulin superfamily often limits the production of bioactive molecules from heterologous cells. To avoid the endoproteolytic processing of Relaxin, the fusion polypeptides of the invention are molecules in which the genetically encoded orientation of the two active chains of Relaxin, designated as A chain and B chain, is reversed wherein the A chain and B chain are connected by a linker peptide. In detail, instead of the genetically determined orientation of the individual DNA segments encoding Relaxin domains, namely, B chainâ€”C chainâ€”A chain, the orientation the DNA segments in the Relaxin variants provided by this invention is: A chainâ€”peptide linkerâ€”B chain. This results in a single chain Relaxin wherein the carboxy-terminus of Relaxin A chain is fused to the amino-terminus of the linker polypeptide L, which carboxy-terminus is fused to the amino-terminus of the Relaxin B chain, designated A-L-B (see FIG. 1 for an illustration). The resulting molecule exhibits biological activity similar to the wild-type Relaxin, but its expression is independent of endo-proteolytic processing.

One embodiment 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 2 A chain polypeptide or a functional variant thereof and the Relaxin B chain polypeptide comprises a Relaxin 2 B chain polypeptide 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 A-L-B comprises a human minimal Relaxin 2 A chain polypeptide (SEQ ID NO: 118) or a functional variant thereof, or comprises a human Relaxin 2 A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof and the Relaxin B chain polypeptide comprises a human Relaxin 2 B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof.

In one embodiment the linker polypeptide L of the aforementioned fusion polypeptides A-L-B consists of a polypeptide which is 6-14 amino acid residues in length. Further preferred are polypeptide linkers L which are 7-13 amino acid residues in length. Further preferred are polypeptide linkers L which are 8-12 amino acid residues in length. Even more preferred are polypeptide linkers L which are 7-11 or 9-11 amino acid residues in length. Even more preferred are polypeptide linkers L which are 9 amino acid residues in length. In a further preferred embodiment, the integer of the length of the polypeptide linker L is selected from the group consisting of the integers 6, 7, 8, 9, 10, 11, 12, 13 and 14.

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. A further preferred embodiment is a linker L which comprises Gly and Ser residues and has a ratio of Gly to Ser of at least 3 to 1.

In a further embodiment the aforementioned linker L comprises at least one attachment site for covalent coupling of a half-life extending moiety. In an embodiment of the invention the aforementioned attachment site is a Lys or a Cys residue.

A preferred embodiment of the invention is a fusion polypeptide comprising A-L-B, wherein

A is a human Relaxin 2 A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof,

B is a human Relaxin 2 B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and

L is a linker polypeptide which is 6-14, 7-13, 8-12, 7-11, 9-11, or 9 amino acid residues in length. 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. A further preferred embodiment is a linker L which comprises Gly and Ser residues and has a ratio of Gly to Ser of at least 3 to 1. In a further embodiment the aforementioned linker L comprises at least one attachment site for covalent coupling of a non-proteinaceous polymer half-life extending moiety. In an embodiment of the invention the aforementioned attachment site is a Lys or a Cys residue.

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

In a further embodiment the aforementioned fusion polypeptides have Relaxin activity. In a further preferred embodiment the Relaxin activity is activation of the relaxin receptor LGR7. In an even further preferred embodiment, the activation of the relaxin receptor LGR7 is determined by a method disclosed in experimental methods.

In another aspect, the invention provides a polynucleotide encoding an aforementioned fusion polypeptide. Such a polynucleotide may further comprise a coding sequence for a signal peptide allowing secretion of the fusion polypeptide. Vectors containing polynucleotides for such fusion polypeptides are included as well. Suitable vectors are for example expression vectors. A further embodiment of the invention is a host cell comprising a polynucleotide, a vector, or expression vector encoding the aforementioned fusion polypeptides. The host cell of the invention can be an eukaryotic cell or a prokaryotic cell. An eukaryotic cell can be a mammalian cell or a yeast or insect cell, preferably a mammalian cell. A prokaryotic cell can be for example an E. coli cell.

In another embodiment the invention provides pharmaceutical compositions comprising the aforementioned fusion polypeptides. The composition may be formulated for intravenous, intraperitoneal or subcutaneous administration.

Another embodiment of the invention provides a pharmaceutical composition or a fusion polypeptide as medicament. A further embodiment is the use of a pharmaceutical composition or a fusion polypeptide in the treatment of cardiovascular diseases, pancreatitis, inflammation, cancer, scleroderma, pulmonary, renal, and hepatic fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic representation of the genetic organization of domains of the wildtype Relaxin and single chain Relaxin as well as their corresponding polypeptides.

FIG. 2 Schematic representation of single chain Relaxin variants.

FIG. 3 Schematic representation of domain organisation of single chain Relaxin fusion protein variants as well as single chain Relaxin variants designed for PEGylation.

FIG. 4a-e Activity in a functional assay of scR 3, scR 4, and scR 5 (FIG. 4a), scR 7, scR 8, scR9, and scR10 (FIG. 4b), scR11 and scE12 (FIG. 4c), human Relaxin 3, scR14, and scR15 (FIG. 4d) and scR17 (FIG. 4e) using the CHO-CRE-LGR7 cell line. As control, hRelaxin 2 (R&D Systems, catalogue number 6586-RN-025) was used. Data are expressed as Relative Light Units, representing the activity of single chain Relaxin variants and Relaxin 2 induced luciferase expression. Symbols represent means, error bars represent S.E.M.

FIG. 5 Activity in a functional assay of scR-Fc 1 by using the CHO-CRE-LGR7 cell line. As control, hRelaxin 2 (R&D Systems, catalogue number 6586-RN-025) was used. Data are expressed as Relative Light Units, representing the activity of scR-Fc 1 and hRelaxin 2 induced luciferase expression. Symbols represent means, error bars represent S.E.M.

FIG. 6 Activity in a functional assay of scR-Fc 5, scR-Fc 6, and scR-Fc 7 using the CHO-CRE-LGR7 cell line. hRelaxin 2 (R&D Systems, catalogue number 6586-RN-025). was used as control. Data are expressed as Relative Light Units, representing the activity of the scR-Fc variants and hRelaxin 2 induced luciferase expression. Symbols represent means, error bars represent S.E.M.

FIG. 7 Activity in a functional assay of scR-Fc 11, scR-Fc 12, and scR-Fc 13 using the CHO-CRE-LGR7 cell line. As control, hRelaxin 2 (R&D Systems, catalogue number 6586-RN-025) was used. Data are expressed as Relative Light Units, representing the activity of scR-Fc variants and hRelaxin 2 induced luciferase expression. Symbols represent means, error bars represent S.E.M.

FIG. 8: Activity in a functional assay of scR-Var 3, scR-Var 4, scR-Var 5, and scR-Var 6 using the CHO-CRE-LGR7 cell line. As control, hRelaxin 2 (R&D Systems, catalogue number 6586-RN-025) was used. Data are expressed as Relative Light Units, representing the activity of scR-Fc variants and hRelaxin 2 induced luciferase expression. Symbols represent means, error bars represent S.E.M.

FIG. 9 In vivo half-life analysis of intravenously administrated hRelaxin 2 or scR-Fc 13. Eight weeks old male Wistar rats (three animals per group) were given a single application of human Relaxin 2 and scR-Fc 13, respectively (0.24 mg/kg). Blood samples were collected at the indicated time points after application and serum levels of each protein were measured by using a quantification ELISA.

FIG. 10: Activity of Relaxin 2 and Relaxin variants in blood samples

Relaxin activity in blood samples obtained from scR-Fc 13 treated rats by using the CHO-CRE-LGR7 cell line was determined. Blood samples collected 3, 5, and 7 days after intravenous administration of scR-Fc 13 were incubated on the CHO-CRE-LGR7 cell line and Relative Lights Units were determined Calibration curves were determined using hRelaxin 2 (R&D Systems, catalogue number 6586-RN-025) and purified scR-Fc 13. The EC50 within the dose response curve is marked by an X. Data are expressed as Relative Light Units, representing the activity of scR-Fc variants and hRelaxin 2 induced luciferase expression. Symbols represent means, error bars represent S.E.M.

FIG. 11: Influence of hRelaxin 2 and scR-Fc 13 on heart rate, coronary flow and contractility in the isolated perfused rat heart model.

At a concentration of 1 nM, application of hRelaxin 2 leads to an increase of heart rate and coronary flow and exhibits a negative inotrophic activity (FIG. 11a-d). Comparable effects were obtained with scR-Fc 13, although at a ten fold higher concentration (FIG. 11e-h).

DETAILED DESCRIPTION OF THE INVENTIONDefinitions

The term â€œamino acid residueâ€ is intended to indicate an amino acid residue contained in the group consisting of 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), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

The term â€œactivity of Relaxinâ€ or â€œRelaxin Activityâ€ is defined by the ability of Relaxin or variants thereof to the activation of the stimulatory G-protein Gs, thus the subsequent generation of the second messenger cyclic AMP, and/or the stimulation of PI3-kinase. Relaxin or variants thereof bind to LGR7 leading to the intracellular activation of the stimulatory G-protein Gs, resulting in the subsequent generation of the second messenger cyclic AMP (cAMP). However, cAMP generation is a time-dependent biphasic response. After an initial short Gs-adenylate cyclase-mediated cAMP response the receptor signal is switching to an inhibitory G protein activation and by this to PI3-kinase-mediated response. (Halls M. L., Bathgate R. A., Summers, R. J. (2005) Signal Switching after Stimulation of LGR7 Receptors by Human Relaxin 2. Ann. N.Y. Acad. Sci. 1041:288-291).

The term â€œhalf-life extending moietyâ€ refers to a pharmaceutically acceptable moiety, domain, or â€œvehicleâ€ covalently linked (â€œconjugatedâ€ ) to the Relaxin fusion polypeptide directly or via a linker, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing chemical modification of the Relaxin fusion polypeptide, increases half-life or other pharmacokinetic properties such as but not limited to increasing the rate of absorption, reduces toxicity, improves solubility, increases biological activity and/or target selectivity of the Relaxin fusion polypeptide, increases manufacturability, and/or reduces immunogenicity of the Relaxin fusion polypeptide, compared to an unconjugated form of the Relaxin fusion polypeptide. The term â€œhalf-life extending moietyâ€ includes non-proteinaceous, half-life extending moieties, such as PEG or HES, and proteinaceous half-life extending moieties, such as serum albumin, transferrin or Fc domain.

â€œPolypeptideâ€ , peptideâ€ and â€œproteinâ€ are used interchangeably herein and include a molecular chain of two or more amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide. The terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included as can be synthesized, or expressed recombinantly using known protein engineering techniques. In addition, inventive fusion proteins can be derivatized as described herein by well-known organic chemistry techniques.

The term â€œfunctional variantâ€ refers to a variant polypeptide which at least retains some of its natural biological activity. In case of the Relaxin 2 variants according to the invention, a functional variant is a variant which shows at least some of its natural activity, such as the activation of the relaxin receptor LGR7. The activation of the relaxin receptor LGR7 can be determined by a method disclosed in experimental methods.

The terms â€œfragment,â€ â€œvariant,â€ â€œderivative,â€ and â€œanalogâ€ when referring to polypeptides of the present invention include any polypeptides that retain at least some of the receptor binding properties of the corresponding wild-type Relaxin polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions, or additions. Variant polypeptides may also be referred to herein as â€œpolypeptide analogs.â€ As used herein a â€œderivativeâ€ of a polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as â€œderivativesâ€ are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

The term â€œfusion proteinâ€ indicates that the protein includes polypeptide components derived from more than one parental protein or polypeptide and/or that the fusion protein includes protein domains derived from one or more parental protein or polypeptide which are not arrayed in their wild type orientation. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is appended in frame with, and optionally separated by a linker or stretcher from, a nucleotide sequence encoding a polypeptide sequence from a different protein. The fusion gene can then be expressed by a recombinant host cell as a single protein.

The term â€œnucleotide sequenceâ€ or â€œpolynucleotideâ€ is intended to indicate a consecutive stretch of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

The term â€œEC₅₀â€ (half maximal effective concentration) refers to the effective concentration of a therapeutic compound which induces a response halfway between the baseline and maximum after some specified exposure time.

The term â€œimmunogenicityâ€ as used in connection with a given substance is intended to indicate the ability of the substance to induce a response from the immune system. The immune response may be a cell or antibody mediated response (see, e.g., Roitt: Essential Immunology (8th Edition, Black-well) for further definition of immunogenicity). Normally, reduced antibody reactivity will be an indication of reduced immunogenicity. The reduced immunogenicity may be determined by use of any suitable method known in the art, e.g. in vivo or in vitro.

The term â€œpolymerase chain reactionâ€ or â€œPCRâ€ generally refers to a method for amplification of a desired nucleotide sequence in vitro, as described, for example, in U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,195. In general, the PCR method involves repeated cycles of primer extension synthesis, using oligonucleotide primers capable of hybridising preferentially to a template nucleic acid.

The term â€œvectorâ€ refers to a plasmid or other nucleotide sequences that are capable of replicating within a host cell or being integrated into the host cell genome, and as such, are useful for performing different functions in conjunction with compatible host cells (a vector-host system): to facilitate the cloning of the nucleotide sequence, i.e. to produce usable quantities of the sequence, to direct the expression of the gene product encoded by the sequence and to integrate the nucleotide sequence into the genome of the host cell. The vector will contain different components depending upon the function it is to perform.

â€œCellâ€ , â€œhost cellâ€ , â€œcell lineâ€ and â€œcell cultureâ€ are used interchangeably herein and all such terms should be understood to include progeny resulting from growth or culturing of a cell.

The term â€œfunctional in vivo half-lifeâ€ is used in its normal meaning, i.e. the time at which 50% of the biological activity of the polypeptide is still present in the body/target organ, or the time at which the activity of the polypeptide is 50% of the initial value.

As an alternative to determining functional in vivo half-life, â€œserum half-lifeâ€ may be determined, i.e. the time at which 50% of the polypeptide circulates in the plasma or bloodstream prior to being cleared. Determination of serum half-life is often more simple than determining the functional in vivo half-life and the magnitude of serum half-life is usually a good indication of the magnitude of functional in vivo half-life. Alternatively terms to serum half-life include â€œplasma half-lifeâ€ , â€œcirculating half-lifeâ€ , â€œserum clearanceâ€ , â€œplasma clearanceâ€ and â€œclearance half-lifeâ€ . The polypeptide is cleared by the action of one or more of the reticuloendothelial systems (RES), kidney, spleen or liver, by tissue factor, SEC receptor or other receptor mediated elimination, or by specific or unspecific proteolysis. Normally, clearance depends on size (relative to the cutoff for glomerular filtration), charge, attached carbohydrate chains, and the presence of cellular receptors for the protein. The functionality to be retained is normally selected from receptor binding or receptor activation. The functional in vivo half-life and the serum half-life may be determined by any suitable method known in the art and may for example generally involve the steps of suitably administering to a mammalian a suitable dose of the amino acid sequence or compound to be treated; collecting blood samples or other samples from said mammalian at regular intervals; determining the level or concentration of the amino acid sequence or compound of the invention in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence or compound of the invention has been reduced by 50% compared to the initial level upon dosing. Reference is for example made to the standard handbooks, such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to â€œPharmacokineticsâ€ , M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).

â€œGlycosylationâ€ is a chemical modification wherein sugar moieties are added to the polypeptide at specific sites. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences Asn-X-Ser and Asn-X-Thr (â€œNâ€”Xâ€”S/Tâ€ ), where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences (or motifs) in a polypeptide creates a potential N-linked glycosylation site. O-linked refers to the attachment of a carbohydrate moiety to the hydroxyl-group oxygen of serine and threonine.

An â€œisolatedâ€ fusion polypeptide is one that has been identified and separated from a component of the cell that expressed it. Contaminant components of the cell are materials that would interfere with diagnostic or therapeutic uses of the fusion polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the fusion polypeptide is purified (1) to greater than 95% by weight of fusion polypeptide as determined e.g. by the Lowry method, UV-Vis spectroscopy or by by SDS-Capillary Gel electrophoresis (for example on a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer device), and in further preferred embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, however, isolated fusion polypeptides will be prepared by at least one purification step.

Overview

The application provides an A-L-B fusion polypeptide, also used terms herein are single chain Relaxin abbreviated as scRelaxin or scR, wherein â€œAâ€ is a Relaxin A chain, â€œBâ€ is a Relaxin B chain and â€œLâ€ is a linker polypeptide. The present application describes an improved Relaxin molecule, wherein the C-terminus of an A chain is linked via a polypeptide linker to the N-terminus of a B chain allowing the fusion polypeptide being expressed as a functional scRelaxin. The application relates, in part, on the surprising discovery that the A-L-B fusion polypeptides can be functionally expressed without the need for endoproteolytic prohormone processing as known for wildtype Relaxin.

Single Chain Versions of RelaxinRelaxin A and B Domains:

A further embodiment 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, wherein Relaxin is selected from the group of Relaxins 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 aforementioned Relaxins are human Relaxins.

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

In a preferred embodiment the Relaxin A chain polypeptide of A-L-B comprises a human minimal Relaxin 2 A chain polypeptide (SEQ ID NO: 118) or a functional variant thereof, or comprises a human Relaxin 2 A 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 further embodiment the Relaxin A chain polypeptide of A-L-B comprises a Relaxin 3 A chain polypeptide or a functional variant thereof. In a further embodiment the Relaxin B chain polypeptide of A-L-B comprises a Relaxin 3 B chain polypeptide or a functional variant thereof.

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

In a preferred embodiment of the aforementioned fusion polypeptides A-L-B a functional variant of the Relaxin A or B chain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions and/or deletions compared to the wild type Relaxin A and B chain, respectively. Further preferred is an aforementioned Relaxin 2 B variant that further comprises the conserved motif Arg-X-X-X-Arg-X-X-Ile/Val-X.

Relaxin A and B chain variants are known in the art. The well characterized binding site geometry of Relaxin provides the skilled person with guidance to design Relaxin A and B chain variants, see for example BÃ¼llesbach and Schwabe J Biol Chem. 2000 Nov. 10; 275(45):35276-80 for variations of the Relaxin B chain and Hossain et al. J Biol Chem. 2008 Jun. 20; 283(25):17287-97 for variations of the Relaxin A chain and the â€œminimalâ€ Relaxin A chain. For example, for the conserved Relaxin 2 B motif (Arg-X-X-X-Arg-X-X-Ile/Val-X) X represents amino acids which are able to form a helical structure example to select appropriate amino acids X in the conserved motif as the three defined amino acids form a triangular contact region on the surface of the Relaxin B chain (BÃ¼llesbach and Schwabe J Biol Chem. 2000 Nov. 10; 275(45)).

In an even more preferred embodiment the Relaxin A chain polypeptide of A-L-B is a human Relaxin 2 A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof and the Relaxin B chain polypeptide is a human Relaxin 2 B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof. In an even more preferred embodiment, the functional variant of human Relaxin 2 A chain polypeptide (SEQ ID NO: 117) is a functional variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and/or insertions compared to SEQ ID NO: 117. Further preferred is a functional variant of human Relaxin 2 B chain polypeptide (SEQ ID NO: 119) wherein the functional variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and/or insertions compared to SEQ ID NO: 119. Even further preferred is an aforementioned human Relaxin 2 B variant that further comprises the conserved motif Arg-X-X-X-Arg-X-X-Ile/Val-X.

In an even more preferred embodiment the Relaxin A chain polypeptide of A-L-B is a human Relaxin 2 A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid exchanges compared to SEQ ID NO: 117 and the Relaxin B chain polypeptide is a human Relaxin 2 B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid exchanges compared to SEQ ID NO:119 and comprising the conserved motif Arg-X-X-X-Arg-X-X-Ile/Val-X.

The person skilled in the art knows how to obtain functional variants. Examples of functional variants are disclosed for the Relaxin A chain in Hossain et al J Biol Chem. 2008 Jun. 20; 283(25):17287-97 or in US Pat. publication No. US2011/0130332 and for the Relaxin B chain in Schwabe and BÃ¼llesbach (2007) Adv Exp Med Biol. 612:14-25 and BÃ¼llesbach and Schwabe J Biol Chem. 2000 Nov. 10; 275(45):35276-80).

Linker L:

In one embodiment the linker polypeptide L of the aforementioned fusion polypeptides A-L-B consists of a polypeptide which is 6-14 amino acid residues in length. Further preferred are polypeptide linkers L which are 7-13 amino acid residues in length. Further preferred are polypeptide linkers L which are 8-12 amino acid residues in length. Even more preferred are polypeptide linkers L which are 7-11, or 9-11 amino acid residues in length. Even more preferred are polypeptide linkers L which are 9 amino acid residues in length. In a further preferred embodiment, the integer of the length of the polypeptide linker L is selected from the group consisting of the integers 6, 7, 8, 9, 10, 11, 12, 13 and 14.

The amino acid composition of the linker can vary, although a linker exhibiting a low immunogenicity score is preferred. Examples of linkers are well known to those skilled in the art and comprise sequences such as (GGGS)n, (GGSG)n, where n are integers. 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 such as for example peptides comprising the sequence [GGGGS]_nas disclosed in U.S. Pat. No. 7,271,149. In other embodiments, a serine-rich linker peptide L is used, as described for example in U.S. Pat. No. 5,525,491.

A further preferred embodiment is a linker L which comprises Gly and Ser residues and has a ratio of Gly to Ser of at least 3 to 1.

In a further embodiment the aforementioned linker L comprises at least one attachment site for covalent coupling of a non-proteinaceous polymer half-life extending moiety. In an embodiment of the invention the aforementioned attachment site is a Lys or a Cys residue.

Examples of such linkers are [GlyGlyGlySerGlyGly] (SEQ ID NO: 137),

[GlyGlyGlySerGlyGlyGly] (SEQ ID NO: 138),

[GlyGlyGlySerGlyGlyGlySerGly] (SEQ ID NO: 139),

[GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer] (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).

It is contemplated that the optimal linker length and amino acid composition can be determined by routine methods known in the art.

A preferred embodiment of the invention is a fusion polypeptide comprising A-L-B, wherein

A is a human Relaxin 2 A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NO: 117,

B is a human Relaxin 2 B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NO:119 and comprising the conserved motif Arg-X-X-X-Arg-X-X-Ile/Val-X, and

L is a linker polypeptide which is 6-14, 7-13, 8-12, 7-11, 9-11, or 9 amino acid residues in length.

In a preferred embodiment the linker polypeptide L of the aforementioned fusion polypeptide A-L-B is 7-11, or 9-11 amino acid residues in length. Even more preferred are polypeptide linkers L which are 9 amino acid residues in length. In a further preferred embodiment, the integer of the length of the polypeptide linker L is selected from the group consisting of the integers 6, 7, 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 flexible linker.

In a preferred embodiment the 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 is consists of amino acid residues selected from the group of amino acids consisting of Gly, Ser, Arg, Leu, Cys, and Lys residues.

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 such as peptides comprising the sequence [GGGGS]_nas disclosed in U.S. Pat. No. 7,271,149. In other embodiments, a serine-rich linker peptide L is used, as described in U.S. Pat. No. 5,525,491.

A further preferred embodiment is a linker polypeptide L which comprises Gly and Ser residues and has a ratio of Gly to Ser of at least 2 to 1.

A further preferred embodiment is a linker polypeptide L which comprises Gly and Ser residues and has a ratio of Gly to Ser of at least 3 to 1.

A further preferred embodiment is a linker polypeptide L which comprises Gly and Ser residues and has a ratio of Gly to Ser of at least 1 to 2.

A further preferred embodiment is a linker polypeptide L which comprises Gly and Ser residues and has a ratio of Gly to Ser of at least 1 to 3.

A further preferred embodiment is a linker polypeptide L with the aforementioned preferred length, wherein all but 4 amino acid residues of the linker L consist of Gly and/or Ser residues and the remaining 4 amino acid residues are selected from the group of natural amino acids.

A further preferred embodiment is a linker polypeptide L with the aforementioned preferred length, wherein all but 3 amino acid residues of the linker L consist of Gly and/or Ser residues and the remaining 3 amino acid residues are selected from the group of natural amino acids.

A further preferred embodiment is a linker polypeptide L with the aforementioned preferred length, wherein all but 2 amino acids residues of the linker L consist of Gly and/or Ser residues and the remaining 2 amino acid residues are selected from the group of natural amino acids.

A further preferred embodiment is a linker polypeptide L with the aforementioned preferred length, wherein all but 1 amino acid residues of the linker L consist of Gly and/or Ser residues and the remaining amino acid residue is selected from the group of natural amino acids.

In a further preferred embodiment the aforementioned group of natural amino acids excludes the amino acid prolin.

A further preferred embodiment is a linker polypeptide L with the aforementioned preferred length, wherein all but 1 amino acid residues of the linker L consist of Gly and/or Ser and the remaining amino acid is selected from the group of Cys and Lys.

In a further preferred embodiment the linker polypeptide L consists of amino acid residues selected from the group of amino acid residues consisting of Gly and Ser residues.

In a further preferred embodiment the linker L consists of amino acid residues selected from the group of amino acids consisting of Gly and Ser residues wherein the ratio of Gly to Ser is at least 2 to 1.

In a further embodiment the aforementioned linker L comprises at least one attachment site for covalent coupling of a nonproteinaceous polymer half-life extending moiety. In an embodiment of the invention the aforementioned attachment site is a Lys or a Cys residue.

Preferred linker polypeptides L are selected from the group of linker polypeptides consisting of

(SEQâ€ƒIDâ€ƒNO:â€ƒ137)[GlyGlyGlySerGlyGly], (SEQâ€ƒIDâ€ƒNO:â€ƒ138)[GlyGlyGlySerGlyGlyGly], (SEQâ€ƒIDâ€ƒNO:â€ƒ139)[GlyGlyGlySerGlyGlyGlySerGly], (SEQâ€ƒIDâ€ƒNO:â€ƒ140)[GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer], (SEQâ€ƒIDâ€ƒNO:â€ƒ141)[GlyGlyGlySerGlyCysGlyGlySerGly], (SEQâ€ƒIDâ€ƒNO:â€ƒ143)[GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGly], (SEQâ€ƒIDâ€ƒNO:â€ƒ144)[LysArgSerLeuSerArgLysLysArg], (SEQâ€ƒIDâ€ƒNO:â€ƒ142)[GlyGlyGlySerGlyLysGlyGlySerGly], (SEQâ€ƒIDâ€ƒNO:â€ƒ145)[GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly],and (SEQâ€ƒIDâ€ƒNO:â€ƒ146)[GlyGlyGlySerGlyGlyGlySerGlyGlyGly].

A preferred embodiment of the invention is a fusion polypeptide comprising A-L-B, wherein

A is a human Relaxin 2 A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof,

B is a human Relaxin 2 B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and

L is a linker polypeptide, which is 7, 8, 9 or 10 amino acids in length.

A preferred embodiment of the invention is a fusion polypeptide comprising A-L-B, wherein

A is a human Relaxin 2 A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof having 1, 2, 3 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NO: 117,

L is a linker polypeptide, which is 7, 8, 9 or 10 amino acids in length.

A preferred embodiment of the invention is a fusion polypeptide comprising A-L-B, wherein

A is a human Relaxin 2 A chain polypeptide (SEQ ID NO: 117),

B is a human Relaxin 2 B chain polypeptide (SEQ ID NO: 119), and

L is a linker polypeptide, which is 7, 8, 9 or 10 amino acids in length. A preferred embodiment of the invention is a fusion polypeptide comprising A-L-B, wherein

A is a human Relaxin 2 A chain polypeptide (SEQ ID NO: 117) or a functional variant thereof,

B is a human Relaxin 2 B chain polypeptide (SEQ ID NO: 119) or a functional variant thereof, and

L is a linker polypeptide, which is 9 amino acids in length.