CN118434437A

CN118434437A - Treatment with heterodimeric relaxin fusion

Info

Publication number: CN118434437A
Application number: CN202280080859.0A
Authority: CN
Inventors: R·T·小乔治; K·M·康诺利; S·A·A·H·奥马尔; A·加布里尔森
Original assignee: MedImmune Ltd
Current assignee: MedImmune Ltd
Priority date: 2021-12-15
Filing date: 2022-12-14
Publication date: 2024-08-02

Abstract

The present invention relates to the use of heterodimeric relaxin fusion polypeptides, particularly for the treatment of heart failure with pulmonary hypertension.

Description

Treatment with heterodimeric relaxin fusion

Technical Field

The present invention relates to methods of treatment using heterodimeric relaxin fusions. In particular, the invention relates to methods of treatment using relaxin-2 fusions.

Background

Relaxin is a peptide hormone belonging to the insulin superfamily. In humans, this relaxin peptide family includes seven peptides with high structural similarity but low sequence similarity: relaxin 1,2 and 3 and insulin-like peptides INSL3, INSL4, INSL5 and INSL6. Naturally occurring relaxin consists of: an a polypeptide chain and a B polypeptide chain covalently linked by two inter-chain disulfide bonds. The a chain has additional intrachain disulfide bonds. Relaxin gene encodes se:Sub>A prohormone having the structure B-C-se:Sub>A (B polypeptide chain and se:Sub>A polypeptide chain linked by C peptide). Prohormones undergo endoproteolytic cleavage by the PC1 and PC2 enzymes, thereby removing the C peptide, followed by secretion of mature relaxin.

Relaxin is a pleiotropic hormone known to mediate systemic hemodynamic changes and renal adaptation changes during pregnancy. Relaxin also has been shown to have anti-fibrotic properties and has beneficial effects in heart failure, such as Acute Decompensated Heart Failure (ADHF). Heart failure is associated with significant morbidity and mortality. It is characterized by complex tissue remodeling involving increased cardiomyocyte death and interstitial fibrosis. Relaxin activates a number of signaling cascades that have proven beneficial in the context of ischemia-reperfusion and heart failure. These signaling pathways include activation of the phosphoinositide 3-kinase pathway and activation of the nitric oxide signaling pathway (Bathgate RA et al (2013) Physiol.Rev. [ physiological review ]93 (1): 405-480; mentz RJ et al (2013) am. HeartJ. [ J. America Heart ]165 (2): 193-199; tietjens J et al (2016) Heart Heart ]102:95-99; wilson SS et al (2015) Pharmacology [ pharmacological ] 35:315-327).

A significant proportion of heart failure patients also suffer from pulmonary arterial hypertension (hf+ph patients). It is estimated that about 50% of heart failure patients with preserved ejection fraction also suffer from pulmonary arterial hypertension, and this proportion of heart failure patients with reduced ejection fraction increases to 60% (Guazzi, (2014) CIRCHEARTFAIL. [ cycle: heart failure ],7:367-377; miller et al, (2013) JACC HeartFail. [ journal of American cardiology: heart failure ],1 (4): 290-299). Patients with heart failure with pulmonary hypertension have been shown to have reduced survival compared to heart failure patients without pulmonary hypertension (Barnett and De Marco, (2012) heart fail. Clin [ heart failure clinic ] 8:447-459). In heart failure patients, an increase or decrease in pulmonary artery diastolic pressure (ePAD) of 3mmHg (corresponding to an increase or decrease in mean pulmonary artery pressure (mPAP) of about 4 mmHg) was estimated to correlate with a 24% increase or 19% decrease in cardiovascular mortality, respectively (Zile MR et al (2017) CIRC HEART FAIL) [ cycle: heart failure ],10:e003594 ]. mPAP a reduction of 4mmHg is also associated with improvement of dyspnea in patients with heart failure and pulmonary arterial hypertension (Solomonica A et al (2013) CIRC HEART FAIL [ cycle: heart failure ], 6:53-60).

Clinical trials have been performed using unmodified recombinant human relaxin-2, i.e., semaphorin (serelaxin). Continuous intravenous administration of celecoxib to hospitalized patients improved cardiac, renal and hepatic injury and congestion markers (Felker GM et al (2014) J.am. Coll. Cardiol. [ journal of American society of cardiology ]64 (15): 1591-1598; metra M et al (2013) J.am. Coll. Cardiol. [ journal of American society of cardiology ]61 (2): 196-206; teerlink JR et al (2013) Lancet [ Lancet ]381 (9860): 29-39). By continuous infusion for about 20 hours, secoisolariciresinol also shows improvement in pulmonary arterial pressure, cardiac output, and systemic and pulmonary vascular resistance in these patients (Ponikowski et al, (2014) European HeartJournal [ J.European heart ] 35:431-441). However, the therapeutic effect is limited due to the rapid clearance of the relaxin from the patient's circulation, and the positive effect rapidly disappears once intravenous injection is stopped. In addition, approximately one third of patients experience significant blood pressure reduction (> 40 mmHg) after intravenous administration of selegiline, and therefore the dose must be reduced by half or even more.

WO2013/004607 and WO 2018/138170 describe recombinant relaxin polypeptides in which relaxin a and relaxin B are single-chain fused to a linker peptide. WO2013/004607 describes recombinant relaxins with linker peptides of at least 5 amino acids and less than 15 amino acids. WO 2018/138170 describes recombinant relaxins with linker peptides of at least 15 amino acids.

In view of the promise of current clinical studies with unmodified recombinant relaxins, there remains a need for additional recombinant relaxins that retain the biological activity of relaxin and provide advantages such as extended half-life, convenient route of administration, and ease of administration.

Disclosure of Invention

The present invention relates to the use of heterodimeric fusion with relaxin activity in the treatment of subjects suffering from heart failure with pulmonary arterial hypertension (hf+ph). In particular, there remains a significant unmet need for treatment of hf+ph subjects.

Accordingly, in one aspect, the invention provides a method of treating a subject suffering from heart failure with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

(i) A first heterodimerization domain linked to at least one relaxin a chain polypeptide or variant thereof; and

(Ii) A second heterodimerization domain linked to at least one relaxin B chain polypeptide or variant thereof,

Wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and wherein the heterodimerization fusion has relaxin activity.

Similarly, the invention provides a heterodimeric fusion for use in treating a subject with heart failure and pulmonary arterial hypertension, the heterodimeric fusion comprising:

Similarly, the invention provides the use of a heterodimeric fusion comprising:

In some embodiments, the relaxin a chain and relaxin B chain of the heterodimeric fusion are covalently bound via one or more (e.g., two) interchain linkages, preferably one or more (e.g., two) interchain disulfide linkages. In some embodiments, the relaxin a chain and the relaxin B chain are not covalently linked to each other by an amino acid linker.

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

In a preferred embodiment, the first and second heterodimerization domains are derived from an immunoglobulin Fc region, such as an immunoglobulin G (IgG) Fc region ("first Fc region" and "second Fc region"). The first and second Fc regions may comprise constant domains CH2 and/or CH3. Preferably, the first and second Fc regions comprise CH2 and CH3.

In alternative embodiments, the first and second heterodimerization domains are derived from an immunoglobulin Fab region.

In yet further alternative embodiments, the first and second heterodimerization domains heterodimerize to form parallel coiled coils.

In some embodiments, the relaxin a chain is linked to a first heterodimerization domain (e.g., a first Fc region) via a linker, and the relaxin B chain is linked to a second heterodimerization domain (e.g., a second Fc region) via a linker. In a preferred embodiment, one or preferably both of the linkers are polypeptides.

In some embodiments, at least one linker is a polypeptide of 6 to 40 amino acids in length. Preferably, both linkers are polypeptides of 6 to 40 amino acids in length. In a preferred embodiment, at least one linker is a polypeptide of 21 amino acids in length. In a particularly preferred embodiment, both linkers are polypeptides of 21 amino acids in length. In certain embodiments, both linkers have the sequence GGGGSGGGGSGGGGSGGGGGS [ SEQ ID NO:5].

In a preferred embodiment, the C-terminus of a first heterodimerization domain (e.g., a first Fc region) is linked to the N-terminus of the relaxin a chain and the C-terminus of a second heterodimerization domain (e.g., a second Fc region) is linked to the N-terminus of the relaxin B chain. In alternative embodiments, the N-terminus of a first heterodimerization domain (e.g., a first Fc region) is linked to the C-terminus of the relaxin a chain and the N-terminus of a second heterodimerization domain (e.g., a second Fc region) is linked to the C-terminus of the relaxin B chain.

In some embodiments, the first and second heterodimerization domains (e.g., the first and second Fc regions) comprise amino acid mutations and/or modifications that promote heterodimerization, preferably amino acid mutations and/or modifications that promote asymmetric heterodimerization. In a preferred embodiment, the amino acid mutations that promote heterodimerization are "Fc pestle" and "Fc mortar" mutations. In particularly preferred embodiments, the "Fc pestle" and "Fc mortar" mutations are present in the CH3 domain. In a preferred embodiment, the first Fc region comprises an "Fc pestle" mutation and the second Fc region comprises an "Fc mortar" mutation. Alternatively, the first Fc region has an "Fc mortar" mutation and the second Fc region has an "Fc pestle" mutation. Preferably, the amino acid mutation that promotes heterodimerization comprises the "Fc mortar" mutations Y349C, T366S, L368A and Y407V in one CH3 domain or conservative substitutions thereof; and "Fc pestle" mutations S354C and T366W or conservative substitutions thereof in another CH3 domain, wherein the amino acid numbering is according to the EU index as in Kabat.

In an embodiment of any aspect of the invention, the relaxin-2A chain polypeptide comprises a sequence as set forth in SEQ ID NO.1 or a variant thereof, and the relaxin-2B chain polypeptide comprises a sequence as set forth in SEQ ID NO.2 or a variant thereof. In some embodiments, the relaxin-2A chain polypeptide comprises the amino acid mutation K9H, K M or K17I, preferably K9H.

The invention also provides a method of treating a subject suffering from heart failure with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

(i) FcX-con-a fusion polypeptides; and

(Ii) FcY-con-B fusion polypeptide,

Wherein:

a is a relaxin a chain or variant thereof, e.g., a relaxin-2A chain or variant thereof;

b is a relaxin B chain or variant thereof, e.g., a relaxin-2B chain or variant thereof;

FcY is an immunoglobulin (e.g., igG 1) Fc region having an "Fc mortar" amino acid mutation and/or modification, preferably comprising a CH3 domain having the amino acid mutation Y349C: T366S: L368A: Y407V, or a conservative substitution thereof;

FcX is an immunoglobulin (e.g., igG 1) Fc region having an "Fc pestle" amino acid mutation and/or modification, preferably comprising a CH3 domain having the amino acid mutation S354C: T366W or conservative substitutions thereof; and

Con is a linker, e.g.a linker polypeptide preferably having the sequence GGGGSGGGGSGGGGSGGGGGS [ SEQ ID NO:5],

Wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX and FcY heterodimerize, and wherein the heterodimeric fusion has relaxin activity.

Similarly, the invention provides a heterodimeric fusion for use in a method of treating a subject with heart failure with pulmonary hypertension, the heterodimeric fusion comprising:

(i) FcX-con-a fusion polypeptides; and

(Ii) FcY-con-B fusion polypeptide,

Wherein:

Similarly, the invention provides the use of a heterodimeric fusion comprising:

(i) FcX-con-a fusion polypeptides; and

(Ii) FcY-con-B fusion polypeptide,

Wherein:

In a particularly preferred embodiment, the heterodimeric fusion comprises a fusion polypeptide having the amino acid sequence of SEQ ID NO. 11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO. 20.

In some embodiments of any aspect of the invention, the heterodimeric fusion further comprises one or more fabs, optionally wherein the heterodimeric fusion comprises one Fab linked to the N-terminus of a first heterodimerization domain (e.g., a first Fc region) and a second Fab linked to the N-terminus of a second heterodimerization domain (e.g., a second Fc region).

In some embodiments of any aspect of the invention, the heterodimeric fusion further comprises a second relaxin a chain polypeptide or variant thereof linked to the N-terminus of a first heterodimerization domain (e.g., a first Fc region) and a second relaxin B chain polypeptide or variant thereof linked to the N-terminus of a second heterodimerization domain (e.g., a second Fc region), optionally wherein the second relaxin a chain is linked to the first heterodimerization domain (e.g., a first Fc region) via a linker polypeptide and the second relaxin B chain is linked to the second heterodimerization domain (e.g., a second Fc region) via a linker polypeptide.

In another aspect, the invention provides a method of treating a subject having heart failure with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

(i) FcX-B-L-A and FcY, optionally FcY-B-L-A Or (b)

(Ii) FcY-B-L-A and FcX, optionally FcX-B-L-A

Wherein:

FcX is an immunoglobulin (e.g., igG 1) Fc region having an "Fc pestle" amino acid mutation and/or modification, preferably comprising a CH3 domain having the amino acid mutation S354C: T366W or conservative substitutions thereof;

a is a relaxin a chain or variant thereof, e.g., a relaxin-2A chain or variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG [ SEQ ID NO:60],

Wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX and FcY heterodimerize, and wherein the heterodimeric fusion has relaxin activity. Alternatively, fcX and FcY are non-Fc heterodimerization domains as described herein. In some embodiments, the relaxin B chain is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

(i) FcX-B-L-A and FcY, optionally FcY-B-L-A Or (b)

(Ii) FcY-B-L-A and FcX, optionally FcX-B-L-A

Wherein:

Wherein amino acid numbering is according to the EU index as in Kabat, wherein FcX and FcY heterodimerize, and wherein the heterodimeric fusion has relaxin activity. Alternatively, fcX and FcY are non-Fc heterodimerization domains as described herein. In some embodiments, the relaxin a chain is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

Similarly, the invention provides the use of a heterodimeric fusion comprising:

(i) FcX-B-L-A and FcY, optionally FcY-B-L-A Or (b)

(Ii) FcY-B-L-A and FcX, optionally FcX-B-L-A

Wherein:

In yet another aspect, the invention provides a method of treating a subject having heart failure with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

(i) FcX-A-L-B and FcY, optionally FcY-A-L-B; or (b)

(Ii) FcY-A-L-B and FcX, optionally FcX-A-L-B;

Wherein:

b is a relaxin B chain or variant thereof, e.g., a relaxin-2B chain or variant thereof; and

(i) FcX-A-L-B and FcY, optionally FcY-A-L-B; or (b)

(Ii) FcY-A-L-B and FcX, optionally FcX-A-L-B;

Wherein:

Similarly, the invention provides the use of a heterodimeric fusion comprising:

(i) FcX-A-L-B and FcY, optionally FcY-A-L-B; or (b)

(Ii) FcY-A-L-B and FcX, optionally FcX-A-L-B;

Wherein:

According to all aspects of the invention, heart failure may be heart failure with a reduced ejection fraction, heart failure with an intermediate ejection fraction or heart failure with a retained ejection fraction.

According to all aspects of the invention, the subject may have an average pulmonary artery pressure of about 25mmHg or greater and/or a right ventricular systolic pressure of about 40mmHg or greater. Typically, this is prior to treatment with the heterodimeric fusion of the invention.

According to all aspects of the invention, the subject may have a pulmonary vascular resistance of less than 3.0 wood units (woodunits). Alternatively, the subject may have pulmonary vascular resistance of 3.0 or more wood units. Typically, this is prior to treatment with the heterodimeric fusion of the invention.

According to all aspects of the invention, the subject may already be equipped with a blood pressure monitoring device, preferably a pulmonary artery pressure monitoring device. Preferably, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device.

In some embodiments of any aspect of the invention, the ratio of relaxin activity of the heterodimeric fusion to relaxin activity of the reference relaxin protein is about 0.001 to about 10.

According to all aspects of the invention, the heterodimeric fusion may be administered as a pharmaceutical composition comprising the heterodimeric fusion of the invention.

Also described herein are nucleic acid molecules (e.g., DNA molecules) encoding the heterodimeric fusions of the invention, vectors comprising the nucleic acid molecules, host cells comprising the vectors or nucleic acids, and methods of producing the heterodimeric fusions of the invention by: culturing the host cell and collecting the fusion protein.

Various aspects and embodiments of the invention are set out in the appended claims. These and other aspects and embodiments of the invention are also described herein.

Brief description of the drawings and sequence Listing

FIG. 1 illustrates an exemplary format of a heterodimeric fusion according to some embodiments of the invention. The format of each fusion polypeptide of the heterodimeric fusion is given in FcX, fcY, A, B, con and L, where FcX ("Fc pestle") and FcY ("Fc mortar") are two Fc regions comprising amino acid mutations and/or modifications that promote heterodimerization; a ("Rlx a") and B ("Rlx B") are relaxin a chain and relaxin B chain polypeptides; "con" is a linker polypeptide; l is a linker polypeptide, the heavy chains of HC X and HC Y-antibodies, the light chains of LC-antibodies, the hinge region of hinge-antibodies, and Fab is a Fab fragment of an antibody.

FIG. 2 shows LC-MS analysis A) of RELAX0019 and RELAX0023 shows the deglycosylation and non-reducing analysis of RELAX0019 and RELAX0023 of the mass of intact molecules; b) RELAX0019 and RELAX0023 deglycosylation and reduction analyses of the masses of the individual Fc fusion chains (pestle-relaxin A chain and mortar-relaxin B chain) are shown.

FIG. 3 shows analysis of C-terminal peptides of RELAX0019 and RELAX0023 by non-reducing peptide mapping using LC-MS. The amino acid sequence of the C-terminal peptide with predicted disulfide bonds represented by lines is shown in the top panel. Panels a and E-extract ion chromatograms of C-terminal peptides in the absence of reducing agent (-DTT). Panels C and G-deconvolution mass spectrum of C-terminal peptide in the absence of reducing agent. Panels B and F-extracted ion chromatograms in the presence of reducing agent (+dtt), and panels D and H-deconvolution mass spectra in the presence of reducing agent. Fig. 3 discloses SEQ ID NOs 75, 77 and 76, respectively, in order of appearance.

Fig. 4 shows the in vitro biological activity of some heterodimeric fusions of the invention as measured by cAMP induction in cells expressing recombinant human RXFP 1.

Fig. 5 shows in vivo Pharmacokinetic (PK) profiles from a series of ELISA experiments in which heterodimeric fusion of the invention was intravenously administered to mice. Data were normalized to% cMax at the 5min time point (T1).

Figure 6 shows reversal of isoprenaline-induced cardiac fibrosis and hypertrophy in mice treated with RELAX0019 and RELAX0023. The following levels of fibrosis and hypertrophy are shown: (1) vehicle (baseline), (2) isoproterenol, (3) isoproterenol+relaxin-2, (4) isoproterenol+relax 0019, and (5) isoproterenol+relax 0023.

FIG. 7 shows in vitro non-specific binding of the heterodimeric fusion of the invention in a Baculovirus (BV) ELISA assay.

Fig. 8 shows the percentage of purity loss, aggregation and fragmentation of RELAX0023, RELAX0127 and RELAX0128 in the solutions after storage.

Figure 9 shows the stability over time of RELAX0023, RELAX0127 and RELAX0128 in solutions assessed by reductive LC-MS analysis. A) Mass spectrum of total ion chromatogram B) reduced molecules

Fig. 10 shows PK profiles of RELAX0023 in cynomolgus monkeys after intravenous and subcutaneous injection.

FIG. 11 shows the nucleotide sequences encoding some of the polypeptides of the invention (SEQ ID NOS 80-140, respectively, in order of occurrence).

Fig. 12 shows the results of a long-term efficacy study of RELAX0023 in cynomolgus monkeys (cynomolgus macaque (Macaca fascicularis)) suffering from heart failure and reduced Left Ventricular Ejection Fraction (LVEF). The effect of RELAX0023 on LVEF, heart Rate (HR) and Mean Arterial Pressure (MAP) was shown in (A), (B) and (C), respectively, in the monkeys treated with relatively low, medium or high doses of RELAX0023 or vehicle control for 20 weeks, followed by the observation period. Each data point represents the mean value of the group (treatment group n=8, vehicle group n=14). The x-axis of each of (A), (B) and (C) represents the number of weeks since the beginning of the treatment period.

Figures 13A-13F show cardiac function, systemic vascular resistance and organ perfusion of MAD cohorts after administration of RELAX 0023: fig. 6A shows the Ejection Fraction (EF) of HFpEF subjects, fig. 6B shows the EF of HFrEF subjects, fig. 6C shows cardiac output of pooled subjects, fig. 6D shows Systemic Vascular Resistance (SVR) of pooled subjects, fig. 6E shows Stroke Volume (SV) of pooled subjects, and fig. 6F shows estimated glomerular filtration rate (evfr) of pooled subjects. HFpEF = heart failure with preserved ejection fraction. HFrEF = heart failure with reduced ejection fraction. Has significance compared to placebo (p < 0.1).

Table 1: the hinge region of the sequence listing is shown in italics, relaxin a is shown underlined, relaxin B is shown in double underlined, and the FC region is shown in bold.

Detailed Description

Relaxin

The present invention is based, at least in part, on the following findings: the heterodimeric fusion described herein may exhibit relaxin activity when the relaxin a chain and relaxin B chain are not covalently linked to each other by an amino acid linker. This is surprising based on the disclosure of WO 2013/004607 and WO 2018/138170, which describe recombinant relaxin, wherein relaxin a and relaxin B are single-chain fused. The inventors of the present invention also found that heterodimerization of the heterodimerization domain induced correct folding and heterodimerization of relaxin a chain and relaxin B chain (see example 2). Furthermore, unlike wild-type relaxin proteins, the fusion polypeptides of the invention do not require endoprotease processing for biological activity.

As used herein, the term "heterodimeric fusion" refers to a heterodimer of fusion polypeptides, wherein one fusion polypeptide comprises a first heterodimerization domain linked to a first subunit of a heterodimeric protein (e.g., relaxin a chain) and the other fusion polypeptide comprises a second heterodimerization domain linked to a second subunit of a heterodimeric protein (e.g., relaxin B chain).

The heterodimeric fusion of the invention may comprise a relaxin a-chain polypeptide and a relaxin B-chain polypeptide from the group of relaxins selected from the group consisting of: relaxin-1, relaxin-2, and relaxin-3. In a preferred embodiment, the relaxin a-chain polypeptide of the present invention is a relaxin-2A-chain polypeptide or variant thereof; and the relaxin B chain polypeptide of the present invention is a relaxin-2B chain polypeptide or variant thereof. In particular embodiments, the relaxin a chain polypeptide comprises a human relaxin-2A chain polypeptide or variant thereof and a human relaxin-2B chain polypeptide or variant thereof.

The terms "chain", "polypeptide" and "peptide" are used interchangeably herein to refer to a chain of two or more amino acids connected by peptide bonds.

In some embodiments, the relaxin-2A chain polypeptide has a sequence as set forth in SEQ ID NO. 1 or a variant thereof, and the relaxin-2B chain polypeptide has a sequence as set forth in SEQ ID NO. 2 or a variant thereof. Variants may comprise one or more amino acid substitutions, deletions and/or insertions. In some embodiments, the relaxin-2A chain polypeptide comprises one or more amino acid mutations selected from the group consisting of: K9E, K9H, K9L, K9M, R E, R H, R22A, R I, R M, R22Q, R S, R22Y, F E, F a and F23I. In a preferred embodiment, the relaxin-2A chain comprises the amino acid mutation K9H.

Relaxin a chain variants and relaxin B chain variants are known in the art. In addition, the skilled artisan can obtain guidance on the design of relaxin a chain variants and relaxin B chain variants. For example, it is understood that variants may retain those amino acids necessary for relaxin function. For example, relaxin-2B chain variants may contain the conserved motifs Arg-X-X-X-Arg-X-X-lle (Claasz AA et al (2002) Eur. J. Biochem. [ European journal of biochemistry ]269 (24): 6287-6293) or Arg-X-X-Val (Bathgate RA et al (2013) PhysiolRev. [ physiological comment ]93 (1): 405-480). Variants may comprise one or more amino acid substitutions and/or insertions. For example, a relaxin-2B chain variant may have one or more additional amino acids compared to SEQ ID NO. 62, such as K30 and R31 and N-terminal V-2, A-1 and M-1. Alternatively or in addition, the variant may comprise one or more amino acid derivatives. For example, the first amino acid of the relaxin-2B chain variant may be pyroglutamic acid.

In a preferred embodiment, the relaxin a chain and relaxin B chain are covalently bound by two interchain disulfide bonds (see example 2).

Relaxin family peptides mediate their biological effects at least in part by activating G Protein Coupled Receptors (GPCRs), and subsequently stimulating or inhibiting cAMP signaling pathways through Gs protein subunits or Gi protein subunits, respectively. Relaxin-2 is known to activate GPCRRXFP1 (also known as LGR 7) and to a lesser extent GPCRRXFP2 (also known as LGR 8), thus stimulating the Gs-cAMP dependent signaling pathway, leading to an increase in cAMP, the second messenger molecule.

As used herein, the term "relaxin activity" refers to the ability of a relaxin molecule to bind to a relaxin receptor, and/or activate the relaxin receptor and/or initiate intracellular signaling cascades. In embodiments in which relaxin activity is that of relaxin-2, relaxin activity may refer to the ability to bind and/or activate the receptors RXFP1 and/or RXFP 2. The term "relaxin activity" is used interchangeably with "biological activity".

Relaxin activity can be determined by measuring the binding of a relaxin molecule to a relaxin receptor and/or by measuring downstream events from binding to a relaxin receptor.

Relaxin activity in vitro and/or in vivo can be assayed. In some embodiments, the in vitro relaxin activity is measured.

Relaxin activity can be determined by measuring the amount and/or presence of molecules from downstream of receptor activation by relaxin. Relaxin activity can be determined, for example, by measuring cAMP production followed by measuring receptor activation by relaxin. Methods for detecting relaxin-induced cAMP production are known in the art. Such methods include CAMP ELISA, HTRF cAMP assay andCAMP assay. In some embodiments, relaxin activity is determined by measuring relaxin-induced cAMP production by HTRF cAMP assay, e.g., as performed in example 3. Relaxin activity can also be determined by measuring Nitric Oxide (NO) production followed by measuring receptor activation by relaxin. Relaxin activity can also be determined by measuring activation of molecules from downstream of receptor activation by relaxin. Relaxin activity can be determined, for example, by measuring activation of the p42/44 MAPK.

Alternatively or in addition, relaxin activity can be determined by measuring activation of a known relaxin target gene. For example, relaxin activity can be determined by measuring activation of transcription of a known relaxin target gene (i.e., VEGF) in THP-1 cells. Methods for determining activation of gene transcription are known in the art and include quantitative PCR analysis of mRNA. Relative expression of VEGF mRNA can be measured by quantitative real-time PCR induction of VEGF transcripts followed by incubation of THP-1 cells with relaxin, as described in Xiao et al (2013) Nat Commun [ Nature communication ] 4:1953.

Alternatively or in addition, relaxin activity may be determined by measuring one or more downstream effects of relaxin. For example, the reduction in cardiac hypertrophy may be measured by echocardiography, left ventricular weight relative to body weight, and/or tibial length according to standard methods. In another example, relaxin activity can be determined by measuring the reduction in fibrosis with a merson trichromatic stain (Masson's Trichrome stain). In another example, relaxin activity can be measured by measuring modulation of connective tissue metabolism, e.g., inhibition of pro-fibrotic factors (e.g., TGF-beta), inhibition of fibroblast proliferation and differentiation, and/or activation of MMP-mediated extracellular matrix degradation (Bathgate RA et al (2013) Physiol Rev. [ physiological comment ]93 (1): 405-480).

In some embodiments, relaxin activity is determined by measuring isoprenaline-induced cardiac hypertrophy (measured as cardiac weight relative to tibial length) and reversal of fibrosis (measured as collagen content relative to cardiac weight), e.g., as performed in example 7.

The activity of the heterodimeric fusion of the invention relative to a reference relaxin protein can be determined. In some embodiments, the reference relaxin protein is a recombinant protein. In a preferred embodiment, the reference relaxin protein is a relaxin protein having an array of relaxin a and relaxin B chains of mature relaxin protein. Recombinant relaxin having arrays of relaxin a and relaxin B chains of mature relaxin proteins is commercially available. For example, recombinant human relaxin-2, murine relaxin-1 and INSL3 are available from R & D Systems (R & D Systems) (catalog numbers 6586-RN, 6637-RN and 4544-NS, respectively).

In some embodiments, the reference relaxin protein has the same relaxin a and B chains as the heterodimeric fusion of the invention, or differs from the relaxin a and B chains of the heterodimeric fusion of the invention by up to 10 amino acids, e.g., 1 or 2 amino acids. In some embodiments, the first amino acid of the B chain of reference relaxin-2 is D, and this amino acid is deleted in the relaxin B chain of the heterodimeric fusion of the present invention.

The reference relaxin protein may be selected from:

(i) Recombinant human relaxin-2 (referred to herein as RELAX 0013); and

(Ii) Recombinant murine relaxin-1 (referred to herein as RELAX 0014); and

(Iii) Recombinant Fc fusion relaxin-2, wherein relaxin a and relaxin B are single chain fused, and wherein Fc is a half-life extending Fc region (referred to herein as RELAX0010 and described in WO 2018/138170); and

(Iv) Recombinant Fc fusion relaxin-2, wherein relaxin a and relaxin B are single chain fused, and wherein Fc is a half-life extending Fc region (referred to herein as RELAX0009 and described in WO 2018/138170); and

(V) Recombinant Fc fusion relaxin-2, wherein relaxin a and relaxin B are single chain fused (referred to herein as RELAX0126 and described in WO 2013/004607); and

(Vi) Recombinant Fc fusion relaxin-2, wherein relaxin a and relaxin B are single chain fused (referred to herein as RELAX0127 and described in WO 2013/004607); and

(Vii) Recombinant Fc fusion relaxin, wherein relaxin a and relaxin B are single chain fused (referred to herein as RELAX0128 and described in WO 2013/004607).

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

Heterodimeric fusions of the invention can be considered to have relaxin activity if they exhibit at least a portion of the activity of a reference relaxin protein. For example, a fusion polypeptide may be considered to have relaxin activity if it has at least about half of the activity of a reference relaxin protein. A heterodimeric fusion of the invention can be considered to have relaxin activity if the ratio of the activity of the fusion polypeptide to the activity of the reference relaxin protein is from about 10 ^-5 to about 1, from about 10 ^-4 to about 1, from about 10 ^-3 to about 1, from about 10 ^-2 to about 1, from about 1/50 to about 1, from about 1/20 to about 1, from about 1/15 to about 1, from about 1/10 to about 1, from about 1/5 to about 1, or from about 1/2 to about 1. Alternatively, a heterodimeric fusion of the invention may be considered to have relaxin activity if the ratio of the activity of the fusion polypeptide to the activity of the reference relaxin protein is from about 1 to about 10 ⁵, from about 1 to about 10 ⁴, from about 1 to about 10 ³, from about 1 to about 100, from about 1 to about 50, from about 1 to about 20, from about 1 to about 15, from about 1 to about 10, from about 1 to about 5, or from about 1 to about 2.

In some embodiments, the relaxin activity of the heterodimeric fusion is about 0.001 to about 10 with the relaxin activity of the reference relaxin protein.

Relaxin activity can be measured as EC50 value. As used herein, the term "EC50" (half maximum effective concentration) refers to the effective concentration of a therapeutic compound that induces a response halfway between the baseline and maximum after a specified exposure time.

Heterodimerization domains

The heterodimeric fusion of the invention comprises a first heterodimerization domain and a second heterodimerization domain. In a preferred embodiment, the first and second heterodimerization domains are derived from an immunoglobulin Fc region.

The term "Fc region" defines the C-terminal region of an immunoglobulin heavy chain that is produced by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain.

The first and second Fc regions may comprise immunoglobulin domains CH2 and/or CH3. In a preferred embodiment, the first and second Fc regions comprise immunoglobulin domains CH2 and CH3.

The Fc region may be derived from an immunoglobulin (e.g., igG) from any species, preferably a human immunoglobulin (e.g., human IgG). In embodiments in which the Fc region is derived from IgG, the Fc region may be derived from IgG of any subclass (e.g., igG1, igG2, igG3, igG 4), preferably IgG1. Preferably, the first and second Fc regions are derived from human IgG1 immunoglobulins. In other embodiments, the first and second Fc regions are derived from human IgG4 immunoglobulins.

In preferred embodiments, the first and second Fc regions comprise amino acid mutations and/or modifications that promote heterodimerization. Such modifications may include introducing asymmetric complementary modifications into each of the first and second Fc regions such that the two chains are compatible with each other and are therefore capable of forming heterodimers, but each chain is not capable of dimerizing with itself. Such modifications may encompass insertions, deletions, conservative and non-conservative substitutions, and rearrangements. Incorporation of such modifications provides a method of increasing the yield of heterodimers produced by recombinant cell culture relative to other unwanted end products (e.g., homodimers).

The first and second Fc regions may comprise any amino acid mutation and/or modification known in the art that promotes heterodimerization. Combinations of modifications can be used to maximize assembly efficiency while minimizing impact on antibody stability.

In the "knob in hole" approach, heterodimerization can be promoted by introducing steric hindrance between the contact residues. "protrusions" are created by replacing one or more small amino acid side chains from the interface of one Fc region ("Fc pestle") with larger side chains (e.g., tyrosine or tryptophan). A compensating "cavity" of the same or similar size as one or more large side chains is created at the interface of another Fc region ("Fc mortar") by replacing an amino acid with a large side chain with an amino acid with a smaller side chain (e.g., alanine or valine). "pestle and mortar" modifications, such as Ridgway JB et al (1996) Protein Eng, [ Protein engineering ]9 (7) 617-621, are described in detail in the following; MERCHANT AM et al (1998) Nat. Biotechnol. [ Nature Biotechnology ]16 (7): 677-681.

Other modifications that can be used to generate heterodimers include, but are not limited to, those that produce favorable electrostatic interactions between the two Fc regions. For example, one or more positively charged amino acids may be introduced into one Fc region, and one or more negatively charged amino acids may be introduced into corresponding positions in another Fc region. Alternatively or in addition, the Fc region may be modified to include mutations that introduce cysteine residues capable of disulfide bond formation. Alternatively or in addition, the Fc region may comprise one or more modifications to hydrophilic and hydrophobic residues at the interface between the chains, such that formation of heterodimers is more advantageous in terms of entropy and enthalpy than formation of homodimers.

Thus, in some embodiments, amino acid mutations and/or modifications that promote heterodimerization create steric hindrance between contacting residues (e.g., by a "mortar and pestle" approach), create favorable electrostatic interactions between the two Fc regions, introduce cysteine residues that are capable of forming disulfide bonds, and/or modify hydrophilic and hydrophobic residues at the interface between the two Fc regions.

In a preferred embodiment, the amino acid mutations that promote heterodimerization are "Fc pestle" and "Fc mortar" mutations. In a preferred embodiment, the "Fc pestle" and "Fc mortar" mutations are present in the CH3 domain.

In some embodiments, the first and second Fc regions are derived from a human IgG1 immunoglobulin and comprise "Fc X" and "Fc Y" having mutations in the CH3 domain, wherein the "Fc X" and "Fc Y" mutations are selected from the combinations shown in table 2 (or conservative substitutions thereof).

Table 2: "FcX" and "Fc Y" mutations

* Wherein the amino acid numbering is according to the EU index as in Kabat.

In a preferred embodiment, "Fc Y" is an "Fc mortar" with mutations Y349C, T366S, L a and Y407V or conservative substitutions thereof, and "Fc X" is an "Fc pestle" with mutations S354C and T366W or conservative substitutions thereof, wherein the amino acid numbering is according to the EU index as in Kabat.

The term "EU index as in Kabat" refers to the numbering system of the human IgG1 EU antibody described in Kabat EA et al (1991) Sequences of Proteins of Immunological Interest [ protein sequence of immunological interest ], 5 th edition Public HEALTH SERVICE [ Public health service ], national Institutes ofHealth [ national institutes of health ], besseda, maryland. All amino acid positions cited herein refer to EU index positions.

In some embodiments, the first Fc region has an "Fc mortar" mutation and the second Fc region has an "Fc pestle" mutation. In alternative and preferred embodiments, the first Fc region has an "Fc pestle" mutation and the second Fc region has an "Fc mortar" mutation.

It will be appreciated that these Fc regions may further comprise other amino acid modifications relative to the wild-type Fc region. The Fc region may be modified, for example, to increase the affinity of IgG molecules for FcRn. WO 02/060919 discloses modified immunoglobulins comprising an Fc region with one or more amino acid modifications and is incorporated herein by reference in its entirety. Methods of preparing Fc regions with one or more amino acid modifications are known in the art.

In some embodiments, the first and/or second Fc region may comprise one or more amino acid modifications, thereby reducing or eliminating effector functions of the Fc region. In some embodiments, amino acid modifications reduce or circumvent cytotoxicity, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

In some embodiments, the first and/or second Fc region may comprise one or more amino acid modifications to increase the half-life of the heterodimeric fusion.

In some embodiments, the first and/or second Fc region comprises at least one of the following combinations of amino acid mutations:

(i) M252Y, S254T and T256E or conservative substitutions thereof;

(ii) L234F, L Q and K322Q or conservative substitutions thereof;

(iii) L234F, L E and P331S or conservative substitutions thereof;

(iv) M252Y, S254T, T256E, L234F, L Q and K322Q or conservative substitutions thereof; or (b)

(V) M252Y, S254T, T256E, L234F, L E and P331S or conservative substitutions thereof,

Wherein the amino acid numbering is according to the EU index as in Kabat.

In some embodiments, the first and/or second Fc region may comprise the amino acid mutations L234F, L E and P331S or conservative substitutions thereof, wherein the amino acid numbering is according to the EU index as in Kabat.

In some embodiments, the Fc region comprising the "Fc mortar" mutation has the sequence shown in SEQ ID NO. 3 or a variant thereof, and the Fc region comprising the "Fc mortar" mutation has the sequence shown in SEQ ID NO. 4 or a variant thereof.

In some embodiments, the Fc region comprises a variant of SEQ ID NO:3 having the amino acid mutation Y349C reverting to Y349 and a variant of SEQ ID NO:4 having the amino acid mutation S354C reverting to S354 such that the Fc region is unable to form stable disulfide bonds.

In some embodiments, the Fc region comprises a variant of SEQ ID NO:3 and/or a variant of SEQ ID NO:4, wherein the first five residues DKTHTCPPC (SEQ ID NO: 69) are modified. In some embodiments, this region is replaced with sequence DKTHTACPPC (SEQ ID NO: 70). In an alternative embodiment, this region is replaced by the sequence GGAGGACPPC (SEQ ID NO: 71). In an alternative embodiment, this region is replaced by the sequence ACPPC (SEQ ID NO: 72).

In alternative embodiments, the first and second heterodimerization domains are derived from an immunoglobulin Fab region. In some embodiments, the heterodimerization domain comprises a CH1 and CL region. The Fab region containing the L and Fd chains has been found to mediate efficient heterodimerization (Schoojans R et al (2000) J.Immunol. [ J.Immunol. ]165 (12): 7050-7057). Thus, in alternative embodiments, the heterodimerization domain comprises an L and Fd chain. In some embodiments, the L and Fd chains heterodimerize to form disulfide bridge stable heterodimers.

In yet further alternative embodiments, the first and second heterodimerization domains heterodimerize to form parallel coiled coils. Heterodimeric coiled coils are described, for example, in the following documents: aronsson et al (2015) Sci.Rep. [ science report ]5:14063. In some embodiments, the heterodimerization domain comprises amino acid mutations and/or modifications that prevent the formation of undesired folding assemblies and/or facilitate the formation of parallel coiled coils.

The first and second heterodimerization domains (e.g., the first and second Fc regions) can form half-life extending moieties. Thus, in some embodiments, the heterodimeric fusion of the invention has an extended half-life compared to reference relaxin.

As used herein, the term "half-life" is used to refer to the time taken for the concentration of fusion protein in plasma to decrease to 50% of its original level. The "half-life" of a protein in plasma may depend on different factors, for example, the size of the protein, its stability, its clearance, turnover rate, proteolytic degradation in vivo, the absorption rate of the body or specific tissue, etc. Methods for determining the half-life of a protein are known in the art and are described in the examples below.

The inventors have demonstrated that the heterodimeric fusion of the invention having first and second heterodimerization domains derived from immunoglobulin Fc has a half-life of at least 5 hours in a mouse model (see example 6). In contrast, after IV administration, human relaxin-2 has a half-life in humans of about 0.09+/-0.04 hours, i.e., 5.4+/-2.4 minutes (Chen SA et al (1993) Pharm. Res. [ drug Ind. 10 (6): 834-838).

It will be appreciated that an extended half-life is advantageous because it allows for the administration of therapeutic proteins according to a safe and convenient dosing regimen (e.g., lower doses that can be administered less frequently). Furthermore, the implementation of lower doses may provide further advantages, for example, providing improved safety features and/or activating multiple in vivo mechanisms of action.

Connector

One or both of the relaxin a and B chains may be linked to their respective heterodimerization domains by a linker polypeptide. In some embodiments, the relaxin a chain is linked to a first heterodimerization domain (e.g., a first Fc region) via a linker polypeptide, and the relaxin B chain is linked to a second heterodimerization domain (e.g., a second Fc region) via a linker polypeptide.

The linker polypeptide may be any suitable length, for example about 6 to 40 amino acids in length, preferably about 6 to 21 amino acids in length. In some embodiments, the linker polypeptide is at least 6 amino acid residues in length, preferably at least 11 amino acids in length, preferably at least 16 amino acids in length. In some embodiments, the linker polypeptide is less than 40 amino acids in length. Linker polypeptides having different or the same length may be used for each arm of the heterodimeric fusion of the invention. In some embodiments, at least one linker polypeptide is 21 amino acids in length. In a preferred embodiment, both linker polypeptides are 21 amino acids in length. The linker polypeptide may have any amino acid sequence. Linker polypeptides having different or identical amino acid compositions may be used for each arm of the heterodimeric fusion of the invention.

In some embodiments, one or preferably both linker polypeptides comprise the proline and alanine repeat sequence (PA) x (SEQ ID NO: 73), preferably wherein x is 3 to 15, preferably wherein the linker polypeptide is greater than 16 amino acids in length, preferably wherein the linker polypeptide consists of: 21 amino acid sequence PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO: 6).

In some embodiments, one or preferably both linker polypeptides comprise glycine and serine repeat sequences, such as those described in the following documents: chen X et al (2013) Adv.drug.Deliv.Rev. [ advanced drug delivery comment ]65 (10): 1357-1369. In some embodiments, one or both linker polypeptides comprise the motif (GGGGS) n (SEQ ID NO: 74), where n may be between 1 and 8, e.g., where n is 4. In some embodiments, one or more linker polypeptides consist of: 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5). In certain embodiments, the two linker polypeptides consist of: 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5).

In some embodiments, one linker polypeptide comprises a proline and alanine repeat sequence as described herein, and the other linker polypeptide comprises a glycine and serine repeat sequence as described herein.

Alternatively, one or both of relaxin a and B chains may be linked to their respective heterodimerization domains by synthesis of a linker polypeptide, such as a polyethylene glycol (PEG) polymer chain. Thus, the relaxin a chain may be linked to the first heterodimerization domain (e.g., the first Fc region) via a synthetic linker, such as a polyethylene glycol (PEG) polymer chain, and the relaxin B chain may be linked to the second heterodimerization domain (e.g., the second Fc region) via a synthetic linker, such as a polyethylene glycol (PEG) polymer chain, wherein the synthetic linker may be covalently or non-covalently attached to the heterodimerization domain (e.g., the Fc region). PEGylation (the process of attaching PEG polymer chains to molecules) can be performed according to methods known in the art.

Stability of

The inventors of the present invention have demonstrated that the heterodimeric fusion of the present invention has unexpectedly superior physical and chemical stability. Thus, in some embodiments, the heterodimeric fusion of the invention has superior physical and/or chemical stability compared to the reference relaxin protein.

The physical stability of relaxin can be determined by measuring purity and aggregation, for example by HP-SEC as in example 9. The chemical stability of relaxin can be determined by measuring fragmentation and modification of the molecule, for example by LC-MS as in example 9.

Surprisingly, the inventors of the present invention have demonstrated superior physical and chemical stability of the heterodimeric fusion of the present invention compared to recombinant Fc fusion relaxin in which relaxin a and relaxin B are single chain fusions (as opposed to relaxin a and B being in separate fusion polypeptides). WO 2013/004607 describes recombinant single chain relaxin fusion polypeptides fused to an immunoglobulin Fc region, e.g., fusion polypeptides referred to herein as RELAX0127 and RELAX 0128. Thus, in some embodiments, the heterodimeric fusion of the invention has superior physical and/or chemical stability compared to RELAX0127 and RELAX 0128.

The heterodimeric fusion can further comprise a half-life extending moiety in addition to the first and second heterodimerization domains. In some embodiments, the half-life extending moiety is a protein half-life extending moiety. The protein half-life extending moiety may be selected from the group consisting of: an Fc region of an immunoglobulin, an albumin binding domain and serum albumin. In further embodiments, the half-life extending moiety is a chemical entity other than a protein or peptide, such as a polyethylene glycol (PEG) polymer chain.

The half-life extending moiety may be attached at the N-terminus or the C-terminus of the first or second heterodimerization domain. In some embodiments, the half-life extending moiety is attached to the N-terminus of the first or second heterodimerization domain. In other embodiments, the half-life extending moiety is attached to the C-terminus of the first or second heterodimerization domain. Methods of attaching half-life extending moieties to heterodimeric fusions are known in the art. For example, the half-life extending moiety may be attached by chemical conjugation or recombinant techniques. The half-life extending moiety may be attached to the heterodimeric fusion directly or through a linker (e.g., a linker polypeptide). The use of linker polypeptides may be particularly suitable when the fusion polypeptide comprises a protein half-life extending moiety, such as an Fc region.

Exemplary embodiments of the invention

The heterodimeric fusion of the invention can have a variety of formats and/or sequences.

The term "fusion polypeptides of the invention (fusion polypeptide of the invention and fusion polypeptides ofthe invention)" may be used to refer to a first heterodimerization domain fused to the relaxin a chain and/or a second heterodimerization domain fused to the relaxin B chain. The fusion polypeptide of the invention may be a recombinant fusion polypeptide, i.e. it has been produced by recombinant DNA technology.

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

In alternative embodiments, the N-terminus of a first heterodimerization domain (e.g., a first Fc region) is linked to the C-terminus of the relaxin a chain and the N-terminus of a second heterodimerization domain (e.g., a second Fc region) is linked to the C-terminus of the relaxin B chain. In some embodiments, the relaxin a-chain polypeptide and/or the relaxin B-chain polypeptide has a free N-terminus.

The heterodimeric fusion of the invention may further comprise one or more Fab. In some embodiments, the heterodimeric fusion comprises one Fab linked to the N-terminus of a first heterodimerization domain (e.g., a first Fc region) and a second Fab linked to the N-terminus of a second heterodimerization domain (e.g., a second Fc region).

The heterodimeric fusion of the invention may further comprise a second relaxin a chain polypeptide or variant thereof and a second relaxin B chain polypeptide or variant thereof. In some embodiments, a second relaxin a chain polypeptide or variant thereof is linked to the N-terminus of a first heterodimerization domain (e.g., a first Fc region) and a second relaxin B chain polypeptide or variant thereof is linked to the N-terminus of a second heterodimerization domain (e.g., a second Fc region), optionally wherein the second relaxin a chain is linked to the first heterodimerization domain (e.g., a first Fc region) via a linker (e.g., a linker polypeptide) and the second relaxin B chain is linked to the second heterodimerization domain (e.g., a second Fc region) via a linker (e.g., a linker polypeptide).

Thus, in some embodiments, the format of the heterodimeric fusion is selected from the group consisting of:

(i) FcX-con-A/FcY-con-B (see, e.g., FIG. 1);

(ii) FcX-con-B/FcY-con-A (see, e.g., FIG. 1);

(iii) A-con-FcX/B-con-FcY (see, e.g., FIG. 1);

(iv) B-con-FcX/A-con-FcY (see, e.g., FIG. 1);

(v) Fab-FcX-con-A/Fab-FcY-con-B (see, e.g., FIG. 1);

(vi)Fab-FcX-con-B/Fab-FcY-con-A；

(vii) A-con-FcX-con-A/B-con-FcY-con-B (see, e.g., FIG. 1);

(viii)B-con-FcX-con-B/A-con-FcY-con-A；

(ix) FcX-con-B-L-A and FcY, optionally FcY-con-B-L-A (see, e.g., FIG. 1)

(X) FcY-con-B-L-A and FcX, optionally FcX-con-B-L-A

(Xi) FcX-con-A-L-B and FcY, optionally FcY-con-A-L-B; and

(Xii) FcY-con-A-L-B and FcX, optionally FcX-con-A-L-B,

Wherein:

FcY is an immunoglobulin Fc region having an "Fc mortar" amino acid mutation and/or modification, preferably comprising a CH3 domain having the amino acid mutation Y349C: T366S: L368A: Y407V, or a conservative substitution thereof;

FcX is an Fc region having an "Fc pestle" amino acid mutation and/or modification, preferably comprising a CH3 domain having the amino acid mutation S354C: T366W or conservative substitutions thereof;

"con" is a linker polypeptide;

B is a relaxin B chain or variant thereof;

a is a relaxin a chain or variant thereof; and

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60).

In another aspect, the invention provides a heterodimeric fusion comprising:

(i) X-B-L-A and Y, optionally Y-B-L-A Or (b)

(Ii) Y-B-L-A and X, optionally X-B-L-A

Wherein:

X and Y are heterodimerization domains as described herein;

L is a linker polypeptide, preferably having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60),

Wherein X and Y heterodimerize, and wherein the heterodimeric fusion has relaxin activity.

In yet another aspect, the invention provides a heterodimeric fusion comprising:

(i) X-A-L-B and Y, optionally Y-A-L-B or

(Ii) Y-A-L-B and X, optionally X-A-L-B,

Wherein:

X and Y are heterodimerization domains as described herein;

L is a linker polypeptide, preferably having amino acid sequence GGGSGGGSGG (SEQ ID NO: 60),

In a particularly preferred embodiment according to all aspects of the invention, the heterodimeric fusion comprises a fusion polypeptide Rlx011DD as shown in SEQ ID NO:11 and Rlx014DD as shown in SEQ ID NO: 20. This heterodimeric fusion may also be referred to as "RELAX0023" or "AZD3427".

In an alternative preferred embodiment, the heterodimeric fusion comprises a fusion polypeptide Rlx013DD as shown in SEQ ID NO:17 and Rlx012DD as shown in SEQ ID NO: 14.

In one aspect of the invention, a heterodimeric fusion is provided comprising a fusion polypeptide combination selected from the group consisting of FcX and FcY combinations shown in table 3.

Table 3: fusion polypeptide combinations in heterodimeric fusions of the invention

* Table 1 shows the sequences of the listed fusion polypeptides.

* In this particular embodiment, the heterodimeric fusion is an IgG and comprises an additional polypeptide corresponding to the light chain shown in SEQ ID No. 54.

According to all aspects of the invention there is provided a heterodimeric fusion comprising the fusion polypeptides shown in SEQ ID NO. 11 and SEQ ID NO. 20 for use in a method of treating a subject suffering from heart failure with pulmonary hypertension, as described herein.

Alternatively, according to all aspects of the invention, there is provided a heterodimeric fusion comprising the fusion polypeptides shown in SEQ ID NO:17 and SEQ ID NO:14 for use in a method of treating a subject suffering from heart failure with pulmonary hypertension, as described herein.

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

Methods known to those of skill in the art may be used to construct expression vectors containing nucleic acid molecules encoding fusion polypeptides of the invention. Suitable vectors include, for example, plasmid, phagemid, phage or viral vectors.

Vectors containing nucleic acid molecules encoding fusion polypeptides of the invention can be transferred to host cells by conventional techniques. Suitable host cells are known in the art. The host cell may be a mammalian cell, such as a HEK293 cell or CHO cell.

Transfected cells may be cultured by conventional techniques to produce fusion polypeptides of the invention.

Once the fusion polypeptides of the invention have been produced, e.g., by recombinant expression, they can be purified by any method known in the art. Exemplary protein purification techniques include chromatography (e.g., ion exchange chromatography, affinity chromatography, and/or size column chromatography), centrifugation, and differential solubility. The present disclosure provides isolated fusion polypeptides, optionally isolated from a cell culture by at least one purification step.

Therapeutic method

The fusion polypeptides of the invention may be provided in pharmaceutical compositions.

The pharmaceutical compositions of the present invention may comprise one or more excipients. Pharmaceutically acceptable excipients are known in the art, see, for example, remington's Pharmaceutical Sciences [ rest pharmaceutical science ] (Joseph p. Remington, 18 th edition, mack Publishing co.), oiston, pa, incorporated herein in its entirety.

The present invention relates to a method of treating a subject suffering from heart failure with pulmonary hypertension by administering a heterodimeric fusion (or a pharmaceutical composition) as described herein, as well as the use of the heterodimeric fusion (or the pharmaceutical composition), and the heterodimeric fusion (or the pharmaceutical composition) for use in the method. In particular, the subject may be an animal, preferably a mammal, more preferably a human.

The use or method may comprise administering a therapeutically effective regimen having a lower frequency of doses of the heterodimeric fusion/fusion polypeptide of the invention than the therapeutically effective dosing regimen of wild-type relaxin molecules.

As used herein, the term "heart failure" includes acute heart failure, chronic Heart Failure (CHF), and Acute Decompensated Heart Failure (ADHF). The term "heart failure" may also include more specific diagnoses, such as heart failure with preserved ejection fraction (HFpEF), heart failure with intermediate ejection fraction or heart failure with reduced ejection fraction (HFrEF). This may also include heart failure caused by hypertrophic cardiomyopathy or dilated cardiomyopathy.

As used herein, the term "pulmonary arterial hypertension" may be defined as a subject having an average pulmonary arterial pressure of about 20mmHg or greater, preferably 25mmHg or greater, typically at rest in the subject. It may also be defined as an average pulmonary artery pressure of about 30mmHg or higher, typically when the subject is exercising or has recently been exercising. Thus, the mean pulmonary artery pressure of the subject may be in the range of about 20mmHg to about 30mmHg, preferably about 25mmHg to about 30mmHg or higher. Alternatively or additionally, the subject may have:

a. Right ventricular systolic pressure of about 40mmHg or greater;

b. Pulmonary Artery Wedge Pressure (PAWP) greater than 15 mmHg; and/or

C. the following pulmonary vascular resistances:

i. less than 3.0 wood units; or (b)

3.0 Or more wood units.

Thus, in some cases, pulmonary arterial hypertension may be categorized as group 2 pulmonary arterial hypertension defined by the world health organization. This may also be referred to as "heart failure due to left heart disease combined with pulmonary hypertension". In other cases, pulmonary arterial hypertension can be categorized as group 1 pulmonary arterial hypertension defined by the world health organization (see Ryan et al 2012, pulm. Circle [ pulmonary circulation ]2 (1): 107-121).

Parameters of pulmonary arterial hypertension and heart failure may be measured or estimated using techniques known in the art. For example, these techniques include echocardiography, pulmonary artery catheters, and implantable monitoring devices. In certain embodiments, the subject may already be equipped with a blood pressure monitoring device, preferably a pulmonary artery pressure monitoring device, as known in the art. In a particular embodiment, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. Typically, the device is provided prior to treatment with the heterodimeric fusion of the invention as described herein. Alternatively, the subject is provided with the device during or after treatment.

As used herein, the term "heart failure combined with pulmonary arterial hypertension" refers to a subset of heart failure subjects (hf+ph subjects) that are concurrently suffering from pulmonary arterial hypertension.

"Treating" refers to ameliorating and/or eliminating one or more symptoms or etiologies of a target disease. In some embodiments, this may involve modulating the level of one or more biomarkers or functions to be within a non-diseased range (as compared to a healthy cohort). For example, the heterodimeric fusion of the invention can reduce Pulmonary Vascular Resistance (PVR) in a subject. For example, post-treatment PVR can be reduced by at least 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, or 1% to 50% or more compared to baseline PVR (prior to administration of the heterodimeric fusion of the invention to a subject). Thus, the heterodimeric fusion of the invention can reduce PVR in a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more compared to baseline PVR (prior to administration of the heterodimeric fusion of the invention to the subject). Additionally or alternatively, the heterodimeric fusion of the invention can reduce the mean pulmonary arterial pressure of the subject (mPAP). For example mPAP may be reduced by at least 1mmHg to 15mmHg or more. Thus, the heterodimeric fusion of the invention can reduce the mean pulmonary arterial pressure of a subject by at least 1mmHg, at least 2mmHg, at least 3mmHg, at least 4mmHg, at least 5mmHg, at least 6mmHg, at least 7mmHg, at least 8mmHg, at least 9mmHg, at least 10mmHg, at least 11mmHg, at least 12mmHg, at least 13mmHg, at least 14mmHg, or at least 15mmHg or more. Likewise, the heterodimeric fusion of the invention can reduce estimated pulmonary artery diastolic pressure (ePAD) in a subject. For example, the ePAD may be reduced by at least 1mmHg to 15mmHg or more. Thus, the heterodimeric fusion of the invention can reduce the estimated pulmonary artery diastolic pressure of a subject by at least 1mmHg, at least 2mmHg, at least 3mmHg, at least 4mmHg, at least 5mmHg, at least 6mmHg, at least 7mmHg, at least 8mmHg, at least 9mmHg, at least 10mmHg, at least 11mmHg, at least 12mmHg, at least 13mmHg, at least 14mmHg, or at least 15mmHg or more. Additionally or alternatively, the heterodimeric fusion of the invention can increase the percent ejection fraction (EF%) of a subject as a measure of cardiac output. For example, EF% may be increased by at least 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, or 1% to 50% or more. Thus, the heterodimeric fusion of the invention can increase the percent ejection fraction (EF%) of a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more. Additionally or alternatively, in a subject, the heterodimeric fusion of the invention can:

a) Increasing Stroke Volume (SV) of the heart;

(b) Decreasing Systemic Vascular Resistance (SVR) and/or increasing estimated glomerular filtration rate (evfr);

(c) Increasing ejection fraction; and/or

(D) Increasing cardiac output;

Compared to baseline levels prior to administration. The combination of a decrease in SVR and an increase in eGFR indicates improved organ perfusion.

Thus, in a subject, the heterodimeric fusion of the invention can:

a) Reducing the PVR;

(b) A decrease mPAP;

(c) Reduce ePAD;

(d) Increasing Stroke Volume (SV) of the heart;

(e) Decreasing Systemic Vascular Resistance (SVR) and/or increasing estimated glomerular filtration rate (evfr);

(f) Increasing ejection fraction; and/or

(G) Increasing cardiac output;

Compared to baseline levels prior to administration. The combination of a decrease in SVR and an increase in eGFR indicates improved organ perfusion. Changes in one or more or all of these parameters may each occur after 1-24 weeks of treatment. In some embodiments, a change in one or more or all of these parameters occurs after 24 weeks of treatment.

In certain embodiments, a decrease of mPAP as described herein may improve dyspnea, such as Solomonica A et al (2013) CIRC HEART FAIL [ cycle: heart failure ] 6:53-60.

The fusion polypeptides (thus including heterodimeric fusions) and/or pharmaceutical compositions of the invention are suitable for parenteral administration to a subject or patient. In some embodiments, the subject or patient is a mammal, particularly a human.

Wild type human relaxin-2 has an in vivo half-life on the order of minutes. It must therefore be administered by continuous intravenous infusion in hospitalized patients and exhibit serious side effects, including blood pressure reduction. In contrast, it will be appreciated that embodiments of the fusion polypeptides (and thus including heterodimeric fusions) and/or pharmaceutical compositions of the invention can be administered to a subject or patient by injection (e.g., by intravenous, subcutaneous, or intramuscular injection). In some embodiments, the fusion polypeptide (and thus the heterodimeric fusion) and/or the pharmaceutical composition is administered by subcutaneous injection. Administration by injection (e.g., by subcutaneous injection) provides better comfort benefits to the subject or patient and provides an opportunity for administration to subjects or patients outside of the hospital setting. In some embodiments, the fusion polypeptide (and thus the heterodimeric fusion) or pharmaceutical composition is administered by self-administration.

In some embodiments, the fusion polypeptides of the invention (thus including heterodimeric fusions) have an increased half-life compared to wild-type relaxin, which allows for lower total exposure on a molar basis. For example, fusion polypeptides of the invention (and thus including heterodimeric fusions) can be administered less frequently than wild-type relaxin, thereby providing a more convenient dosing regimen.

Kits comprising the pharmaceutical compositions of the invention may be provided. The kit may comprise a package containing the pharmaceutical composition of the invention and instructions. In some embodiments, the pharmaceutical compositions of the present invention are formulated in a single dose vial or container closure system (e.g., a prefilled syringe). Optionally, associated with such one or more containers may be an announcement in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which announcement reflects approval of the agency for human administration for manufacture, use or sale.

As used herein, the article "a" or "an" may refer to one or more than one (e.g., at least one) of the grammatical object of the article.

"About" may generally mean an acceptable degree of error in the measured quantity given the nature or accuracy of the measured value. Exemplary degrees of error are within a percentage (%) of a given value or range of values, typically within 10%, and more typically within 5%.

Embodiments described herein as "comprising" one or more features may also be considered as a disclosure of a corresponding embodiment "consisting of" such features.

The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia, european pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

Concentrations, amounts, volumes, percentages, and other numerical values may be presented in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

The above embodiments are to be understood as illustrative examples. Additional embodiments are contemplated. It is to be understood that any feature described in relation to any one embodiment may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments, or any combination of any other of the embodiments. Furthermore, equivalents and modifications not described above may also be employed without departing from the scope of the invention, which is defined in the accompanying claims.

Other examples and variations of fusion polypeptides and methods described herein will be apparent to those of skill in the art in the context of the present disclosure.

Other examples and variations are within the scope of the disclosure, as indicated in the appended claims. All documents cited herein are incorporated by reference in their entirety, including all data, tables, figures, and text presented in the cited documents.

Examples

Example 1: production of recombinant heterodimeric Fc relaxin-2 fusion proteins

The heterodimerization properties of the pestle-mortar Fc domains (Fc pestle and Fc mortar) have been used to design the Fc relaxin-2 fusion proteins described herein to induce proper folding and heterodimerization of the a and B chains of relaxin-2.

More precisely, relaxin-2A and B chains have been genetically fused to two complementary Fc's (at the N-and/or C-terminus of the Fc's) via linkers, as shown in FIG. 1. CHO cells are then co-transfected with two expression vectors comprising each of the single Fc-relaxin chains (a and/or B). The two complementary Fc portions assemble in CHO cells and thus promote the assembly and correct folding of relaxin-2. As demonstrated in example 2 below, disulfide bonds are then formed between the complementary Fc strands and between the a and B strands, thereby regenerating the native relaxin-2 structure.

The heterodimeric Fc relaxin-2 fusion protein is secreted in the supernatant and then purified by affinity chromatography using an automated system, wherein the Fc region of the protein is bound to the column matrix.

Example 2: LC-MS analysis of Fc relaxin-2 pestle heterodimers

LC-MS analysis was performed on both non-reduced and reduced deglycosylated Fc-relaxin-2 heterodimers. For deglycosylation, the samples were diluted to 1mg/ml and buffered with 10mM Tris-Cl at pH 7.80. peptide-N-glycosidase F (PNGase F) (Roche) was added to the sample at a concentration of 1 unit of enzyme per 50. Mu.g of Fc-relaxin-2 and incubated overnight at 37 ℃. For non-reducing analysis, samples were diluted to 0.05mg/ml in water and 20. Mu.L was loaded into LC-MS certified total recovery bottles with pre-slit caps (Waters part number: 186005663 CV). For the reduction analysis, 10mM TCEP was added and the samples were incubated at 37℃for an additional 30 minutes before analysis.

Experiments were performed using an ACQUITY I-Class UPLC (Vortight corporation, milford (Milford), mass.) coupled to a Xex G2-XS Q-TOF instrument, both operating using UNIFI scientific information systems. For LC systems, solvent a is water containing 0.1% formic acid and solvent B is acetonitrile containing 0.1% formic acid (both UPLC-MS grade, all clear company (BioSolve)). The UV detector was set to measure at wavelengths of 220nm and 280nm and the vial was placed in a sample chamber maintained at a temperature of 4 ℃. A volume of 1. Mu.L was injected into a BEH C4 column of the reverse phase ACQUITY UPLC protein,On a column (Wattshi part number: 186004495) and using an increasing gradient of solvent B from 5% to 75% over 6 minutes.

The mass spectrometer was calibrated from 500-5000m/z by infusing 2. Mu.g/. Mu.L sodium iodide in 50% 2-propanol and lockspray was 200 pg/. Mu.L leucine enkephalin (Leucine Enkephalin). The instrument is operated according to a positive ionization action mode and a sensitivity analyzer mode, and the key settings are as follows: capillary voltage = 3.0V; sample cone voltage = 40V; source temperature = 120 ℃; desolvation temperature = 450 ℃; taper hole airflow = 120L/h; desolvation gas flow = 1000L/h; mass range = 500-5000m/z, scan time = 1.0sec.

The data is processed in UNIFI software. The spectra were pooled from retention times in the chromatogram from which the protein of interest was eluted. The raw data is the background subtracted and deconvolved using the MaxEnt1 algorithm for macromolecules. Experimental data were compared to the mass of theoretical sequences in which disulfide bonds were considered for non-reducing analysis and free cysteines were considered for reducing analysis. Deamidation of asparagine (+1 Da) is also considered after deglycosylation of peptide-N-glycosidase F.

LC-MS analysis confirmed that disulfide bonds were formed between the complementary Fc strands and between the a and B strands, thereby regenerating the native relaxin-2 structure. As an example, fig. 2A shows LC-MS data for RELAX0019 and RELAX 0023. Non-reducing analysis confirmed that heterodimers of expected masses 58932Da and 59361Da were formed for RELAX0019 and RELAX0023, respectively: no homodimers were detected. The reduction analysis (fig. 2B) confirmed the sequence identity of both strands and showed that they did not have modifications.

Non-reducing peptide mapping to identify disulfide bonds

Heterodimeric Fc-relaxin (50 μg) was placed into a clean sample tube and diluted in 17 μl of 100mM sodium phosphate (pH 7.0). Alkylation of free cysteine was achieved by adding 0.5 μl of 5mg/ml iodoacetamide followed by incubation at room temperature for 20 minutes. After alkylation, 2.5. Mu.L of 100mM sodium phosphate buffer (pH 7.0) and 2.5. Mu.L of sodium chloride were added. The protein was denatured by adding 40 μl of 8.0M guanidine hydrochloride and incubating at 37 ℃ for 30 min. Dilution was achieved by adding 125. Mu.L of 100mM sodium phosphate buffer (pH 7.0) followed by 0.5. Mu.L of 40mM EDTA. Endoprotease Lys-C (and photochemistry Co., wako Chemicals)) was reconstituted in water at a concentration of 1mg/ml and 5. Mu.L was added to Fc-relaxin-2. Digestion was carried out at 37℃for 2 hours, after which time an additional 5. Mu.L of Lys-C was added and incubation was continued for an additional 2 hours. For peptide analysis, 42.5 μl of sample was transferred to a UPLC vial and 2.5 μl of water was added. For disulfide bond reduction, 2.5 μl of 500mM DTT was added to another 42.5 μl aliquot of the sample and left at room temperature for 15 minutes before LC-MS analysis.

Peptide analysis was performed using an ACQUITY I-Class UPLC (Wolter, milford, mass.) coupled to a Xex G2-XS Q-TOF instrument, both operating using UNIFI scientific information systems. For LC systems, solvent a is water containing 0.1% formic acid and solvent B is acetonitrile containing 0.1% formic acid (both UPLC-MS grade, all clear). The UV detector was set to measure at a wavelength of 214nm and the vial was placed in a sample chamber maintained at a temperature of 4 ℃. A volume of 10. Mu.l was injected into the reversed phase ACQUITY BEH C18The proteins were eluted on a column (Waters company part number 186003687) and using an increasing gradient of solvent B from 5% to 37% B over 73.5 minutes, then increasing to 60% B over an additional 2.5 minutes. After 77.5 minutes, the column was held at 95% b for 5 minutes.

The mass spectrometer was calibrated from 100-2600m/z by infusing 2. Mu.g/. Mu.L sodium iodide in 50% 2-propanol and lockspray is 200 pg/. Mu.L leucine enkephalin. The instrument is operated according to a positive ionization action mode and a sensitivity analyzer mode, and the key settings are as follows: capillary voltage = 3.0V; sample cone voltage = 25V; source temperature = 100 ℃; desolvation temperature = 250 ℃; taper hole airflow = 0L/h; desolvation gas flow = 500L/h; mass range = 100-2600m/z, scan time = 0.5sec.

Data were processed in UNIFI software by entering the sequence with the expected disulfide bond and retrieving the peptide for matching Lys-C production. The chromatograms obtained in the absence and presence of the reducing agent were overlaid to verify that the identified disulfide-bonded peptides were no longer observed upon reduction.

Peptides were identified that matched the expected mass of disulfide-bonded relaxin-2 peptides incorporated into both the a and B chains, as shown at the top of fig. 3 (SLSLSPGGGGGSGGGGSGGGGSGGGGGSQLYSALANKCCHVGCTK = LCGRELVRAQIAICGMSTWS = RSLARFC (SEQ ID NOs 75-77, respectively), the expected mass of 6836.23Da, including 3 disulfide bonds). Fig. 3 (a-D) shows the identification of this peptide for RELAX0019 and demonstrates that this peptide is no longer observed when reducing agent is added: panels a and B show extracted ion chromatograms in the absence and presence of DTT, and panels C and D show the corresponding mass spectra of peptides. Fig. 3 (E-H) shows the identification of the same peptide as RELAX0023 and demonstrates that the peptide is no longer observed when reducing agent is added: panels E and F show extracted ion chromatograms in the absence and presence of DTT, and panels G and H show the corresponding mass spectra of peptides. These data demonstrate that relaxin a and B chains interact through disulfide bonds within the heterodimers RELAX0019 and RELAX 0023.

Example 3: in vitro Activity of Fc-relaxin-2 fusion proteins (cell-based cAMP Activity assay)

The relaxin-2 fusion polypeptides produced as described above are tested for biological activity, e.g., stimulation of one or more cellular receptor responses, by the following method.

Stable cell lines expressing human or mouse receptors produced in CHO cells were purchased from dieshi Wo Kesi company (DiscoverX).

-CAMP Hunter ^TM CHO-K1 RXFP1 Gs, cell line (Dishi Wo Kesi company catalog number 95-0127C 2)

-CAMP Hunter ^TM CHO-K1 RXFP2 Gs cell line (Dishi Wo Kesi company catalog number 95-0140C 2)

-CAMP Hunter ^TM CHO-K1 mRXFP Gs cell line (Dishi Wo Kesi company catalog number 95-0180C 2)

Activation of these receptors results in the production of downstream cAMP second messengers, which can be measured in a functional activity assay.

Conventional cAMP assays were performed using the following Bovine Serum Albumin (BSA) based assay buffers: hank's balanced salt solution (sigma #h8264) supplemented with 0.1% bsa (sigma #a9418) and 0.5mM IBMX (sigma #i7018) was adjusted to pH 7.4 with 1M NaOH.

Frozen vials of cells expressing the receptor of interest were thawed rapidly in a water bath, transferred to pre-warmed cell culture medium and spun at 240xg for 5 minutes. Cells were resuspended in cell culture medium at an optimized concentration (e.g., 3.33x 10 ⁴ cells/mL hRXFP 1), and 30 μl of the cell suspension was added to poly-D-lysine coated 384-well plates (Greiner) # 781946) and allowed to adhere overnight. The next day, the medium was removed from the plate and replaced with 5uL assay buffer. An 11-point serial dilution of the test recombinant peptide or Fc fusion sample was added to the cells using a non-contact liquid dispenser (ECHO ^TM, labcyto corporation (labcyto)). All sample dilutions were made in duplicate. An additional 5 μl of assay buffer was added to each well and the plate incubated for 30 minutes at room temperature.

CAMP levels were measured using a commercially available cAMP dynamic G _S HTRF kit (Cisbio, inc. (Cisbio), catalog No. 62AM4 PEJ) following a two-step protocol according to manufacturer's recommendations. Briefly, anti-cAMP cryptates (donor fluorophore) and cAMP-d2 (acceptor fluorophore) are made separately by diluting them 1/20 each in conjugation and lysis buffer provided in the kit. mu.L of anti-cAMP cryptate was added to all wells of the assay plate, and 5. Mu.L of cAMP-d2 was added to all wells except for non-specific binding (NSB) wells, to which conjugation and lysis buffer was added. Plates were incubated for one hour at room temperature and then read on an Envision (perkin elmer) using excitation wavelengths of 320nm and emission wavelengths of 620nm and 665 nm. Data were converted to% Δf and then to activation of the maximal natural agonist response as described in manufacturer's guidelines and analyzed by 4-parameter logistic fit to determine EC50 values. In the case of hRXFP cells, these results were compared with the corresponding results for recombinant h relaxin-2 (R & D systems, cat# 6586 RN); in the case of mRXFP cells, a comparison was made with the corresponding results for m relaxin-1 (R & D systems, cat# 6637 RN); and in the case of hRXFP cells, with the corresponding results of INSL3 (R & D systems, catalog number 4544 NS).

Data analysis was performed using statistical analysis software (GRAPHPAD PRISM, 6 th edition).

The biological activity of the tested constructs is provided in table 4 and figure 4. Table 4 has summarized the average EC50 measurements from several assays for both recombinant human relaxin-2 and fusion polypeptides.

RELAX0013, RELAX0014 and RELAX0010 are reference proteins, wherein RELAX0013 is recombinant human relaxin-2, RELAX0014 is recombinant murine relaxin-1, and RELAX0010 is a single chain fusion protein comprising an A chain, a 15 amino acid linker, a B chain, a 15 amino acid linker and an Fc comprising the amino acid sequence of SEQ ID NO.8 is described in WO 2018/138170.

Table 4: the biological activity (n: the number of repeat sequences) of the heterodimeric Fc relaxin fusion polypeptide.

From the results presented in table 4, it can be concluded that the tested heterodimeric Fc relaxin fusion proteins were not as potent as the single chain fusion RELAX0010 or recombinant human relaxin-2 peptide, but they still retained high levels of biological activity (ranging from about 10pM to about 80pM in the human RXFP1 cell line).

These results show that relaxin a and B chains can be fused to either/both ends (the linker can be attached to the N or C terminus of the relaxin chain) as well as either chain of the heterodimer Fc (X or Y) and retain biological activity. Thus, the format of the heterodimeric Fc relaxin fusion proteins described herein constitutes a robust format for producing long half-life active relaxin.

The presence of disulfide bonds to stabilize the heterodimeric Fc did not affect the potency of the fusion proteins (compare RELAX0023 to RELAX0021 and RELAX0024 to RELAX 0022).

The two upper hinge regions used (GGAGGA (SEQ ID NO: 78) and natural DKTHT (SEQ ID NO: 79)) did not affect efficacy (compare RELAX0023 to RELAX0019 and RELAX0024 to RELAX 0020). The exact amino acid sequence of the upper hinge is not important for the activity of the fusion protein.

Example 4: effect of linker composition and Length in heterodimeric relaxin-2 Fc fusion proteins

The linker may consist of glycine and serine residues (GS), or may consist of proline and alanine repeats (PA). The length of the linker used herein is 6 to 21 residues. Examples of long GS linkers are: GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5) (21 amino acids). Examples of long PA linkers are: PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO: 6) (21 amino acids).

Linkers of different lengths and compositions may be placed on each Fc chain of the heterodimeric relaxin-2 Fc fusion polypeptide.

Examples of heterodimeric relaxin-2 Fc fusion proteins with various linkers are shown in table 5. This table also shows information about: developability/manufacturability (expression yield and percentage of monomeric/non-aggregated relaxin-2 Fc fusion protein after protein a capture from cell culture supernatant) and bioactivity.

Table 5: effect of linker on the bioactivity and developability characteristics of the heterodimeric Fc relaxin-2 fusion protein during small scale expression.

The length and composition of the linker does have an impact on the developability of the molecule. As shown in table 5, heterodimeric relaxin-2 Fc fusion polypeptides with PA linkers of less than or equal to 16 amino acids are not well expressed. In contrast, a 21 residue long PA linker significantly increases expression yield. The expression yield of the constructs with the GS linker was more consistent.

Heterodimeric relaxin-2 Fc fusion proteins with short and asymmetric (different) linkers retain potency. Reduced bioactivity was observed only in fusion proteins with low monomer content (RELAX 0109, RELAX0110 and RELAX 0111).

Example 5: point mutations in relaxin-2 sequences

Relaxin single point mutant analogs were prepared as heterodimeric Fc relaxin-2 fusion proteins. Table 6 shows examples of such molecules that retain potency and advantageous developability characteristics.

The targeted natural residues are positively charged and may be susceptible to proteolysis, but are not involved in binding relaxin to its receptor.

For example, the R22X analog of a heterodimeric Fc relaxin-2 fusion protein appears to consistently have improved developability/manufacturability characteristics.

Table 6: examples of relaxin-2 analogs that retain potency and favorable developability characteristics during small scale expression.

The results presented in table 6 demonstrate that some variability in the amino acid sequence of the relaxin-2A chain is tolerated without loss of potency while preserving the favorable developability characteristics.

Example 6: PK profile of Fc-relaxin-2 fusion proteins

Pharmacokinetic (PK) profiles of relaxin-2 fusion polypeptides were determined using a relaxin ELISA assay and/or cAMP assay. Relaxin-2 fusion polypeptides were administered at 6mg/kg to 6-10 week old male C57BL/6J (Jax) mice (Jackson laboratories (Jackson Laboratories)) via the Subcutaneous (SC) and/or Intravenous (IV) route. For IV route administration, serum samples were collected 5, 30 and 60 minutes after drug administration, followed by 3 and/or 6 and/or 8 and 24 hours, followed by a series of minimum 1 day intervals up to 21 days. For SC route administration, a similar schedule was followed, with collection occurring less frequently during the first 8 hours; for example, the first sample is collected at 30 minutes, followed by 3 hours, 8 hours, 24 hours, 30 hours, and 48 hours, followed by a series of collection at least 1 day intervals up to 21 days. Samples were collected into serum tubes by cardiac puncture and kept at room temperature for 15 to 30 minutes, then centrifuged at 10000rpm for 10 minutes within 30 minutes of collection. Aliquots were stored at < -80℃and later tested by ELISA or cAMP activity assay.

For most molecules, PK samples were tested in ELISA using anti-h relaxin-2 capture (pre-coated human relaxin-2 Quantikine ELISA kit, R & D systems company, catalog No. DRL 200) and anti-human Fc detection antibody (AU 003 labeled with HRP), except for RELAX0010 (described in WO 2018/138170), which was tested in ELISA using anti-human Fc capture and anti-h relaxin-2 detection (using polyclonal HRP labeled antibody from human relaxin-2 ELISA kit (R & D systems company, catalog No. DRL 200). In both assays, the capture antibody coated plates were blocked with 100 μl of RD1-19 assay diluent at room temperature for one hour. mu.L of standard or sample was added to each well and incubated for two hours at room temperature. The sample was aspirated and the wells were washed three times with assay wash buffer. 50 μl of HRP-labeled detection antibody was added to each well, diluted 1:1000 in PBS/1% BSA in the case of anti-human Fc-specific detection, or used undiluted in the case of anti-h-relaxin-2 detection. After 1 hour incubation at room temperature and three washes, 50. Mu.L/well TMB (SureBlue RESERVE KPL-00-03) was added and once the color change had occurred, the reaction was stopped by adding 50. Mu.L/well TMB stop solution (KPL 50-85-06).

Biological activity of PK samples in cell-based cAMP activity assay.

Serum samples collected from animals as described above were tested for biological activity to measure functional relaxin-2 to assess the integrity of the Fc-relaxin-2 fusion polypeptide. Stable cell lines expressing the human RXFP1 receptor produced in CHO cells were purchased from dieshi Wo Kesi company. Activation of this receptor results in the production of a downstream cAMP second messenger that can be measured in a functional activity assay.

CAMP assays were performed using the following Bovine Serum Albumin (BSA) based assay buffers: hank's balanced salt solution (sigma #h8264) supplemented with 0.1% bsa (sigma #a9418) and 0.5mM IBMX (sigma #i7018) was adjusted to pH 7.4 with 1M NaOH.

A dosage solution of relaxin-2 fusion polypeptide or recombinant relaxin-2 peptide (R & D systems, cat. No. 6586-RN) was diluted in assay buffer and a non-contact liquid dispenser (ECHO, labcyte Co.) was used to generate 11-point standard curves of four matrix concentrations. The matrix used was blank serum from a simulated dose of animals and was manually added to the wells at twice the required concentration and cells were allowed to be added. The test sample was transferred from the serum tube to 384 Kong Yuanban and was used to establish four dilutions in assay buffer by a non-contact liquid dispenser (ECHO, labcyte corporation). All sample dilutions were made in duplicate.

Frozen vials of hRXFP expressing cells were thawed quickly in a water bath, transferred to pre-warmed cell culture medium and spun at 240xg for 5 minutes. Cells were resuspended in 8mL of cell culture medium, inoculated in T75 flasks containing 10mL of medium, and allowed to adhere overnight. The next day, cells were isolated using accutase and spun at 240xg for 5 minutes. The resulting cell pellet was resuspended at an optimized concentration and 2.5 μl of cell suspension was added to each well of the assay plate using a combined droplet dispenser (Combi-drop dispenser).

CAMP levels were measured using a commercially available cAMP dynamic 2HTRF kit (Cisbio corporation, catalog No. 62AM4 PEJ) following a two-step protocol according to manufacturer's recommendations. Briefly, anti-cAMP cryptates (donor fluorophore) and cAMP-d2 (acceptor fluorophore) are made separately by diluting them 1/20 each in conjugation and lysis buffer provided in the kit. 2.5. Mu.L of anti-cAMP cryptate was added to all wells of the assay plate, and 2.5. Mu.L of cAMP-d2 was added to all wells except for non-specific binding (NSB) wells, to which conjugation and lysis buffer was added. Plates were incubated for one hour at room temperature and then read on an Envision (perkin elmer) using excitation wavelengths of 320nm and emission wavelengths of 620nm and 665 nm. The data was converted to% Δf as described in the manufacturer's guidelines and sample values were calculated from the linear portion of the standard curve.

Results and conclusions

Fig. 5 shows a summary of data from a series of in vivo PK experiments, in which Fc-relaxin-2 polypeptide IV was administered to mice. Data were normalized for the 5 minute time point.

Following IV administration, the half-life of human relaxin-2 in humans is about 0.09+/-0.04 hours, i.e., 5.4+/-2.4 minutes (Chen et al 1993). Recombinant relaxin Fc fusion polypeptides all showed improved half-life compared to native relaxin-2. Fc-relaxin polypeptides (exemplified by RELAX0019, RELAX0023, RELAX0034, RELAX0046, and RELAX 0117) in which relaxin a and B chains were linked to different heterodimeric Fc chains have improved PK properties compared to those in which the relaxin chains were linked to a linker (exemplified by RELAX0010 and RELAX 0009). However, since both linker-containing molecules RELAX0088 and RELAX0122 show good in vivo stability, the presence of the linker between relaxin A and B chains is not directly related to the rapid elimination of Fc-relaxin polypeptides in vivo.

It was unexpected in this study that the heterodimeric Fc-relaxin fusion polypeptides (RELAX 0019, RELAX0023, RELAX0034, RELAX0046, RELAX0117, RELAX0088 and RELAX 0122) all had significantly improved pharmacokinetic properties compared to the Fc-relaxin fusion polypeptides RELAX0010 and RELAX 0009.

Example 7: reversing established hypertrophy and fibrosis by RELAX0019 and RELAX0023

Isoprenaline (15 mg/kg/day) was infused via micropump into C57B6 mice for 10 days to induce cardiac hypertrophy and fibrosis. Mice infused with vehicle for the same duration were used as baseline controls. After 10 days, the micropump was removed and mice were given a new micropump containing r-relaxin-2 (500 ug/kg/day), or mice received the first of two weekly (QW) subcutaneous injections of RELAX0019 (20 mg/kg) or RELAX0023 (20 mg/kg). After a 14 day treatment period, mice were sacrificed and their hearts were collected for analysis of hypertrophy and fibrosis. Hearts from baseline control mice were collected after removal of the vehicle micropump. Hypertrophy is determined as a measure of cardiac weight relative to tibial length, and fibrosis is established by quantifying the collagen content relative to cardiac weight. In this model, infusion of isoproterenol significantly induced both hypertrophy and fibrosis. QW administration of RELAX0019 and RELAX0023 returned isoprenaline-induced hypertrophy to baseline levels as with constant infusion of r-relaxin-2. All relaxin treatments also reduced cardiac fibrosis by more than 50%. For each group, n=8. * P <0.01, p <0.001, p <0.0001

Recombinant relaxin Fc fusion proteins RELAX0019 and RELAX0023 were able to reverse hypertrophy and fibrosis in a similar manner to native h-relaxin-2 (fig. 6).

Example 8: nonspecific binding of Fc-relaxin-2 proteins was assessed using baculovirus ELISA.

RELAX protein was expressed in CHO cells and purified as described above. The baculovirus ELISA developed to evaluate non-specific binding of monoclonal antibodies (ref: hotzel et al, 2012mabs 4:6, 753-760) was adapted to determine non-specific binding of Fc-relaxin polypeptides with modifications, whereby instead of calculating the "BV score" (baculovirus plate absorbance/blank absorbance), non-specific binding was calculated as a signal to background (where background is the value obtained in the absence of Fc-relaxin polypeptide) for baculovirus plates and blanks, respectively. This measure was introduced to reflect the increased non-specific binding of some Fc peptides to coated and uncoated (blank) plates (compared to monoclonal antibodies). Formulations of each protein were prepared at 100nM or 10nM in PBS (Gibco company 14190-086) +0.5% BSA (Sigma company A9576) and used in duplicate for ELISA assays on 96-well Nunc Maxisorp F plates coated with 1% baculovirus extract in 50. Mu.L/well 50mM sodium carbonate (BV plates) or with 50mM sodium carbonate (blank plates) overnight at 4 ℃. After washing with PBS, the plates were blocked with 300 μl/well of pbs+0.5% bsa for 1 hour at room temperature and washed three times with PBS. 50. Mu.L/well of PBS+0.5% BSA (background) or RELAX protein dilutions were added and incubated for 1h at room temperature. After three washes in PBS, detection antibody (anti-human Fc-specific-HRP sigma A0170) diluted 1:5000 in PBS+0.5% BSA was added at 50. Mu.L/well. Samples were incubated for 1 hour at room temperature and plates were washed three times in PBS. HRP substrate-TMB (SureBlue RESERVE KPL-00-03) was then added at 50. Mu.L/well, and after a color change, the reaction was stopped by adding 50. Mu.L/well of 0.5M sulfuric acid. Absorbance was measured at 450nm and non-specific binding was determined for each sample. Nonspecific binding (fold binding relative to background) is defined as the ratio of nonspecific binding in the presence of Fc relaxin-2 protein and in the absence of Fc relaxin-2 protein (background). Data for Fc-relaxin-2 proteins tested at 2 different concentrations of 100nM or 10nM are shown in table 7.

Table 7: binding of Fc-relaxin fusion proteins in baculovirus ELISA at 100nM and 10nM (-001, 002, 003 refers to different batches of the same protein)

As shown in table 7 and fig. 7, the heterodimeric relaxin-2 Fc fusion polypeptides exhibited lower non-specific binding when the GS linker was used to attach the relaxin chain to the C-terminus. The localization of some asymmetric PA linkers, some point mutations and relaxin chains at the N-terminus, especially in the case of bivalent molecules (RELAX 0117), increases the non-specific binding to both blank and BV coated plates. At both high (100 nM) and low (10 nM) concentrations, some Fc-relaxin proteins with particularly high non-specific binding exhibited greater non-specific binding to the blank plate compared to BV-coated plates. Although the control molecules-bivalent RELAX0009, RELAX0010, RELAX0126, RELAX0127 and RELAX0128 all demonstrated high non-specific binding, neither the presence of the linker between the A and B chains of relaxin nor the bivalent itself driven high non-specific binding, as can be demonstrated by the low non-specific binding of RELAX 0122.

Example 9: stability in solution

The stability of RELAX0023 was evaluated using high performance size exclusion chromatography (HP-SEC) and liquid chromatography-mass spectrometry (LC-MS) and compared to RELAX0127 and RELAX 0128. HP-SEC, which detects absorbance at 280nm, can be used to measure purity, aggregation and fragmentation. The molecular buffer is exchanged into the optimized formulation composition and then concentrated up to 10mg/mL. All samples were placed under stress temperature conditions (40 ℃) for up to 4 weeks. At time points 1, 2 and 4 weeks, samples were collected and injected into size exclusion columns and eluted with aqueous flow equality at a fixed flow rate. Larger molecules are excluded to a greater extent from the pores of the size exclusion column than smaller molecules and therefore elute earlier. Peaks eluting earlier than the monomer peaks were recorded as aggregates. Peaks eluting after the monomer peak (excluding buffer related peaks) were recorded as fragments. The results are reported as percent purity, percent aggregate, and percent fragment, and are shown in fig. 8. RELAX0023 is the most stable molecule with a purity loss of only 0.1%/month, whereas RELAX0128 and RELAX0127 are 7.7% and 9.3%, respectively. Both RELAX0127 and RELAX0128 showed signs of aggregation, however, the aggregation level of RELAX0023 did not increase, indicating better physical solution stability. Fragmentation appears to be a major factor in loss of purity, with RELAX0127 having 6.6% fragmentation per month and RELAX0128 of 6.8%. RELAX0023 had a fragmentation rate of only 0.7%/month. Meanwhile, after 4 weeks of storage at 40 ℃, the total peak area of RELAX0128 decreased from 22403 to 18216 (19% decrease), while RELAX0127 decreased from 22225 to 18823 (15% decrease). This significant loss of total peak area and high fragmentation rate indicate that there may be a high degree of chemical degradation of both molecules. It should be noted that this loss of total area has a large impact on the chromatogram curves of the two molecules. This explains why RELAX0128 and RELAX0127 show a lower percentage of aggregates at 4 weeks compared to the previous time points, despite the apparent increase in aggregate peak area after storage. In contrast, the total peak area of RELAX0023 was only reduced by 0.03% from 21828 to 21761, indicating a better stability profile compared to RELAX0128 and RELAX 0127.

Fragmentation of the molecules was further verified by LC-MS using a reductive mass analysis, indicating an increase in intensity of the fragment peaks of RELAX0127 and RELAX0128 after storage at 40 ℃ (figure 9A). In contrast, the fragment peak of RELAX0023 remained unchanged after stress. Mass spectra under reducing conditions also showed modifications of RELAX0127 and RELAX0128 over time as evidenced by shift of the peak to larger mass and broadening of the peak, indicating greater heterogeneity (FIG. 9B). In contrast, the complete mass spectrum of RELAX0023 remained unchanged, indicating that no modification had occurred. This study showed that RELAX0023 has superior physical and chemical stability compared to RELAX0127 and RELAX 0128.

Example 10: PK profile of RELAX0023 in cynomolgus monkey

The Pharmacokinetic (PK) profile of RELAX0023 in cynomolgus monkeys was determined using an ELISA-based sandwich immunoassay. RELAX0023 was administered to a total of 12 female cynomolgus monkeys randomly assigned to 4 groups (3 animals/group). Animals SC in groups 1,2 and 3 were administered 0.1, 1 and 10mg/kg of RELAX0023, respectively. Animals in group 4 were given 10mg/kg RELAX0023 by IV bolus injection. Serum samples were collected 0.25 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 96 hours, 7 days, 14 days, and 21 days after drug administration.

Assay plates were coated with goat anti-human IgG antibodies and incubated with cynomolgus monkey serum from animals of groups 1-4. Plate-bound RELAX0023 was detected by an anti-relaxin antibody conjugated to HRP. Cynomolgus monkey serum was diluted 1:10 prior to addition to the plates. In 100% serum, the lower limit of quantitation was 0.010. Mu.g/mL, and the upper limit of quantitation was 0.300. Mu.g/mL.

Results and conclusions

Figure 10 shows the mean serum concentration versus time curve of RELAX0023 in cynomolgus monkeys after a single dose. RELAX0023 showed linear PK in the dose range of 0.01 to 10mg/kg after single dose administration of SC. A proportional increase in the dose of C _max was observed. The average C _max values were 0.400, 4.69, 34.8 μg/mL for the 0.1, 1 and 10mg/kg SC dose groups, respectively. A dose-proportional increase in the AUC _0-last value was also observed from the 0.1mg/kg to 10mg/kg SC group. The average AUC _0-last values were 2.01, 25.5, 193 μg days/mL for the 0.1, 1, and 10mg/kg SC dose groups, respectively. In general, RELAX0023 PK was linear in the range of 0.1mg/kg to 10mg/kg with an average CL/F of 51.0 mL/day/kg and an average t _1/2 of 3.07 days. SC bioavailability of RELAX0023 was estimated to be 88.2%.

Example 11: evaluation of long term efficacy of RELAX0023 in cynomolgus monkeys (cynomolgus macaques) suffering from heart failure and reduced Left Ventricular Ejection Fraction (LVEF)

The long term efficacy of RELAX0023 on cardiac function was evaluated in obese and aged cynomolgus monkeys (cynomolgus macaques). Cynomolgus monkey is chosen as the test species of choice, and not other lower mammalian species, as it is phylogenetically and physiologically closely related to humans. Elderly cynomolgus monkeys fed with a high fat diet for at least 2 years have the same risk factors as human patients predisposed to cardiovascular disease and develop metabolic syndrome that can progress characteristically to heart failure and reduced LVEF. The effect of RELAX0023 on LVEF was evaluated when administered at different dose levels by Subcutaneous (SC) injection for 20 weeks, with the first dose administered at week 1 of the study followed by an observation period of 18 weeks. Of a collection of approximately 100 obese and aged cynomolgus monkeys, aged 12-20 years and weighing 6-15kg, that had been fed a high fat diet for at least 2 years, 38 were identified by 2D echocardiography screening as having a LVEF of 30% -60%. Healthy monkeys of this age had a body weight of 5-8kg and a LVEF of 70% -75% and thus a LVEF of 60% or less represented the HFrEF model. The identified animals were selected and randomly assigned to 3 treatment groups of 8 monkeys each, and vehicle group 14 monkeys. The dosing period consisted of subcutaneous administration of RELAX0023 at 3 ascending dose levels (each below 10mg/kg; referred to as "low", "medium" and "high" doses, respectively) once a week.

Cardiac function measurements were determined 9 times by 2D echocardiography at baseline week-2 and at weeks 5, 9, 13, 17, 21, 25, 29, 33 of dosing and post dosing observation period. Further 2D echocardiography was scheduled at week 39 (end of study). Parameters including LVEF are based on the apex two-and four-chamber section and biplane approach. Parameters including Mean Arterial Pressure (MAP) and Heart Rate (HR) were measured using HDO (high definition oscillometric).

Results and conclusions

RELAX0023 was able to significantly improve LVEF at all RELAX0023 dose levels at weeks 5, 9, 13, 17 and 21 without affecting heart rate or blood pressure compared to vehicle controls (FIG. 12). A significant improvement in LVEF after treatment with RELAX0023 compared to week 0 (baseline) was observed throughout the elution period from the end of treatment to week 33 of the study. These dramatic results indicate a significant improvement in the hemodynamics of the treated animals and clearly demonstrate the efficacy of RELAX0023 in treating heart failure in this model. Furthermore, to the best of the inventors' knowledge, the extent of sustained response following treatment is largely unattainable by any other previously known compounds targeting this mechanism of action pathway. The monkeys were monitored continuously until week 39 of the study.

Example 12: phase 1 (Ph 1) study in healthy volunteers and heart failure patients

Study D8330C00001 is a phase Ia/b, randomized, single blind, placebo controlled, first human (FTIH) study (ClinicalTrials gov identifier NCT 04630067). The primary objective of this study was to assess the safety and tolerability of single and multiple escalated doses of RELAX0023 (also known as "AZD 3427"), and the secondary objective was to assess the (i) Pharmacokinetic (PK) and (ii) immunogenicity of single and multiple escalated doses of AZD 3427.

The study was performed in 2 parts (part a and part B). Part a is a single escalation dose (SAD) study with healthy participants (non-fertility men and women) and part B is a multiple escalation dose (MAD) study with HF participants (non-fertility men and women).

Part B included 48 patients in 6 cohorts (8 participants per cohort). Of these, 3 consisted of HFrEF participants (queues 1b, 3b and 5 b), and 3 consisted of HF participants with EF+.41% (queues 2b, 4b and 6 b). The dosage levels for HFrEF and HF cohorts with EF+.gtoreq.41% were 5mg (cohorts 1b, 2 b), 15mg (cohorts 3b, 4 b) and 45mg (cohorts 5b, 6 b), administered once a week (QW) for 5 weeks (i.e., a total of 5 doses).

For part B of 48 patients including 6 cohorts, inclusion criteria included: (i) all queues: known to be clinically diagnosed as phase C HF (NYHA class I to III) and to receive stable drug therapy for at least 12 weeks prior to screening, during which there is no significant dose change or no new drug added, (ii) cohorts 1b, 3b, 5b: patients diagnosed with HFrEF (defined as EF+.ltoreq.40%) (iii) cohorts 2b, 4b, 6b: patients diagnosed with HF with ef≡41% (including patients diagnosed with HFpEF (defined as ef+.50%)), (iv) all cohorts: BMI is between 18 and 40kg/m ² (inclusive) and body weight is at least 55kg and not more than 120kg (inclusive), and (v) all queues: NT-proBNP >125pg/mL or BNP >35pg/mL were previously recorded.

Example 13: results of Ph1 AZD3427 MAD study in HF patients

In part B MAD cohort, data from HFpEF and HFrEF patients were pooled. Trends indicate that AZD3427 improved cardiac function, including improved cardiac output and Stroke Volume (SV), reduced Systemic Vascular Resistance (SVR), and improved organ perfusion (SVR and evfr) (fig. 13A-6F).

Although the hypertensive status of subjects in the MAD cohort has not been determined, the observed improvement in cardiac output and Stroke Volume (SV), reduction in Systemic Vascular Resistance (SVR), increase in estimated glomerular filtration rate (evfr), and thus improvement in organ perfusion (SVR and evfr) are expected to be beneficial for hf+ph patients.

Example 14: ph2b study-random, placebo-controlled, multicenter, dose-range study of AZD3427 in participants with heart failure due to left heart disease combined with pulmonary arterial hypertension (world health organization [ WHO ], group 2).

This study (study ID number: D8330C 00003) was intended to assess the ability of AZD3427 to reduce Pulmonary Vascular Resistance (PVR) 24 weeks after treatment in participants with Heart Failure (HF) combined with 2 groups of pulmonary arterial hypertension (PH).

Approximately 220 participants were randomly assigned to 4 treatment groups (at a ratio of 1:1:1), receiving Subcutaneous (SC) injections of AZD3427 or placebo every 2 weeks for 24 weeks. This study will evaluate 3 dose levels of AZD 3427: dose a, dose B and dose C. Dose adjustment was not applicable to this study. The study will be conducted in about 60 study centers in 15 countries estimated. The study will include approximately 16 study visits: 2 visits during the screening period, 13 visits during the treatment period, and one visit during the follow-up period. The expected total duration of the study is 32 to 37 weeks, depending on the length of the screening period.

The participants will receive a single subcutaneous dose of AZD3427 (dose A, B or C) or placebo every 2 weeks from day 1 to 155 for 24 weeks.

The primary outcome measure would be the change in Pulmonary Vascular Resistance (PVR) from baseline after 24 weeks of treatment. The effect of AZD3427 on PVR parameters compared to placebo will also be assessed as measured by Right Heart Catheterization (RHC) after 24 weeks of treatment in participants with HF and group 2 PH.

The secondary outcome measures include:

Changes in mean pulmonary arterial pressure (mPAP) from baseline

Changes in Pulmonary Artery Wedge Pressure (PAWP) from baseline

Changes in cardiac output from baseline

Changes in Stroke Volume (SV) from baseline

Changes in Ejection Fraction (EF) from baseline

Changes in Left Ventricular Global Longitudinal Strain (LVGLS) from baseline

Changes in Pulmonary Arterial Systolic Pressure (PASP) from baseline

Changes in right ventricular/left ventricular (RV/LV) ratio from baseline

Changes in right ventricular outflow tract acceleration time (RVOT AT) from baseline

Changes in Tricuspid Regurgitation Velocity (TRV) from baseline

TAPSE/PASP [ tricuspid annulus plane systolic deviation/pulmonary systolic pressure ] changes from baseline

Changes in right ventricular strain/pulmonary arterial systolic pressure (RVS/PASP) from baseline

Changes in Inferior Vena Cava (IVC) diameter with inspiratory collapse relative to baseline

Changes in systemic vascular resistance from baseline

Change in 6 minute walk distance (6 MWD) from baseline

Changes in the total symptom score (KCCQ TSS) of the Kansas City cardiomyopathy questionnaire from baseline

Changes in New York Heart Association functional Classification (NYHA FC) from baseline

Serum creatinine changes from baseline

Variations of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) from baseline

Changes in cystatin C from baseline

Changes in eGFR (estimated glomerular filtration rate) from baseline

Inclusion criteria: 1. participants must be more than or equal to 18 years old (inclusive). 2. According to the guidelines of the european society of cardiology/respiratory society (ESC/ESR) for pulmonary hypertension (PH-LHD) caused by left heart disease in 2022, participants must be pre-diagnosed with HF, NYHA functional grade (FC) from grade II to IV, and pre-diagnosed with PH-LHD or pulmonary hypertension caused by left heart disease with a high or moderate probability. Participants must receive stable HF standard-of-care medications, including diuretics. 3. According to the 2022ESC/ERS guidelines, the participants must have a combination of echocardiographic parameters that show a moderate or high probability of suffering from PH. 4. At the screening visit 2, participants must have elevated pulmonary artery pressure in the study in the RHC according to the RHC manual provided by the sponsor: (a) PAWP is not less than 15mmHg (b) mPAP is not less than 20mmHg 5 and the minimum weight is 50kg (inclusive). 6. Can sign informed consent.

Exclusion criteria 1. PH was diagnosed as World Health Organization (WHO) group 1, WHO 3, WHO 4, or WHO 5. 2. There is historical or current evidence of a clinically significant disease or disorder. 3. Decompensated HF or any hospitalization. 4. There is any contraindication of RHC. 5. There is a history of hypersensitivity to SC injections or devices. 6. There is a history of hypersensitivity reactions to drugs having a chemical structure or class similar to AZD3427 or any component of the AZD3427 drug product, or clinically significant allergies/hypersensitivity reactions are occurring. 7. Lung diseases with forced expiratory volume/vital capacity (FEV 1/VC) <30% of the first seconds are known. 8. Congenital long QT syndrome. 9. Cardiac ventricular arrhythmias in need of treatment. Participants with atrial fibrillation or flutter and controlled ventricular rate are permitted. 10. There is a history of or an expectation of heart transplantation or ventricular assist device implantation. 11. Any known planned (predetermined) highly invasive Cardiovascular (CV) procedure (e.g., coronary revascularization, atrial fibrillation/atrial flutter ablation, valve repair/replacement, aortic aneurysm surgery, etc.). 12. Participants who had previously received AZD 3427.

Claims

1. A method of treating a subject having heart failure with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

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

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

4. The method according to any one of the preceding claims, wherein the relaxin a chain of the heterodimeric fusion is a relaxin-2A chain and the relaxin B chain of the heterodimeric fusion is a relaxin-2B chain.

5. The method according to any one of the preceding claims, wherein the relaxin a-chain of the heterodimeric fusion is linked to the first heterodimerization domain via a linker and the relaxin B-chain of the heterodimeric fusion is linked to the second heterodimerization domain via a linker, optionally wherein one or preferably both linkers are polypeptides.

6. The method according to claim 5, wherein one or preferably both linkers of the heterodimeric fusion are 6 to 40 amino acids in length, e.g. one or preferably both linkers are 21 amino acids in length.

7. The method according to any one of the preceding claims, wherein the first and second heterodimerization domains of the heterodimeric fusion are derived from an immunoglobulin Fc region (a "first Fc region" and a "second Fc region", respectively), optionally wherein the first and second Fc regions comprise constant domains CH2 and CH3.

8. The method of claim 7, wherein the C-terminus of the first Fc region is linked to the N-terminus of the relaxin a chain and the C-terminus of the second Fc region is linked to the N-terminus of the relaxin B chain.

9. The method according to claim 7 or 8, wherein the first and second Fc regions comprise amino acid mutations and/or modifications that promote heterodimerization, optionally wherein the amino acid mutations that promote heterodimerization are "Fc pestle" and "Fc mortar" mutations, such as the "Fc pestle" and "Fc mortar" mutations present in the CH3 domain.

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

11. The method according to claim 10, wherein the amino acid mutations that promote heterodimerization comprise:

a. The "Fc mortar" mutations Y349C, T366S, L a and Y407V in one CH3 domain; and

B. the "Fc pestle" mutations S354C and T366W in the other CH3 domain,

Wherein the amino acid numbering is according to the EU index as in Kabat.

12. The method according to claim 11, wherein:

a. The first Fc region comprises an "Fc pestle" mutation and the second Fc region comprises an "Fc mortar" mutation; or (b)

B. the second Fc region comprises an "Fc pestle" mutation and the first Fc region comprises an "Fc pestle" mutation.

13. The method according to any one of claims 10 to 12, wherein the first and/or second Fc region comprises the amino acid mutations L234F, L E and P331S, wherein the amino acid numbering is according to the EU index as in Kabat.

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

15. The method of claim 14, wherein the relaxin-2A chain polypeptide of the heterodimeric fusion comprises the amino acid mutation K9H, K M or K17I.

16. The method according to any one of claims 5 to 15, wherein both linkers of the heterodimeric fusion have the sequence GGGGSGGGGSGGGGSGGGGGS [ SEQ ID NO:5].

17. A method of treating a subject having heart failure with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

(i) FcX-con-a fusion polypeptides; and

(Ii) FcY-con-B fusion polypeptide,

Wherein:

FcY is an Fc region comprising the constant domains CH2 and CH3 of a human IgG1 immunoglobulin and comprises "Fc mortar" amino acid mutations and/or modifications, preferably amino acid mutations Y349C:T366S:L368A:Y407V;

FcX is an Fc region having an "Fc pestle" amino acid mutation and/or modification, preferably comprising constant domains CH2 and CH3 of a human IgG1 immunoglobulin, and comprising an "Fc pestle" amino acid mutation and/or modification, preferably amino acid mutation S354C: T366W; and

Con is a linker polypeptide, preferably having the sequence GGGGSGGGGSGGGGSGGGGGS [ SEQ ID NO:5],

18. The method according to any of the preceding claims, wherein the heterodimeric fusion comprises a fusion polypeptide having the amino acid sequence of SEQ ID No. 11 and a fusion polypeptide having the amino acid sequence of SEQ ID No. 20.

19. The method according to any one of claims 8 to 18, wherein the heterodimeric fusion further comprises one or more fabs, optionally wherein the heterodimeric fusion comprises one Fab linked to the N-terminus of the first Fc region and a second Fab linked to the N-terminus of the second Fc region.

20. The method of any one of claims 8-19, wherein the heterodimeric fusion further comprises a second relaxin a chain polypeptide or variant thereof linked to the N-terminus of the first Fc region and a second relaxin B chain polypeptide or variant thereof linked to the N-terminus of the second Fc region, optionally wherein the second relaxin a chain is linked to the first Fc region via a linker polypeptide and the second relaxin B chain is linked to the second Fc region via a linker polypeptide.

21. A method of treating a subject having heart failure with pulmonary hypertension, the method comprising administering to the subject an effective amount of a heterodimeric fusion comprising:

(i) FcX-B-L-A and FcY, optionally FcY-B-L-A Or (b)

(Ii) FcY-B-L-A and FcX, optionally FcX-B-L-A

Wherein:

FcY is an immunoglobulin Fc region having an "Fc mortar" amino acid mutation and/or modification, preferably comprising a CH3 domain having the amino acid mutation Y349C: T366S: L368A: Y407V;

FcX is an immunoglobulin Fc region having an "Fc pestle" amino acid mutation and/or modification, preferably comprising a CH3 domain having the amino acid mutation S354C: T366W;

b is a relaxin B chain or variant thereof, e.g., a relaxin 2B chain or variant thereof;

a is a relaxin a chain or variant thereof, e.g., a relaxin 2A chain or variant thereof; and

22. The method according to claim 21, wherein the relaxin B chain of the heterodimeric fusion is linked to FcX and/or FcY via a linker, optionally a linker polypeptide of 6 to 40 amino acids in length, e.g. 21 amino acids in length.

23. The method according to any one of the preceding claims, wherein the ratio of relaxin activity of the heterodimeric fusion to relaxin activity of the reference relaxin protein is about 0.001 to about 10.

24. The method according to any one of the preceding claims, wherein the heart failure is heart failure with a reduced ejection fraction, heart failure with an intermediate ejection fraction or heart failure with a retained ejection fraction.

25. The method according to any one of the preceding claims, wherein the subject has an average pulmonary artery pressure of about 25mmHg or greater, a pulmonary artery wedge pressure (panp) of greater than 15mmHg and/or a right ventricular systolic pressure of about 40mmHg or greater.

26. The method according to any one of the preceding claims, wherein the subject has a pulmonary vascular resistance of less than 3.0 wood units.

27. The method according to any one of claims 1-25, wherein the subject has pulmonary vascular resistance of 3.0 or more wood units.

28. The method according to any of the preceding claims, wherein the subject is equipped with a blood pressure monitoring device, preferably a pulmonary artery pressure monitoring device.

29. The method of claim 28, wherein the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device.

30. The method according to any one of the preceding claims, wherein the heterodimeric fusion is administered as a pharmaceutical composition comprising the heterodimeric fusion and a pharmaceutically acceptable excipient.

31. The method according to any one of the preceding claims, wherein the heterodimeric fusion or pharmaceutical composition is administered to the subject by subcutaneous injection.

32. The method according to any one of the preceding claims, wherein the heterodimeric fusion or pharmaceutical composition is administered by self-administration.

33. The method according to any one of the preceding claims, wherein administration of the heterodimeric fusion or pharmaceutical composition achieves one or more of the following:

a) PVR reduction;

(b) mPAP decrease;

(c) Decreased ePAD;

(d) Increased Stroke Volume (SV) of the heart;

(e) Systemic Vascular Resistance (SVR) decreases and/or glomerular filtration rate (evfr) increases is estimated;

(f) An increase in ejection fraction; and/or

(G) Increased cardiac output;

compared to baseline levels prior to administration.