Pharmaceutical formulations comprising heterodimer relaxin fusion proteins and uses thereof

Abstract

The present disclosure relates to pharmaceutical formulations comprising heterodimeric relaxin fusion polypeptides, particularly heterodimeric relaxin 2 fusion polypeptides, and uses thereof. Thus, the present disclosure provides pharmaceutical formulations comprising relaxin fusion polypeptides and kits comprising the pharmaceutical formulations as well as uses thereof, including methods of treatment. The pharmaceutical formulations of the present disclosure are particularly useful in the treatment of cardiovascular diseases, such as in the treatment of heart failure.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of European patent application No. 23160008.1 filed on March 3, 2023, the contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is hereby incorporated by reference in its entirety. The sequence listing was created on February 16, 2024, is named 201258-WO-PCT Sequence listing.xml, and is 82,163 bytes in size. Technical Field

[0005] The present disclosure relates to the field of pharmaceutical preparations. Specifically, a stable pharmaceutical preparation of a peptide-Fc fusion protein is provided. Background Art

[0006] Biotherapeutics are a class of drugs in which the active ingredient is derived from a biological source. Thus, biotherapeutics include macromolecular therapeutics (such as proteins, antibodies, peptides, and nucleic acids) as well as cell-based therapeutics. Due to their high specificity for their target and superior safety profile compared to small molecule therapeutics, biotherapeutics offer healthcare practitioners an effective and safe alternative to treat a wide range of diseases and conditions. Consequently, biotherapeutics represent a rapidly growing segment of therapeutic agents available to healthcare practitioners for the treatment of a wide range of diseases and conditions.

[0007] However, the structural complexity and size of biotherapeutics make them prone to instability in formulations. Biotherapeutics are susceptible to physical and / or chemical degradation, which not only leads to reduced efficacy and drug product shelf life but also causes safety issues. Chemical degradation can encompass deamination, isomerization, oxidation, hydrolysis, and glycation, while physical degradation can include aggregation, particle formation, precipitation, surface adsorption, and denaturation. Both forms of degradation can negatively impact both the efficacy and safety of biotherapeutics.

[0008] Well-designed manufacturing and purification methods can often produce highly pure products. However, many biotherapeutics can still degrade over time during storage, transport, and administration. In some cases, the stability of a biotherapeutic is inherent to its molecular sequence. In other cases, exogenous factors, such as host cell proteins co-purified with the target biotherapeutic or impurities from some excipients, can act as catalysts to trigger chemical or physical degradation.

[0009] Relaxin is a peptide hormone belonging to the insulin superfamily. In humans, the relaxin peptide family comprises seven peptides that are structurally highly similar but have low sequence similarity: relaxin-1, relaxin-2, relaxin-3, and the insulin-like peptides INSL3, INSL4, INSL5, INSL6. Naturally occurring relaxin is composed of A- and B-chain polypeptides covalently linked by two inter-chain disulfide bonds. The A-chain has an additional intra-chain disulfide bond. In females, relaxin-2 expression peaks during pregnancy and relaxin is believed to play a role in placental development and fetal implantation. However, relaxin has also been found to have anti-fibrotic properties. A heterodimeric fusion, such as HFUS1 (also known as RELAX0023), a recombinant fusion protein consisting of the Fc portion of human IgG1 linked to human relaxin-2, has been shown to retain the anti-fibrotic effects of human relaxin and has thus been shown to exhibit efficacy as a heart failure therapy, as described in WO 2021 / 255127.

[0010] There remains a need to develop formulations of these heterodimeric fusion proteins with relaxin activity, such as HFUS1, to meet the shelf-life requirements of their drug products. SUMMARY

[0011] During formulation development of the heterodimeric fusions described herein, several challenges were encountered related to the shelf-life and stability of the molecules. These challenges included a tendency to self-associate leading to aggregation; a tendency for amino acid clipping and very easy particle formation in certain pH ranges.

[0012] Accordingly, it is an object of the present disclosure to provide stable formulations of heterodimeric fusions with relaxin activity, such as HFUS1, to meet the shelf-life requirements of their drug products and address one or more of the challenges described above.

[0013] Accordingly, the present disclosure relates to pharmaceutical formulations of heterodimeric fusions with relaxin activity, such as HFUS1.

[0014] In one aspect, there is provided a pharmaceutical formulation comprising a heterodimeric fusion and a lipase-resistant surfactant, wherein the heterodimeric fusion comprises:

[0015] (i) a first heterodimerization domain linked to at least one relaxin A-chain polypeptide or variant thereof; and

[0016] (ii) a second heterodimerization domain linked to at least one relaxin B-chain polypeptide or variant thereof,

[0017] wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and wherein the heterodimeric fusion has relaxin activity.

[0018] In some embodiments, the relaxin A chain and the relaxin B chain are covalently bound by one or more (e.g., two) interchain linkages, optionally 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.

[0019] In some embodiments, the relaxin A chain is a relaxin-2 A chain and the relaxin B chain is a relaxin-2 B chain.

[0020] In particular embodiments, the first and second heterodimerization domains are derived from an immunoglobulin Fc region, e.g., an immunoglobulin G (IgG) Fc region (“first Fc region” and “second Fc region”). The first and second Fc regions can comprise constant domains CH2 and / or CH3. In particular embodiments, the first and second Fc regions comprise CH2 and CH3.

[0021] In alternative embodiments, the first and second heterodimerization domains are derived from an immunoglobulin Fab region.

[0022] In further alternative embodiments, the first and second heterodimerization domains heterodimerize to form a parallel coiled coil.

[0023] In some embodiments, the relaxin A chain is linked to the first heterodimerization domain (e.g., first Fc region) via a linker, and the relaxin B chain is linked to the second heterodimerization domain (e.g., second Fc region) via a linker. In particular embodiments, one or both linkers is a polypeptide.

[0024] In some embodiments, at least one linker is a polypeptide between 6 and 40 amino acids in length. In particular embodiments, both linkers are polypeptides between 6 and 40 amino acids in length. In particular embodiments, at least one linker is a polypeptide 21 amino acids in length. In particular embodiments, both linkers are polypeptides 21 amino acids in length. In certain embodiments, both linkers have the amino acid sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5].

[0025] In particular embodiments, the C-terminus of the first heterodimerization domain (e.g., first Fc region) is linked to the N-terminus of the relaxin A chain, and the C-terminus of the second heterodimerization domain (e.g., second Fc region) is linked to the N-terminus of the relaxin B chain. In alternative embodiments, the N-terminus of the first heterodimerization domain (e.g., first Fc region) is linked to the C-terminus of the relaxin A chain, and the N-terminus of the second heterodimerization domain (e.g., second Fc region) is linked to the C-terminus of the relaxin B chain.

[0026] 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, which can be asymmetric amino acid mutations and / or modifications that promote heterodimerization. In particular embodiments, the amino acid mutations that promote heterodimerization are “Fc knob” and “Fc hole” mutations. In specific embodiments, the “Fc knob” and “Fc hole” mutations are present in the CH3 domains. In some embodiments, the first and second Fc regions are derived from a human IgGl immunoglobulin, optionally wherein the C-terminal lysine (K447, according to the EU index as in Kabat) can be absent in the CH3 domain of the first and / or second Fc region. In particular embodiments, the first Fc region comprises the “Fc knob” mutations and the second Fc region comprises the “Fc hole” mutations. Alternatively, the first Fc region has the “Fc hole” mutations and the second Fc region has the “Fc knob” mutations. In particular embodiments, the amino acid mutations that promote heterodimerization comprise the “Fc hole” mutations Y349C, T366S, L368A, and Y407V, or conservative substitutions thereof, in one CH3 domain; and the “Fc knob” mutations S354C and T366W, or conservative substitutions thereof, in the other CH3 domain, wherein the amino acid numbering is according to the EU index in Kabat. In particular embodiments, the first and / or second Fc region comprises the amino acid mutations L234F, L235E, and P331S, wherein the amino acid numbering is according to the EU index in Kabat.

[0027] In embodiments of any aspect of the disclosure, the relaxin-2 A-chain polypeptide comprises the sequence set forth in SEQ ID NO: 1 or a variant thereof, and the relaxin-2 B-chain polypeptide comprises the sequence set forth in SEQ ID NO: 2 or a variant thereof. In some embodiments, the relaxin-2 A-chain polypeptide comprises the amino acid mutation K9H, K17M, or K17I.

[0028] In some embodiments, both linkers have the amino acid sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5].

[0029] The present disclosure also provides a pharmaceutical formulation comprising a heterodimeric fusion and a fat-tolerant surfactant, wherein the heterodimeric fusion comprises:

[0030] (i) an FcX-con-A fusion polypeptide; and

[0031] (ii) an FcY-con-B fusion polypeptide, wherein:

[0032] A is a relaxin A chain or variant thereof, e.g., relaxin-2 A chain or variant thereof;

[0033] B is a relaxin B chain or variant thereof, e.g., relaxin-2 B chain or variant thereof;

[0034] FcY is an immunoglobulin (e.g., IgGl) Fc region having "Fc knob" amino acid mutations and / or modifications, optionally comprising a CH3 domain having amino acid mutations S354C:T366W or conservative substitutions thereof;

[0035] FcX is an immunoglobulin (e.g., IgGl) Fc region having "Fc knob" amino acid mutations and / or modifications, optionally comprising a CH3 domain having amino acid mutations S354C:T366W or conservative substitutions thereof; and

[0036] con is a linker, e.g., a linker polypeptide, such as the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5],

[0037] wherein the amino acid numbering is according to the EU index in Kabat, wherein FcX heterodimerizes with FcY, and wherein the heterodimeric fusion has relaxin activity.

[0038] In particular embodiments, the heterodimeric fusion comprises or consists of 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.

[0039] In some embodiments of any aspect of the disclosure, 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 heterodimerization domain (e.g., first Fc region) and a second Fab linked to the N-terminus of the second heterodimerization domain (e.g., second Fc region).

[0040] In some embodiments of any aspect of the disclosure, the heterodimeric fusion further comprises a second relaxin A chain polypeptide or variant thereof linked to the N-terminus of the first heterodimerization domain (e.g., first Fc region) and a second relaxin B chain polypeptide or variant thereof linked to the N-terminus of the second heterodimerization domain (e.g., second Fc region), optionally wherein the second relaxin A chain is linked to the first heterodimerization domain (e.g., first Fc region) via a linker polypeptide and the second relaxin B chain is linked to the second heterodimerization domain (e.g., second Fc region) via a linker polypeptide.

[0041] The present disclosure also provides a pharmaceutical formulation comprising a heterodimeric fusion and a fatase-tolerant surfactant, wherein the heterodimeric fusion comprises:

[0042] (i) FcX-B-L-A and FcY, optionally FcY-B-L-A; or

[0043] (ii) FcY-B-L-A and FcX, optionally FcX-B-L-A;

[0044] wherein:

[0045] FcY is an immunoglobulin (e.g., IgGl) Fc region having an “Fc Wrench” amino acid mutation and / or modification, optionally comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V or conservative substitutions thereof;

[0046] FcX is an immunoglobulin (e.g., IgGl) Fc region having an “Fc Knob” amino acid mutation and / or modification, optionally comprising a CH3 domain having the amino acid mutations S354C:T366W or conservative substitutions thereof;

[0047] B is a relaxin B chain or variant thereof, e.g., a relaxin-2 B chain or variant thereof;

[0048] A is a relaxin A chain or variant thereof, e.g., a relaxin-2 A chain or variant thereof; and

[0049] L is a linker polypeptide, optionally having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60], wherein the amino acid numbering is according to the EU index in Kabat, wherein FcX heterodimerizes with FcY, and wherein the heterodimeric fusion protein 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, which is optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

[0050] The present disclosure also provides a pharmaceutical formulation comprising a heterodimeric fusion and a fatase-tolerant surfactant, wherein the heterodimeric fusion comprises:

[0051] (i) FcX-A-L-B and FcY, optionally FcY-A-L-B; or

[0052] (ii) FcY-A-L-B and FcX, optionally FcX-A-L-B;

[0053] wherein:

[0054] FcY is an immunoglobulin (e.g., IgGl) Fc region having an “Fc knob” amino acid mutation and / or modification, optionally comprising a CH3 domain having the amino acid mutations S354C:T366W, or conservative substitutions thereof;

[0055] FcX is an immunoglobulin (e.g., IgGl) Fc region having an “Fc knob” amino acid mutation and / or modification, optionally comprising a CH3 domain having the amino acid mutations S354C:T366W, or conservative substitutions thereof;

[0056] A is a relaxin A chain or variant thereof, e.g., a relaxin-2 A chain or variant thereof;

[0057] B is a relaxin B chain or variant thereof, e.g., a relaxin-2 B chain or variant thereof; and

[0058] L is a linker polypeptide, optionally having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60], wherein the amino acid numbering is according to the EU index in Kabat, wherein FcX heterodimerizes with FcY, and wherein the heterodimeric fusion protein 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, which is optionally a linker polypeptide of 6 to 40 amino acids in length, e.g., 21 amino acids in length.

[0059] In some embodiments of any aspect of the disclosure, the ratio of the relaxin activity of the heterodimeric fusion relative to the relaxin activity of a reference relaxin protein is about 0.001 to about 10.

[0060] In some embodiments of any aspect of the disclosure, the formulation comprises less than about 10,000, about 6000, about 5,000, about 1,000, about 750, about 600, about 500, about 250, about 150, about 100, or about 50 / mL of particles greater than 2 pm, 5 pm, 10 pm, 15 pm, 20 pm, or 25 pm in diameter.

[0061] In some embodiments of any aspect of the disclosure, the concentration of the lipase-resistant surfactant is 0.001% (w / v) to 1% (w / v). In certain embodiments, the concentration of the lipase-resistant surfactant is 0.005% (w / v) to 0.2% (w / v). In certain embodiments, the concentration of the lipase-resistant surfactant is 0.02% (w / v) to 0.06% (w / v). In specific embodiments, the concentration of the lipase-resistant surfactant is 0.04% (w / v).

[0062] In some embodiments of any aspect of the disclosure, the lipase-resistant surfactant is not enzymatically hydrolysable by lipoprotein lipase (LPL), lipase 9, phospholipase 2, or phospholipase 2A. In particular embodiments, the lipase-resistant surfactant is not enzymatically hydrolysable by LPL enzyme. In some embodiments, the lipase-resistant surfactant does not comprise an ester linkage that is enzymatically hydrolysable by lipoprotein lipase, lipase 9, phospholipase 2, or phospholipase 2A. In some embodiments, the lipase-resistant surfactant does not comprise an ester linkage that is enzymatically hydrolysable by lipoprotein lipase. In some embodiments, the lipase-resistant surfactant is a water-soluble non-ionic triblock copolymer formed from polyethylene oxide (PEO) and polypropylene oxide (PPO) blocks. In particular embodiments, the water-soluble non-ionic triblock copolymer is poloxamer 188 (P188). Alternatively, the lipase-resistant surfactant is D-a-tocopheryl polyethylene glycol succinate (TPGS). In further embodiments, the lipase-resistant surfactant is selected from P188, TPGS, Kolliphor HS15, Kolliphor EL, Kolliphor RH40, PEG 300, PEG 400, Brij 58, and Brij 35.

[0063] In some embodiments of any aspect of the disclosure, the formulation further comprises a buffer having a pH of about 3 to about 10, optionally about 5.5 to about 7.5. In particular embodiments, the formulation has a pH in the range of 6 to 7. In particular embodiments, the formulation has a pH of 6.5.

[0064] In some embodiments of any aspect of the disclosure, the concentration of the buffer is 0.1 mM to 100 mM, such as 5 mM, 10 mM, 15 nM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. In particular embodiments, the concentration of the buffer is 10 mM to 30 mM. In particular embodiments, the concentration of the buffer is 20 mM.

[0065] In some embodiments, the buffering agent is selected from acetate, acetic acid, succinate, succinic acid, phosphate, phosphoric acid, ascorbate, ascorbic acid, lactate, lactic acid, tartaric acid, maleic acid, glycine, gluconate, citrate, histidine, imidazole, bicarbonate and carbonic acid, sodium benzoate, benzoic acid, edetate, malate, tris, glycinylglycine, and mixtures thereof. In particular embodiments, the buffering agent is selected from citrate buffer and histidine buffer. In particular embodiments, the buffering agent is histidine, histidine hydrochloride, or a histidine / histidine hydrochloride buffer. In particular embodiments, the buffering agent is a histidine / histidine hydrochloride buffer (i.e., a combination of histidine and histidine hydrochloride). In one embodiment, the buffering agent is L-histidine / L-histidine hydrochloride monohydrate.

[0066] In some embodiments of any aspect of the disclosure, the formulation additionally comprises an excipient, optionally wherein the excipient is an ionic excipient. In some embodiments, the concentration of the excipient is 100 mM to 300 mM. In particular embodiments, the concentration of the excipient is 140 mM to 240 mM, e.g., 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, or 240 mM. In particular embodiments, the concentration of the excipient is 190 mM. Ionic excipients for use in the formulations described herein include salts and charged amino acids. The ionic excipient can comprise a combination of salts and charged amino acids. Exemplary charged amino acids include arginine and lysine. Exemplary salts include chloride, succinate, acetate, and sulfate, as well as carbonate, gluconate, lactate, and malate. In particular embodiments, the ionic excipient is a charged amino acid hydrochloride (HC1).

[0067] In some embodiments, the excipient is an ionic excipient selected from arginine salts or lysine salts. In some embodiments, the ionic excipient is selected from arginine HC1 or lysine HC1. In particular embodiments, the ionic excipient is arginine HC1. It is also understood that the buffering agent itself can be an ionic excipient as described herein. Thus, in some embodiments, the buffering agent is an ionic excipient.

[0068] In some embodiments of any aspect of the disclosure, the formulation further comprises a sugar, optionally wherein the sugar is sucrose. Other sugars that can be used include, but are not limited to, trehalose, lactose, mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, polyols such as trihydric or higher sugar alcohols (e.g., glycerol, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol), glucose, maltose, maltulose, isomaltulose, lactulose, and cyclodextrins.

[0069] In some embodiments of any aspect of the disclosure, the concentration of the heterodimeric fusion is 0.1 mg / mL to 100 mg / mL, optionally 0.2 mg / mL to 50 mg / mL, optionally 1 mg / mL to 30 mg / mL. In some embodiments, the formulation comprises 0.2 mg / mL - 50 mg / mL of the heterodimeric fusion, 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5. In some embodiments, the formulation comprises 50 mg / mL of the heterodimeric fusion. In other embodiments, the formulation comprises 30 mg / mL of the heterodimeric fusion. In other embodiments, the formulation comprises 33 mg / mL of the heterodimeric fusion. In other embodiments, the formulation comprises 5 mg / mL of the heterodimeric fusion. In other embodiments, the formulation comprises 1 mg / mL of the heterodimeric fusion. In other embodiments, the formulation comprises 1.1 mg / mL of the heterodimeric fusion. In some embodiments, the heterodimeric fusion comprises or consists of 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.

[0070] In one aspect, the disclosure also provides a pharmaceutical formulation as described herein for use in therapy. The disclosure also provides a method of treating a subject having a disease or disorder, the method comprising administering to the subject a pharmaceutical formulation as described herein.

[0071] In one aspect, the disclosure also provides a pharmaceutical formulation as described herein for use in the treatment of a subject having heart failure, including heart failure with pulmonary arterial hypertension (e.g., type 2 pulmonary arterial hypertension). The disclosure also provides a method of treating a subject having heart failure, including heart failure with pulmonary arterial hypertension (e.g., type 2 pulmonary arterial hypertension), the method comprising administering to the subject a pharmaceutical formulation as described herein. In some embodiments, the heart failure is heart failure with reduced ejection fraction, heart failure with mid-range ejection fraction, or heart failure with preserved ejection fraction. In some embodiments, the subject has a mean pulmonary arterial pressure of about 25 mmHg or greater, a pulmonary arterial wedge pressure (PAWP) higher than 15 mmHg, and / or a right ventricular systolic pressure of about 40 mmHg or greater. In some embodiments, the subject has a blood pressure monitoring device installed, which can be a pulmonary arterial pressure monitoring device. In some embodiments, the pulmonary arterial pressure monitoring device is a CardioMEMS pressure monitoring device. In some embodiments, the subject has a pulmonary vascular resistance of less than 3.0 wood units. In other embodiments, the subject has a pulmonary vascular resistance of 3.0 or greater wood units.

[0072] In some embodiments, the fusion polypeptide or pharmaceutical formulation is suitable for subcutaneous injection and / or administration to a subject by subcutaneous injection. In some embodiments, the fusion polypeptide or pharmaceutical formulation is suitable for self-administration and / or administration by self-administration.

[0073] In some embodiments, administration of the pharmaceutical formulation results in one or more of the following: a decrease in PVR; a decrease in mPAP; a decrease in ePAD; an increase in stroke volume (SV) of the heart; a decrease in systemic vascular resistance (SVR) and / or an increase in estimated glomerular filtration rate (eGFR); an increase in ejection fraction; and / or an increase in cardiac output, as compared to baseline levels prior to administration.

[0074] In one aspect, the present disclosure also provides a kit comprising a pharmaceutical formulation as described herein.

[0075] Aspects and embodiments of the present disclosure are set forth in the appended claims. These and other aspects and embodiments of the present disclosure are also described herein. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 shows purity loss of 50 mg / mL (A) and 10 mg / mL (B) HFUS1 after 3 months, 3 months, and 1 month storage at 5°C, 25°C, and 40°C, respectively.

[0077] Figure 2 Figure 3 shows the unfolding temperature curve of HFUS1 measured by differential scanning calorimetry thermogram.

[0078] Figure 3 Figure 4 shows the self-diffusion coefficient and hydrodynamic radius of HFUS1 at 0.005 g / mL, 0.008 g / mL, 0.011 g / mL, 0.016 g / mL, and 0.020 g / mL concentrations.

[0079] Figure 4A Figure 5 is a bar graph showing C-terminal amino acid cleavage of the Relaxin B chain of HFUS1 assessed by mass spectrometry. F4, F5, F6, and F9 correspond to Formulations 4, 5, 6, and 9, respectively, as described in Table 4.

[0080] Figure 4B Figure 6 is a bar graph showing % change in the tri-sulfide bond of HFUS1 assessed by mass spectrometry. F4, F5, F6, and F9 correspond to Formulations 4, 5, 6, and 9, respectively, as described in Table 4.

[0081] Figure 4C Figure 7 is a bar graph showing methionine 271 (M271) oxidation at the Relaxin B chain of HFUS1 assessed by mass spectrometry. F4, F5, F6, and F9 correspond to Formulations 4, 5, 6, and 9, respectively, as described in Table 4.

[0082] Figure 5 illustrates purity profile of HFUS1 in histidine-arginine HC1 at pH 5.5 to 7.0 indicating monthly change rate (%) in monomer, aggregation and fragmentation during storage at 40°C (A), 25°C (B) and 5°C (C).

[0083] Figure 6 is a bar graph showing C-terminal amino acid cleavage of relaxin at pH ranging from 5.5 to 7.0 at 0°C, 5°C, 25°C and 40°C.

[0084] Figure 7 shows purity loss of AZ3427 with protease inhibitors (PI) assessed by HPSEC.

[0085] Figure 8 is a bar graph showing amino acid cleavage of AZ3427 with protease inhibitors (PI) assessed by mass spectrometry.

[0086] Figure 9 illustrates particle formation after storage at 5°C. (A) shows pH screen samples of HFUS1 formulation in histidine-arginine HC1 at 6 month storage time point, (B) shows pH screen samples of HFUS1 formulation in histidine-arginine HC1 at 12 month storage time point, (C) shows AZ3427 formulation optimization samples (various buffers, excipients and pH) after 12 months of storage, formulation 1 to 8 represent formulation 1 to 8 respectively as described in Table 4.

[0087] Figure 10 FTIR spectra of HFUS1 particles compared to protein and PS80 references are demonstrated. Highlighted boxes indicate similar IR features to protein references, and traces of features of PS80.

[0088] Figure 11 shows visual inspection of HFUS1 formulation in 2R vials after storage at 5°C for (A) 9 months and (B) 12 months.

[0089] Figure 12 shows visual inspection of HFUS1 formulation in 1 mL prefilled syringes after storage at 5°C for (A) 9 months and (B) 12 months.

[0090] Figure 13 shows particle count / mL of HFUS1 formulation measured by microflow imaging for (A) particles with diameter equal to or greater than 1 pm but less than 2 pm; (B) particles with diameter equal to or greater than 2 pm; and (C) particles with diameter equal to or greater than 10 pm.

[0091] Figure 14Figure 5 is the total ion LC-MS chromatogram of PS-80 species in stressed and unstressed samples (POE is polyoxymethylene).

[0092] Figure 15 shows HPSEC of HFUS1 formulations with PS80 and P188 (A) stored at 40°C for 3 months and (B) stored at 5°C for 12 months (PS80 sample) or 18 months (P188 sample). MPP is the main product peak, which is the sum of the monomer and the shoulder.

[0093] Figure 16 Shown are changes in the isoelectric point of HFUS1 formulations with PS80 and P188 stored at 5°C for 12 months (PS80 sample) and 18 months (P188 sample) as measured by capillary isoelectric focusing (cIEF).

[0094] FIG17 shows HPSEC of HFUS1 formulations stored at (A) 40° C., (B) 40° C., (C) 25° C., and (D) 5° C. The percentage change in ratio for each month is indicated. "50 L DEV LOT (P1) 0.04% P188 Target" corresponds to formulation F1 (target) in Table 6. The x-axis indicates the change in the test formulation relative to F1 (target) and is related to the formulations listed in Table 6.

[0095] Figure 18 shows capillary gel electrophoresis (CGE) of HFUS1 formulations after storage for 0 and 1 month at (A) 40°C, (B) 25°C, and (C) 5°C. "50L DEV LOT (P1) 0.04% P188 Target" corresponds to formulation F1 (Target) in Table 6. The x-axis indicates the change in the test formulation relative to F1 (Target) and is related to the formulations listed in Table 6.

[0096] FIG19 shows capillary isoelectric focusing (CIEF) of HFUS1 formulations stored at (A) 40° C., (B) 25° C., and (C) 5° C. The percentage change in ratio for each month is indicated. “50LDEV LOT (P1) 0.04% P188 Target” corresponds to formulation F1 (target) in Table 6. The x-axis indicates the change in the test formulation relative to F1 (target) and is related to the formulations listed in Table 6.

[0097] FIG20 shows microfluidic imaging (MFI) of HFUS1 formulations stored for 3 months at (A) 40° C. for particles with a diameter of 2 μm or greater, (B) 40° C. for particles with a diameter of 10 μm or greater, (C) 5° C. for particles with a diameter of 2 μm or greater, and (D) 5° C. for particles with a diameter of 10 μm or greater. “F0” to “F8” correspond to the formulations listed in Table 6.

[0098] Figure 21 shows HPSEC (monomer percentage) for HFUS1 low concentration formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C. “Target (0.25 MG / ML),” “Worst case (0.25 MG / ML),” “Target (1 MG / ML),” and “Worst case (1 MG / ML)” correspond to Formulations PI to P4 in Table 7, respectively.

[0099] Figure 22 shows capillary gel electrophoresis (CGE) for HFUS1 low concentration formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C. PI to P4 correspond to Formulations PI to P4 in Table 7, respectively.

[0100] Figure 23 shows capillary isoelectric focusing (CIEF) (major peak %) for HFUS1 low concentration formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C. The percent change in ratio is indicated per month. PI to P4 correspond to Formulations PI to P4 in Table 7, respectively.

[0101] Figure 24 shows microflow imaging (MFI) for HFUS1 low concentration formulations stored at 5°C, 25°C, and 40°C for (A) particles with diameters equal to or greater than 25 pm, and (B) particles with diameters equal to or greater than 10 pm. “Target (0.25 MG / ML),” “Worst case (0.25 MG / ML),” “Target (1 MG / ML),” and “Worst case (1 MG / ML)” correspond to Formulations PI to P4 in Table 7, respectively.

[0102] Figure 25 Exemplary formats of heterodimeric fusions are shown, in accordance with some embodiments of the disclosure. The format of each fusion polypeptide of the heterodimeric fusion is given with FcX, FcY, A, B, con, and L, where FcX (“Fc Knob”) and FcY (“Fc Hole”) 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, HC X and HC Y - heavy chains of an antibody, LC - a light chain of an antibody, Hinge - a hinge region of an antibody, and Fab - a Fab fragment of an antibody.

[0103] Figure 26 shows particle counts via microflow imaging (MFI) for various HFUS1 formulations stored at (A) 5°C for particles with diameters equal to or greater than 2 pm, (B) 5°C for particles with diameters equal to or greater than 10 pm, (C) 5°C for particles with diameters equal to or greater than 25 pm, (D) 25°C for particles with diameters equal to or greater than 2 pm, (E) 25°C for particles with diameters equal to or greater than 10 pm, (F) 25°C for particles with diameters equal to or greater than 25 pm, (G) 40°C for particles with diameters equal to or greater than 2 pm, (H) 40°C for particles with diameters equal to or greater than 10 pm, and (I) 40°C for particles with diameters equal to or greater than 25 pm. Data correspond to the formulations listed in Table 8, where "33 mg / ml" = PFS- control; "33 mg / mL pH 6" = PFS-1; "33 mg / mL pH 7" = PFS-2; "33 mg / mL low Arg HCl" = PFS-3; "33 mg / mL high Arg HCl" = PFS-4; "33 mg / mL 0.02% P188" = PFS-5; "33 mg / mL 0.06% P188" = PFS-6.

[0104] Figure 27 shows particle counts via light blockage (HIAC) for HFUS1 formulations stored at (A) 5°C for particles with diameters equal to or greater than 2 pm, (B) 5°C for particles with diameters equal to or greater than 10 pm, (C) 5°C for particles with diameters equal to or greater than 25 pm, (D) 25°C for particles with diameters equal to or greater than 2 pm, (E) 25°C for particles with diameters equal to or greater than 10 pm, (F) 25°C for particles with diameters equal to or greater than 25 pm, (G) 40°C for particles with diameters equal to or greater than 2 pm, (H) 40°C for particles with diameters equal to or greater than 10 pm, and (I) 40°C for particles with diameters equal to or greater than 25 pm. Data correspond to the formulations listed in Table 8, where "33 mg / ml" = PFS- control; "33 mg / mL pH 6" = PFS-1; "33 mg / mL pH 7" = PFS-2; "33 mg / mL low Arg HCl" = PFS-3; "33 mg / mL high Arg HCl" = PFS-4; "33 mg / mL 0.02% P188" = PFS-5; "33 mg / mL 0.06% P188" = PFS-6.

[0105] Figure 28 shows HPSEC of HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C. The percent change in monomer ratio is indicated monthly. Data is related to the formulations listed in Table 8, where “33 mg / ml” = PFS-control; “33 mg / mL pH 6” = PFS-1; “33 mg / mL pH 7” = PFS-2; “33 mg / mL low Arg HC1” = PFS-3; “33 mg / mL high Arg HC1” = PFS-4; “33 mg / mL 0.02% P188” = PFS-5; “33 mg / mL 0.06% P188” = PFS-6.

[0106] Figure 29 shows capillary isoelectric focusing (cIEF) of HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C. Data is related to the formulations listed in Table 8, where “33 mg / ml” = PFS-control; “33 mg / mL pH 6” = PFS-1; “33 mg / mL pH 7” = PFS-2; “33 mg / mL low Arg HC1” = PFS-3; “33 mg / mL high Arg HC1” = PFS-4; “33 mg / mL 0.02% P188” = PFS-5; “33 mg / mL 0.06% P188” = PFS-6.

[0107] Figure 30 shows potency of HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C. Data is related to the formulations listed in Table 8, where “33 mg / ml” = PFS-control; “33 mg / mL pH 6” = PFS-1; “33 mg / mL pH 7” = PFS-2; “33 mg / mL low Arg HC1” = PFS-3; “33 mg / mL high Arg HC1” = PFS-4; “33 mg / mL 0.02% P188” = PFS-5; “33 mg / mL 0.06% P188” = PFS-6.

[0108] Figure 31 shows amino acid cleavage at the C-terminus of the B chain of relaxin for HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C, assessed by RP-HPLC. Data are related to the formulations listed in Table 8, where “33 mg / ml” = PFS- control; “33 mg / mL pH 6” = PFS-1; “33 mg / mL pH 7” = PFS-2; “33 mg / mL low Arg HC1” = PFS-3; “33 mg / mL high Arg HC1” = PFS-4; “33 mg / mL 0.02% P188” = PFS-5; “33 mg / mL 0.06% P188” = PFS-6. DETAILED DESCRIPTION

[0109] Definitions

[0110] Unless otherwise defined, all specialized terms, symbols and other technical and scientific terms or terminology used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for the convenience of the reader. The inclusion of a definition herein should not be construed as an acknowledgement that the term is not equally applicable to other embodiments or aspects of the claimed subject matter.

[0111] Concentrations, amounts, volumes, percentages, and other numerical values in this disclosure can be presented in a range format. It is to be understood that the use of such range formats is merely for convenience and brevity and that all values within and ranging from the stated range are contemplated unless otherwise described. Concentrations, amounts, volumes, percentages, and other numerical values in this disclosure can be presented in a range format. It is to be understood that the use of such range formats is merely for convenience and brevity and that all values within and ranging from the stated range are contemplated unless otherwise described.

[0112] All publications, including patents, patent applications, publications, and databases, mentioned in this specification are herein incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication, patent, patent application, publication, and database was specifically and individually indicated to be incorporated by reference. To the extent publication incorporates definitions from the Dictionary of Chemical Terms, the definitions herein control.

[0113] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. One skilled in the art will recognize that the scope of the present disclosure is not limited to the particular embodiments described, and one will recognize the many applications of the concepts disclosed. The following description illustrates the disclosure and, of course, should not be construed as limiting the scope of the intended application.

[0114] Reference to a subject matter disclosed or described herein anywhere in this application relates to a subject matter disclosed or described anywhere in this application.

[0115] SUMMARY

[0116] During the development of the formulation of the heterodimeric fusion, HFUS1, several challenges were encountered related to the shelf-life and stability of the molecule. These challenges included: the tendency of HFUS1 to self-associate leading to aggregation; the tendency of amino acid clipping within certain pH ranges and the extreme ease of particle formation.

[0117] Accordingly, the present disclosure describes some deliberately designed studies to identify the root cause of HFUS1 instability, as well as formulation development and optimization work to identify stable liquid formulations of the molecule to meet its drug product shelf-life requirements.

[0118] The present inventors have demonstrated that HFUS1 tends to self-associate (see Example 1). High levels of self-association of the molecule can lead to the formation of soluble aggregates that can become precursors of insoluble large size aggregates and eventually particles, significantly impacting the stability profile of the molecule. The present inventors identified the need for formulation optimization to reduce this self-association and aggregation tendency. Surprisingly, it was determined that surfactants in the formulation play a key role in particle formation associated with free fatty acids (FFAs), which can act as nuclei to trigger HFUS1 protein aggregation. Optimization of surfactants significantly reduced particle formation and decreased the aggregation tendency (see Example 3). It was also found that optimization of the pH, buffers and excipients used in the formulation of HFUS1 further reduced aggregation. Overall, a histidine-arginine HC1 system (i.e., a histidine-based buffer system with arginine hydrochloride ionic-type excipient) was identified to provide the highest colloidal and conformational stability to HFUS1 (see Example 2).

[0119] The present inventors have also identified that proteases can be the cause of fragmentation and clipping of HFUS1. This enzymatic activity is more likely to occur at lower pH, where proteases are most effective in cleaving the molecule. Therefore, it is important to maintain the pH of the formulation in a higher range, as demonstrated in the pH optimization studies (see Example 2). The optimal pH range of 5.5 to 7.5, particularly 6 to 7, more particularly 6.5 minimizes chemical degradation impacts such as fragmentation of the molecule and amino acid (AA) clipping.

[0120] As mentioned above, the inventors identified the formation of particles over time in the HFUS1 formulation (see Example 3). As explained in Example 3, several underlying causes for particle formation were hypothesized. Surprisingly, the inventors identified the presence of the surfactant PS80 as the cause for particle formation. The inventors identified that the enzymatic hydrolysis of the ester bond of PS80 by the lipoprotein lipase (LPL) present in the formulation can be the cause for the degradation of PS80, which in turn leads to the formation of impurities such as free fatty acids (FFA), which can act as nuclei to trigger aggregation of the HFUS1 protein, resulting in particles. At the same time, due to the degradation, the PS80 levels decrease, thus losing its protective / surfactant effect to prevent particle formation during storage. The use of lipase-resistant surfactants, such as poloxamer 188 (P188) and D-a-tocopheryl polyethylene glycol succinate (TPGS), enables to mitigate particle formation in the HFUS1 formulation.

[0121] The effect of HFUS1 concentration on stability was also evaluated and HFUS1 showed good stability in the formulations described herein at a wide range of HFUS1 concentrations, with data for HFUS1 concentrations ranging from 0.25 mg / mL to 50 mg / mL, especially for formulations with HFUS1 concentrations of 0.25 mg / mL, 1 mg / mL, 5 mg / mL, 33 mg / mL and 50 mg / mL (see Examples 4, 5 and 6).

[0122] In summary, the histidine-arginine HC1 system was chosen because of its effectiveness in reducing the self-association tendency of the molecule and the identification of an optimized pH range that effectively prevents AA cleavage and fragmentation of the molecule (see Example 2). In addition, a detailed research investigation was carried out to understand the cause of the particle formation problem and identified PS80 as the root cause. This led to the optimization of the surfactant in the formulation system and the replacement of the surfactant such as P188 and TPGS mitigated the particle formation problem (see Example 3). P188 was chosen as the leading surfactant for the formulation. Finally, a comprehensive formulation stability study was carried out to evaluate the robustness of the formulation system. The histidine / histidine HC1-arginine HC1 formulation showed an excellent stability profile and robustness. The effect of HFUS1 concentration on stability was also evaluated and HFUS1 showed good stability at a wide range of tested concentrations as well (see Examples 4 to 6).

[0123] Accordingly, provided herein is a pharmaceutical formulation comprising a heterodimeric fusion (e.g., HFUS1) and a lipase-resistant surfactant, wherein the heterodimeric fusion comprises:

[0124] (i) a first heterodimerization domain linked to at least one relaxin A chain polypeptide or variant thereof; and

[0125] (ii) a second heterodimerization domain linked to at least one relaxin B chain polypeptide or variant thereof,

[0126] wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and wherein the heterodimeric fusion has relaxin activity.

[0127] Also provided herein are pharmaceutical formulations as described herein for use in therapy. Also provided is a method of treating a subject having a disease or condition, the method comprising administering to the subject a pharmaceutical formulation as described herein.

[0128] Also provided herein are pharmaceutical formulations as described herein for use in the treatment of a subject having heart failure, optionally heart failure with pulmonary arterial hypertension (e.g. pulmonary arterial hypertension type 2). Also provided herein is a method of treating a subject having heart failure, optionally heart failure with pulmonary arterial hypertension (e.g. pulmonary arterial hypertension type 2), the method comprising administering to the subject a pharmaceutical formulation as described herein.

[0129] Also provided herein is a kit comprising a pharmaceutical formulation as described herein.

[0130] Heterodimeric fusion with relaxin activity

[0131] Relaxin

[0132] The pharmaceutical formulations of the present disclosure comprise a heterodimeric fusion having relaxin activity, e.g. HFUS1.

[0133] As described in WO2021 / 255127, the heterodimeric fusions described herein, e.g. HFUS1, can exhibit relaxin activity when the relaxin A chain and the relaxin B chain are not covalently linked to each other by an amino acid linker. Advantageously, heterodimerization of the heterodimerization domains induces correct folding and heterodimerization of the relaxin A chain and the relaxin B chain (see Example 2 of WO2021 / 255127). In addition, unlike wild-type relaxin proteins, the heterodimeric fusions, e.g. HFUS1, do not require endoproteolytic processing to obtain biological activity.

[0134] 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., a relaxin A chain), and the other fusion polypeptide comprises a second heterodimerization domain linked to a second subunit of the heterodimeric protein (e.g., a relaxin B chain). In some embodiments, the heterodimeric fusion is HFUS1 (also known as RELAX0023). HFUS1 is a heterodimeric fusion consisting of 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.

[0135] The heterodimeric fusion used in the formulations of the present disclosure can comprise a relaxin A chain polypeptide and a relaxin B chain polypeptide from a relaxin group selected from the group consisting of relaxin-1, relaxin-2, and relaxin-3. In particular embodiments, the relaxin A chain polypeptide is a relaxin-2 A chain polypeptide or a variant thereof; and the relaxin B chain polypeptide is a relaxin-2 B chain polypeptide or a variant thereof. In particular embodiments, the relaxin A chain polypeptide comprises a human relaxin-2 A chain polypeptide or a variant thereof and a human relaxin-2 B chain polypeptide or a variant thereof.

[0136] The terms “chain,” “polypeptide,” and “peptide” are used interchangeably herein to refer to a chain of two or more amino acids linked by peptide bonds.

[0137] In some embodiments, the relaxin-2 A chain polypeptide has the sequence set forth in SEQ ID NO: 1 or a variant thereof, and the relaxin-2 B chain polypeptide has the sequence set forth in SEQ ID NO: 2 or a variant thereof. Variants can comprise one or more amino acid substitutions, deletions, and / or insertions. In some embodiments, the relaxin-2 A chain polypeptide comprises one or more amino acid mutations selected from the group consisting of K9E, K9H, K9L, K9M, R18E, R18H, R22A, R22I, R22M, R22Q, R22S, R22Y, F23E, F23A, and F23I. In a particular embodiment, the relaxin-2 A chain comprises the amino acid mutation K9H.

[0138] Relaxin A chain and B chain variants are known in the art. In addition, guidance on the design of relaxin A chain and B chain variants is available to the skilled person. For example, it will be appreciated that variants can retain those amino acids required for relaxin function. For example, relaxin-2 B chain variants can comprise the conserved motif Arg-X-X-X-Arg-X-X-lle (Claasz AA et al. (2002) Eur. J. Biochem. 269(24):6287-6293) or Arg-X-X-X-Arg-X-X-Val (Bathgate RA et al., (2013) Physiol Rev. 93(1):405-480). Variants can comprise one or more amino acid substitutions and / or insertions. For example, a relaxin-2 B chain variant can have one or more additional amino acids compared to SEQ ID NO: 62, for example K30 and R31 and N-terminal V-2, A-1 and M-1. Alternatively or additionally, variants can comprise one or more amino acid derivatives. For example, the first amino acid of a relaxin-2 B chain variant can be pyroglutamate.

[0139] In particular embodiments, the relaxin A chain and the relaxin B chain are covalently bound via two interchain disulfide bonds (see Example 2 of WO 2021 / 255127).

[0140] Peptides of the relaxin family mediate their biological effects at least in part through activation of G protein-coupled receptors (GPCRs) and subsequent stimulation or inhibition of the cAMP signaling pathway through Gs or Gi protein subunits, respectively. Relaxin-2 is known to activate the GPCR RXFP1 (also known as LGR7) and to a lesser extent the GPCR RXFP2 (also known as LGR8), thereby stimulating the Gs-cAMP dependent signaling pathway leading to an increase in the second messenger molecule cAMP.

[0141] As used herein, the term “relaxin activity” refers to the ability of a relaxin molecule to bind to a relaxin receptor and / or to activate said relaxin receptor and / or to initiate an intracellular signaling cascade. In embodiments where the relaxin activity is relaxin-2 activity, the relaxin activity can refer to the ability to bind and / or activate the receptors RXFP1 and / or RXFP2. The term “relaxin activity” can be used interchangeably with “biological activity”.

[0142] Relaxin activity can be determined by measuring the binding of a relaxin molecule to a relaxin receptor and / or by measuring downstream events following binding to a relaxin receptor.

[0143] Relaxin activity can be determined in vitro and / or in vivo. In some embodiments, relaxin activity is determined in vitro.

[0144] Relaxin activity can be determined by measuring the amount and / or presence of molecules downstream of relaxin activation of receptors. For example, relaxin activity can be determined by measuring the level of cAMP production following relaxin activation of receptors. Methods for detecting relaxin-induced cAMP production are known in the art. Such methods include cAMP ELISA, HTRF cAMP assay and cAMP assay. In some embodiments, relaxin activity is determined by measuring relaxin-induced cAMP production by HTRF cAMP assay, for example as performed in Example 3 of WO2021 / 255127. Relaxin activity can be determined by measuring the level of nitric oxide (NO) production following relaxin activation of receptors. Relaxin activity can also be determined by measuring the level of activation of downstream molecules following relaxin activation of receptors. For example, relaxin activity can be determined by measuring the level of activation of p42 / 44 MAPK.

[0145] Alternatively or in addition, relaxin activity can be determined by measuring the level of activation of known relaxin target genes. For example, relaxin activity can be determined by measuring the level of transcriptional activation of the known relaxin target gene VEGF in THP-1 cells. Methods for determining the level of gene transcriptional activation are known in the art, including quantitative PCR analysis of mRNA. The relative amount of VEGF mRNA can be measured by real-time quantitative PCR to detect the level of induced expression of VEGF transcript following incubation of THP-1 cells with relaxin, as described in Xiao et al., (2013) Nat Commun. 4: 1953.

[0146] Alternatively or in addition, relaxin activity can be determined by measuring one or more downstream effects of relaxin. For example, according to standard methods, the reduction of cardiac hypertrophy can be measured by echocardiography, the ratio of left ventricular weight to body weight and / or tibial length. In another example, relaxin activity can be determined by measuring the reduction of fibrosis by Masson’s trichrome staining. In another example, relaxin activity can be determined by measuring the modulation of connective tissue metabolism, such as inhibition of profibrotic factors (such as TGF-b), inhibition of fibroblast proliferation and differentiation, and / or activation of MMP-mediated extracellular matrix degradation (Bathgate RA et al., (2013) Physiol Rev. 93(1):405-480).

[0147] In some embodiments, relaxin activity is determined by measuring the reversal of isoprenaline-induced cardiac hypertrophy (measured as the ratio of heart weight to tibial length) and fibrosis (measured as the ratio of collagen content to heart weight), for example as performed in Example 7 of WO2021 / 255127.

[0148] The activity of the heterodimeric fusion proteins of the disclosure (e.g. HFUS1) can be determined relative to a reference relaxin protein. In some embodiments, the reference relaxin protein is a recombinant protein. In particular embodiments, the reference relaxin protein is a relaxin protein having the relaxin A chain and B chain arrangement of the mature relaxin protein. Recombinant relaxin having the A chain and B chain structural arrangement of the mature relaxin protein is commercially available. For example, recombinant human relaxin-2, murine relaxin-1 and INSL3 are available from R&D systems (catalogue numbers 6586-RN, 6637-RN and 4544-NS respectively).

[0149] In some embodiments, the reference relaxin protein has the same relaxin A and B chains as the heterodimeric fusion protein of the disclosure, or differs from the relaxin A and B chains of the heterodimeric fusion protein of the disclosure by up to 10 amino acids, for example 1 or 2 amino acids. In some embodiments, the first amino acid of the B chain of the reference relaxin-2 is D, and this amino acid is deleted in the relaxin B chain of the heterodimeric fusion of the disclosure.

[0150] The reference relaxin protein can be selected from:

[0151] (i) recombinant human relaxin-2 (referred to herein as RELAX0013); and

[0152] (ii) recombinant murine relaxin-1 (referred to herein as RELAX0014); and

[0153] (iii) recombinant Fc-fused relaxin-2, wherein the relaxin A and B are fused in a single chain and wherein the Fc is a half-life extended Fc region (referred to herein as RELAX0010 and described in WO2018 / 138170); and

[0154] (iv) recombinant Fc-fused relaxin-2, wherein the relaxin A and B are fused in a single chain and wherein the Fc is a half-life extended Fc region (referred to herein as RELAX0009 and described in WO2018 / 138170); and

[0155] (v) recombinant Fc-fused relaxin-2, wherein the relaxin A and B are fused in a single chain (referred to herein as RELAX0126 and described in WO 2013 / 004607);

[0156] and

[0157] (vi) recombinant Fc-fused relaxin-2, in which relaxin A and relaxin B are fused in a single chain (referred to herein as RELAX0127 and described in WO 2013 / 004607);

[0158] as well as

[0159] (vii) Recombinant Fc fusion relaxin in which relaxin A and relaxin B are fused in a single chain (referred to herein as RELAX0128 and described in WO 2013 / 004607).

[0160] In a specific embodiment, the reference relaxin protein is a relaxin-2 protein having the relaxin-2A chain and relaxin-2B chain arrangement of the mature relaxin-2 protein as disclosed in UniProtKB / Swiss-Prot Accession No. P04090.1.

[0161] Heterodimeric fusions of the present disclosure (e.g., HFUS1) 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 can be considered to have relaxin activity if it has at least about half the activity of a reference relaxin protein. A heterodimeric fusion of the present disclosure can be considered to have relaxin activity if the ratio of its activity to the activity of the reference relaxin protein is about 10:1. -5 to about 1, about 10 -4 to about 1, about 10 -3 to about 1, about 10 -2 The fusion polypeptide may be considered to have relaxin activity if the ratio of the activity of the heterodimeric fusion of the present disclosure to the activity of the reference relaxin protein is from about 1 to about 10, 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, the fusion polypeptide may be considered to have relaxin activity if the ratio of the activity of the heterodimeric fusion of the present disclosure to the activity of the reference relaxin protein is from about 1 to about 10. 5 , about 1 to about 10 4 , about 1 to about 10 3 , about 1 to about 100, about 1 to about 50, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 2, then the fusion polypeptide is considered to have relaxin activity.

[0162] In some embodiments, the ratio of relaxin activity of the heterodimeric fusion (eg, HFUS1) to the relaxin activity of a reference relaxin protein is from about 0.001 to about 10.

[0163] Relaxin activity can be determined as an EC50 value. As used herein, the term "EC50" (half maximal effective concentration) refers to the effective concentration of a therapeutic compound that induces a response halfway between baseline and maximum after a specified exposure time.

[0164] Heterodimerization domain

[0165] The heterodimeric fusion used in the formulations of the present disclosure (e.g., HFUS1) comprises a first heterodimerization domain and a second heterodimerization domain. In particular embodiments, the first heterodimerization domain and the second heterodimerization domain are derived from an immunoglobulin Fc region.

[0166] The term“Fc region” defines a C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain.

[0167] The first Fc region and the second Fc region can comprise immunoglobulin domains CH2 and / or CH3. In particular embodiments, the first Fc region and the second Fc region comprise immunoglobulin domains CH2 and CH3.

[0168] The Fc region can be derived from an immunoglobulin of any species (e.g., IgG), particularly from a human (e.g., human IgG). In embodiments where the Fc region is derived from an IgG, the Fc region can be derived from an IgG of any subclass (e.g., IgG1, IgG2, IgG3, IgG4), particularly IgG1. In particular embodiments, the first Fc region and the second Fc region are derived from a human IgG1 immunoglobulin. In other embodiments, the first Fc region and the second Fc region are derived from a human IgG4 immunoglobulin.

[0169] In particular embodiments, the first Fc region and the second Fc region comprise amino acid mutations and / or modifications that promote heterodimerization. Such modifications can include the introduction of asymmetric complementary modifications into each of the first c region and the second Fc region, such that the two chains are compatible with each other and thus able to form a heterodimer, but each chain is not able to homodimerize with itself. Such modifications can encompass insertions, deletions, conservative substitutions, and non-conservative substitutions, and rearrangements. Incorporation of such modifications provides a means for increasing the yield of heterodimers produced by recombinant cell culture over other undesired end products, such as homodimers.

[0170] The first Fc region and the second Fc region can comprise any amino acid mutations and / or modifications that promote heterodimerization known in the art. Combinations of modifications can be used to maximize assembly efficiency while minimizing the impact on antibody stability.

[0171] In the "knobs-in-holes" approach, heterodimerization can be promoted by introducing steric hindrance between the contacting residues. A "knob" is created by replacing one or more small amino acid side chains from one Fc region ("Fc knob") with a larger side chain (e.g., tyrosine or tryptophan) onto the interface of the other Fc region ("Fc hole"). A compensating "hole" of the same or similar size as the large side chain is created by replacing the amino acid with the large side chain with an amino acid having a smaller side chain (e.g., alanine or valine) onto the interface of the other Fc region. "Knobs-in-holes" modifications are described in detail, for example, in Ridgway JB et al. (1996) Protein Eng. 9(7) 617-621; Merchant AM et al. (1998) Nat. Biotechnol. 16(7):677-681.

[0172] Other modifications that can be used to produce 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 can be introduced into one Fc region, and one or more negatively charged amino acids can be introduced into the corresponding position in the other Fc region. Alternatively or additionally, the Fc regions can be modified to include mutations that introduce cysteine residues capable of forming disulfide bonds. Alternatively or additionally, the Fc regions can comprise one or more modifications to hydrophilic and hydrophobic residues at the interface between the chains so as to make the formation of a heterodimer more favorable in entropy and enthalpy than the formation of a homodimer.

[0173] Thus, in some embodiments, the amino acid mutations and / or modifications that promote heterodimerization create steric hindrance between the contacting residues (e.g., by "knobs-in-holes"), create favorable electrostatic interactions between the two Fc regions, introduce cysteine residues capable of forming disulfide bonds, and / or modify hydrophilic and hydrophobic residues at the interface between the two Fc regions.

[0174] In particular embodiments, the amino acid mutations that promote heterodimerization are "Fc knob" and "Fc hole" mutations. In particular embodiments, the "Fc knob" and "Fc hole" mutations are present in the CH3 domain.

[0175] In some embodiments, the first Fc region and the second Fc region are derived from a human IgGl immunoglobulin and comprise "Fc X" and "Fc Y" mutations in the CH3 domain, wherein the "Fc X" and "Fc Y" mutations are selected from the combinations (or conservative substitutions thereof) shown in Table 1.

[0176] Table 1 : "Fc X" and "Fc Y" mutations

[0177]

[0178]

[0179] *Amino acid numbering is according to the EU index as in Kabat.

[0180] In a specific embodiment, "Fcγ" is an "Fc hole" having mutations Y349C, T366S, L368A, and Y407V, or conservative substitutions thereof, and "FcX" is an "Fc knob" having mutations S354C and T366W, or conservative substitutions thereof, wherein amino acid numbering is according to the EU index as in Kabat.

[0181] The term "EU index as in Kabat" refers to the numbering system for the human IgG1 EU antibody as described in Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service. National Institutes of Health. Bethesda, MD. All amino acid positions mentioned in this application refer to EU index positions.

[0182] In some embodiments, the first Fc region has an "Fc hole" mutation and the second Fc region has an "Fc knob" mutation. In alternative embodiments, the first Fc region has an "Fc knob" mutation and the second Fc region has an "Fc hole" mutation.

[0183] It will be appreciated that the Fc region may also 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 the IgG molecule for FcRn. WO 02 / 060919 discloses modified immunoglobulins comprising an Fc region having one or more amino acid modifications, and is incorporated herein by reference in its entirety. Methods for preparing Fc regions having one or more amino acid modifications are known in the art.

[0184] In some embodiments, the first Fc region and / or the second Fc region may comprise one or more amino acid modifications that reduce or eliminate the effector functions of the Fc region. In some embodiments, these amino acid modifications reduce or circumvent cytotoxicity, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0185] In some embodiments, the first Fc region and / or the second Fc region may comprise one or more amino acid modifications that increase the half-life of the heterodimeric fusion (eg, HFUS1).

[0186] In some embodiments, the first Fc region and / or the second Fc region comprises at least one of the following combinations of amino acid mutations:

[0187] (i) M252Y, S254T, and T256E, or conservative substitutions thereof;

[0188] (ii) L234F, L235Q, and K322Q, or conservative substitutions thereof;

[0189] (iii) L234F, L235E, and P331S, or conservative substitutions thereof;

[0190] (iv) M252Y, S254T, T256E, L234F, L235Q, and K322Q, or conservative substitutions thereof; or

[0191] (v) M252Y, S254T, T256E, L234F, L235E, and P331S, or conservative substitutions thereof

[0192] (vi) M252Y, S254T, T256E, L234F, L235Q, K322Q, and P331S, or conservative substitutions thereof.

[0193] wherein the numbering of amino acids is according to the EU index in Kabat.

[0194] In some embodiments, the first Fc region and / or the second Fc region can comprise the amino acid mutations L234F, L235E, and P331S, or conservative substitutions thereof, wherein the numbering of amino acids is according to the EU index in Kabat.

[0195] In some embodiments, the Fc region comprising the“Fc knob” mutation has the sequence set forth in SEQ ID NO: 3 or a variant thereof, and the Fc region comprising the“Fc hole” mutation has the sequence set forth in SEQ ID NO: 4 or a variant thereof.

[0196] In some embodiments, the Fc region comprises a variant of SEQ ID NO: 3 with the amino acid mutation Y349C reverted to Y349 and a variant of SEQ ID NO: 4 with the amino acid mutation S354C reverted to S354, such that the Fc region is unable to form a stable disulfide bond.

[0197] 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 the sequence DKTHTACPPC (SEQ ID NO: 70). In alternative embodiments, this region is replaced with the sequence GGAGGACPPC (SEQ ID NO: 71). In alternative embodiments, this region is replaced with the sequence ACPPC (SEQ ID NO: 72).

[0198] 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 region and a CL region. It has been found that a Fab region comprising an L chain and an Fd chain mediates efficient heterodimerization (Schoonjans R et al. (2000) J. Immunol. 165(12):7050-7057). Thus, in alternative embodiments, the heterodimerization domain comprises an L chain and an Fd chain. In some embodiments, the L chain and Fd chain heterodimerize to form a disulfide bridge stabilized heterodimer.

[0199] In further alternative embodiments, the first and second heterodimerization domains heterodimerize to form a parallel coiled coil. Heterodimeric coiled coils are described in, for example, Aronsson et al. (2015) Sci. Rep. 5: 14063. In some embodiments, the heterodimerization domains comprise amino acid mutations and / or modifications to prevent the formation of undesirable folding assemblies and / or to promote the formation of a parallel coiled coil.

[0200] The first and second heterodimerization domains (e.g., first and second Fc regions) can form a half-life extending moiety. Thus, in some embodiments, the heterodimeric fusion of the disclosure (e.g., HFUS1) has an extended half-life compared to a reference relaxin.

[0201] As used herein, the term “half-life” is used to refer to the time taken for the concentration of a fusion protein in the plasma to fall to 50% of its original level. The “half-life” of a protein in the plasma can depend on different factors such as the size of the protein, its stability, its clearance rate, turnover rate, proteolytic degradation in vivo, rate of absorption by the body or particular tissues, etc. Methods of determining the half-life of a protein are known in the art and are described in the Examples below.

[0202] As shown in WO 2021 / 255127, a heterodimeric fusion as described herein (e.g., HFUS1) having first and second heterodimerization domains derived from an immunoglobulin Fc has a half-life in a mouse model of at least 5 hours (see Example 6 of WO 2021 / 255127). In contrast, human relaxin-2 has a half-life after IV administration of about 0.09 + / - 0.04 hours, i.e., 5.4 + / - 2.4 minutes in humans (Chen SA et al. (1993) Pharm. Res. 10(6):834-838).

[0203] It will be appreciated that an extended half-life is advantageous because it allows therapeutic proteins to be administered according to a safe and convenient dosing regimen, e.g., according to a lower dose that can be administered less frequently. Moreover, the achievement of a lower dose can provide further advantages, such as providing an improved safety profile and / or activating multiple in vivo mechanisms of action.

[0204] Linker

[0205] One or both of the Relaxin A chain and B chain can be linked to their respective heterodimerization domain 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.

[0206] The linker polypeptide can have any suitable length, e.g., a length of between about 6 and 40 amino acids, such as a length of between about 6 and 21 amino acids. In some embodiments, the linker polypeptide is at least 6 amino acid residues in length, particularly at least 11 amino acids in length, particularly at least 16 amino acids in length. In some embodiments, the linker polypeptide is less than 40 amino acids in length. Linker polypeptides of different or the same length can be used for each arm of the heterodimeric fusion as described herein (e.g., HFUS1). In some embodiments, at least one linker polypeptide is 21 amino acids in length. In particular embodiments, both linker polypeptides are 21 amino acids in length. The linker polypeptide can have any amino acid sequence. Linker polypeptides of different or the same amino acid formulation can be used for each arm of the heterodimeric fusion as described herein (e.g., HFUS1).

[0207] In some embodiments, one or both linker polypeptides comprises a proline and alanine repeat sequence (PA)x(SEQ ID NO: 73). In particular embodiments, x is 3 to 15, optionally wherein the linker polypeptide has a length of greater than 16 amino acids, optionally wherein the linker polypeptide consists of the 21 amino acid sequence PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO: 6).

[0208] In some embodiments, one or both of the linker polypeptides comprises a glycine and serine repeat sequence, such as those described in Chen X, et al. (2013) Adv. Drug. Deliv. Rev. 65(10): 1357-1369. In some embodiments, one or both of the linker polypeptides comprises the motif (GGGGS)n(SEQ ID NO: 74), where n can be between 1 and 8, for example where n is 4. In some embodiments, one or more of the linker polypeptides consists of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5). In certain embodiments, both of the linker polypeptides consist of the 21 amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5).

[0209] 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.

[0210] Alternatively, one or both of the relaxin A and B chains can be linked to their respective heterodimerization domains by a synthetic linker polypeptide, such as a polyethylene glycol (PEG) polymer chain. Thus, the relaxin A chain can be linked to a first heterodimerization domain (e.g., a first Fc region) via a synthetic linker, such as a polyethylene glycol (PEG) polymer chain, and the relaxin B chain can be linked to a second heterodimerization domain (e.g., a second Fc region) via a synthetic linker, such as a polyethylene glycol (PEG) polymer chain, where the synthetic linker can be covalently or non-covalently attached to the heterodimerization domain (e.g., Fc region). PEGylation, the process of attaching PEG polymer chains to molecules, can be performed according to methods known in the art.

[0211] Stability

[0212] As shown in WO2021 / 255127, the heterodimeric fusions as described herein (e.g., HFUS1) have unexpectedly superior physical and chemical stability. Thus, in some embodiments, the heterodimeric fusions as described herein (e.g., HFUS1) have superior physical and / or chemical stability compared to reference relaxin proteins.

[0213] Physical stability of relaxin can be determined by measuring purity and aggregation, for example by HP-SEC as in Example 9 of WO2021 / 255127. Chemical stability of relaxin can be determined by measuring fragmentation and modification of the molecule, for example by LC-MS as in Example 9 of WO2021 / 255127.

[0214] Surprisingly, as shown in WO 2021 / 255127, the heterodimeric fusion as described herein (e.g., HFUS1) has superior physical and chemical stability compared to recombinant Fc-fused relaxins, where relaxin A and relaxin B are fused in a single chain (as opposed to relaxin A and B in separate fusion polypeptides). WO 2013 / 004607 describes recombinant single-chain relaxin fusion polypeptides fused to an immunoglobulin Fc region, such as the fusion polypeptides referred to herein as RELAX0127 and RELAX0128. Thus, in some embodiments, the heterodimeric fusion as described herein (e.g., HFUS1) has superior physical and / or chemical stability compared to RELAX0127 and RELAX0128.

[0215] In addition to the first heterodimerization domain and the second heterodimerization domain, the heterodimeric fusion (e.g., HFUS1) can comprise a half-life extension moiety. In some embodiments, the half-life extension moiety is a protein half-life extension moiety. The protein half-life extension moiety can 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 extension moiety is a chemical entity that is not a protein or peptide, such as a polyethylene glycol (PEG) polymer chain.

[0216] The half-life extension moiety can be attached at the N-terminus or the C-terminus of the first heterodimerization domain or the second heterodimerization domain. In some embodiments, the half-life extension moiety is attached at the N-terminus of the first heterodimerization domain or the second heterodimerization domain. In other embodiments, the half-life extension moiety is attached at the C-terminus of the first heterodimerization domain or the second heterodimerization domain. Methods for attaching a half-life extension moiety to a heterodimeric fusion (e.g., HFUS1) are known in the art. For example, the half-life extension moiety can be attached by chemical conjugation or recombinant techniques. The half-life extension moiety can be attached directly or through a linker (e.g., a linker polypeptide) to the heterodimeric fusion (e.g., HFUS1). The use of a linker polypeptide can be particularly appropriate when the fusion polypeptide comprises a protein half-life extension moiety, such as an Fc region.

[0217] Exemplary embodiments

[0218] The heterodimeric fusion (e.g., HFUS1) used in the formulations of the present disclosure can have a variety of formats and / or sequences.

[0219] The terms "a fusion polypeptide" and "fusion polypeptides" can be used to refer to a first heterodimerization domain fused to a relaxin A chain, and / or a second heterodimerization domain fused to a relaxin B chain. The fusion polypeptides used in the formulations of the present disclosure can be recombinant fusion polypeptides, i.e., they have been produced by recombinant DNA technology.

[0220] In particular embodiments, the C-terminus of the first heterodimerization domain (e.g., first Fc region) is connected to the N-terminus of the relaxin A chain, and the C-terminus of the second heterodimerization domain (e.g., second Fc region) is connected 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.

[0221] In alternative embodiments, the N-terminus of the first heterodimerization domain (e.g., first Fc region) is connected to the C-terminus of the relaxin A chain, and the N-terminus of the second heterodimerization domain (e.g., second Fc region) is connected 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.

[0222] In alternative embodiments, the N-terminus of the first heterodimerization domain (e.g., first Fc region) is connected to the C-terminus of the relaxin A chain, and the N-terminus of the second heterodimerization domain (e.g., second Fc region) is connected 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.

[0223] In alternative embodiments, the N-terminus of the first heterodimerization domain (e.g., first Fc region) is connected to the C-terminus of the relaxin A chain, and the N-terminus of the second heterodimerization domain (e.g., second Fc region) is connected 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.

[0224] Thus, in some embodiments, the format of the heterodimeric fusion (e.g., HFUS1) is selected from:

[0225] (i) FcX-con-A / FcY-con-B (see, e.g., Figure 1A); Figure 25 );

[0226] (ii) FcX-con-B / FcY-con-A (see, e.g., Figure 1B); Figure 25 );

[0227] (iii) A-con-FcX / B-con-FcY (see, e.g., Figure 1C); Figure 25

[0228] (iv) B-con-FcX / A-con-FcY (see, e.g., Figure 1D); Figure 25

[0229] (v) Fab-FcX-con-A / Fab-FcY-con-B (see, e.g., Figure 1E); Figure 25

[0230] (vi) Fab-FcX-con-B / Fab-FcY-con-A;

[0231] (vii) A-con-FcX-con-A / B-con-FcY-con-B (see, e.g., Figure 1F); Figure 25

[0232] (viii) B-con-FcX-con-B / A-con-FcY-con-A;

[0233] (ix) FcX-con-B-L-A and FcY, optionally FcY-con-B-L-A (see, e.g., Figure 1G); Figure 25

[0234] (x) FcY-con-B-L-A and FcX, optionally FcX-con-B-L-A;

[0235] (xi) FcX-con-A-L-B and FcY, optionally FcY-con-A-L-B; and

[0236] (xii) FcY-con-A-L-B and FcX, optionally FcX-con-A-L-B, wherein:

[0237] FcY is an immunoglobulin Fc region having "Fc hole" amino acid mutations and / or modifications, optionally comprising a CH3 domain having amino acid mutations Y349C:T366S:L368A:Y407V or conservative substitutions thereof;

[0238] FcX is an Fc region having "Fc knob" amino acid mutations and / or modifications, optionally comprising a CH3 domain having amino acid mutations S354C:T366W or conservative substitutions thereof;

[0239] "con" is a linker polypeptide;

[0240] B is a relaxin B chain or variant thereof;

[0241] ​​​​​A is a relaxin A chain or variant thereof; and

[0242] L is a linker polypeptide, optionally having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60).

[0243] In another aspect, the heterodimeric fusion used in the formulations of the present disclosure comprises

[0244] (i) X-B-L-A and Y, optionally Y-B-L-A; or

[0245] (ii) Y-B-L-A and X, optionally X-B-L-A,

[0246] wherein:

[0247] X and Y are heterodimerization domains as described herein;

[0248] B is a relaxin B chain or variant thereof, e.g., a relaxin-2 B chain or variant thereof;

[0249] A is a relaxin A chain or variant thereof, e.g., a relaxin-2 A chain or variant thereof; and

[0250] L is a linker polypeptide, optionally having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60),

[0251] wherein X and Y heterodimerize, and wherein the heterodimeric fusion has relaxin activity.

[0252] In yet another aspect, the heterodimeric fusion used in the formulations of the present disclosure comprises

[0253] (i) X-A-L-B and Y, optionally Y-A-L-B or

[0254] (ii) Y-A-L-B and X, optionally X-A-L-B,

[0255] wherein:

[0256] X and Y are heterodimerization domains as described herein;

[0257] A is a relaxin A chain or variant thereof, e.g., a relaxin-2 A chain or variant thereof;

[0258] B is a relaxin B chain or variant thereof, e.g., a relaxin-2 B chain or variant thereof; and

[0259] L is a linker polypeptide, optionally having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60),

[0260] wherein X and Y heterodimerize, and wherein the heterodimeric fusion has relaxin activity.

[0261] In particular embodiments, the heterodimeric fusion comprises fusion polypeptides, i.e., Rlx011DD as set forth in SEQ ID NO: 11 and Rlx014DD as set forth in SEQ ID NO: 20. In particular embodiments, the heterodimeric fusion consists of fusion polypeptides, i.e., Rlx011DD as set forth in SEQ ID NO: 11 and Rlx014DD as set forth in SEQ ID NO: 20 (referred to as “HFUS1” or “RELAX0023”). In alternative embodiments, the heterodimeric fusion comprises fusion polypeptides, i.e., Rlx013DD as set forth in SEQ ID NO: 17 and Rlx012DD as set forth in SEQ ID NO: 14.

[0262] In one aspect of the disclosure, the heterodimeric fusion used in the formulations of the disclosure (e.g., HFUS1) comprises a combination of fusion polypeptides selected from the FcX and FcY combinations set forth in Table 2.

[0263] Table 2: Fusion polypeptide combinations in heterodimeric fusions of the disclosure

[0264]

[0265]

[0266] The sequences of the listed fusion polypeptides are set forth in Table 3.

[0267] **In this particular embodiment, the heterodimeric fusion is an IgG, and comprises an additional polypeptide corresponding to the light chain set forth in SEQ ID NO: 54

[0268] In one aspect, a heterodimeric fusion comprising fusion polypeptides set forth in SEQ ID NO: 11 and SEQ ID NO: 20 is provided.

[0269] In one alternative aspect, a heterodimeric fusion comprising fusion polypeptides set forth in SEQ ID NO: 17 and SEQ ID NO: 14 is provided.

[0270] The fusion polypeptides used in the formulations of the disclosure can be produced by any method known in the art and as described in WO 2021 / 255127. In some embodiments, the fusion polypeptides used in the formulations of the disclosure are produced by recombinantly expressing a nucleic acid molecule encoding the fusion polypeptide in a host cell.

[0271] Methods known to those of skill in the art can be used to construct expression vectors containing nucleic acid molecules. Suitable vectors include, for example, plasmids, phagemids, bacteriophages, or viral vectors.

[0272] Vectors containing nucleic acid molecules can be transferred into host cells by conventional techniques. Suitable host cells are known in the art. In some embodiments, the host cell is a mammalian cell, such as a HEK293 cell or a CHO cell.

[0273] Transfected cells can be cultured by conventional techniques to produce the fusion polypeptides used in the formulations of the disclosure.

[0274] Once a fusion polypeptide of the disclosure has been produced, e.g., by recombinant expression, it can be purified by any method known in the art. Exemplary protein purification techniques include chromatography (e.g., ion exchange, affinity, and / or size- fractionation column chromatography), centrifugation, and differential solubility. WO 2021 / 255127 provides isolated fusion polypeptides that have been separated from the cell culture optionally by at least one purification step.

[0275] Stable pharmaceutical formulations

[0276] The disclosure provides a pharmaceutical formulation comprising a heterodimeric fusion (e.g., HFUS1) and a fatase-tolerant surfactant, wherein the heterodimeric fusion comprises:

[0277] (i) a first heterodimerization domain linked to at least one relaxin A chain polypeptide or variant thereof; and

[0278] (ii) a second heterodimerization domain linked to at least one relaxin B chain polypeptide or variant thereof,

[0279] wherein the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and wherein the heterodimeric fusion has relaxin activity.

[0280] In some embodiments, the pharmaceutical formulation further comprises a buffer. In some embodiments, the pharmaceutical formulation further comprises an excipient.

[0281] Lipase-resistant surfactants

[0282] During the development of the pharmaceutical formulations of the present disclosure, the inventors identified the formation of particles visible over time in the HFUS1 formulations (see Example 3). Surprisingly, the inventors identified the presence of the surfactant PS80 as the cause of particle formation. The inventors identified that the enzymatic hydrolysis of the ester bonds of polysorbate 80 (PS80) by lipase (host cell protein) present in the formulation (due to co-purification with HFUS1 during recombinant manufacturing) can be the cause of PS80 degradation, which in turn leads to the formation of impurities such as free fatty acids (FFAs), which can act as nuclei to trigger aggregation of the HFUS1 protein, resulting in particles. Additionally, the degradation of PS80 over time can result in an effective reduction in the amount of PS80 in the formulation, adversely affecting the protective effect of the surfactant. The use of lipase-resistant surfactants, such as poloxamer 188 (P188) and D-a-tocopheryl polyethylene glycol succinate (TPGS), enables the mitigation of particle formation in HFUS1 formulations.

[0283] A "surfactant" refers to a surface-active agent that lowers the surface tension of a liquid in which it is dissolved. Surfactants can be included in a pharmaceutical formulation for various reasons, including, for example, to prevent or control aggregation, particle formation, or surface adsorption in a liquid formulation, or to prevent or control these phenomena during lyophilization or reconstitution of a lyophilized formulation. Surfactants include, for example, amphoteric organic compounds that exhibit partial solubility in both organic solvents and aqueous solutions. General properties of surfactants include their ability to lower the surface tension of water, lower the interfacial tension between oil and water, and form micelles. Surfactants can be anionic, non-ionic, cationic, amphoteric, zwitterionic, and combinations thereof.

[0284] Surfactants are generally amphiphilic molecules that contain a hydrophilic group and a lipophilic group. The hydrophilic-lipophilic balance (HLB) value can be used as a measure of the ratio between these groups, and can have a value between 0-60, which defines the affinity of the surfactant for water or oil. Molecules with an HLB value greater than 10 have an affinity for water (hydrophilicity), and molecules with an HLB value less than 10 have an affinity for oil (lipophilicity). Non-ionic surfactants have an HLB value ranging from 0-20.

[0285] The critical micelle concentration (CMC) is the concentration at which, or above which, surfactants form micelles. Below the CMC, the surface tension decreases with increasing concentration of surfactant. Above the CMC, additional surfactant added to the system forms micelles.

[0286] Lipase is an enzyme that can hydrolyze triglycerides into their constituent fatty acids and glycerol. Lipases include: lipoprotein lipase, lipase 9, phospholipase 2, phospholipase 2A, oropharyngeal lipase, hepatic lipase, pancreatic lipase, endothelial lipase, bile salt-dependent lipase, lysosomal lipase, hormone-sensitive lipase, gastric lipase, and lingual lipase.

[0287] In some embodiments, the lipase-resistant surfactant is not enzymatically hydrolyzable by lipoprotein lipase, lipase 9, phospholipase 2, phospholipase 2A, oropharyngeal lipase, hepatic lipase, pancreatic lipase, endothelial lipase, bile salt-dependent lipase, lysosomal lipase, hormone-sensitive lipase, gastric lipase, or lingual lipase. In some embodiments, the lipase-resistant surfactant is not enzymatically hydrolyzable by lipoprotein lipase.

[0288] In some embodiments, the lipase-resistant surfactant does not comprise an ester linkage that is enzymatically hydrolyzable by lipoprotein lipase, lipase 9, phospholipase 2, phospholipase 2A, oropharyngeal lipase, hepatic lipase, pancreatic lipase, endothelial lipase, bile salt-dependent lipase, lysosomal lipase, hormone-sensitive lipase, gastric lipase, or lingual lipase. In some embodiments, the lipase-resistant surfactant does not comprise an ester linkage that is enzymatically hydrolyzable by lipoprotein lipase.

[0289] In some embodiments, the lipase-resistant surfactant is a water-soluble non-ionic triblock copolymer formed from polyethylene oxide (PEO) and polypropylene oxide (PPO) blocks. In specific embodiments, the water-soluble non-ionic triblock copolymer is poloxamer 188 (P188).

[0290] Poloxamer 188 (P188; CAS Number 9003-11-6) or Pluronic F68 is a non-ionic triblock copolymer with a defined number of PEO and PPO repeating sequences with a molecular weight of approximately 7680 Da - 9510 Da. P188 has amphiphilic properties due to the presence of two hydrophilic side chains (PPO) attached to a hydrophobic central core (PEO). The HLB value of poloxamer is 29. See Chen et al. (2022). Poloxamer 188 (P188), A Potential Polymeric Protective Agent for Central Nervous System Disorders: A Systematic Review. Curr Neuropharmacol. 20(4):799-808. The melting point of P188 is about 51 °C to about 53 °C. P188 has a CMC of about 24 mg / mL to about 32 mg / mL at 37 °C. See Moghimi et al. (2004). Causative factors behind poloxamer 188 (Pluronic F68, Flocor TM )-induced complement activation in human sera. A protective role against poloxamer-mediated complement activation by elevated serum lipoprotein levels. Biochimica et Biophysica Acta 1689; 103-113.

[0291] Alternatively, the lipase-resistant surfactant is D-a-tocopherol polyethylene glycol succinate (TPGS). Formulations comprising TPGS have been described in WO 2022 / 101826.

[0292] As described in WO 2022 / 101826, TPGS (also known as tocophersolan) is a water-soluble synthetic derivative of natural a-tocopherol (vitamin E) formed by covalently linking a tocopherol succinate (an ester formed by esterification of tocopherol and succinic acid) to a polyethylene glycol (PEG) moiety via an esterification reaction. The general structure of TPGS is:

[0293]

[0294] TPGS has amphiphilic properties due to the presence of a polar hydrophilic head (polyethylene glycol) and a lipophilic tail (phytyl chain of d-a-tocopherol). TPGS surfactants can include PEG moieties of various molecular weights. In one aspect, the PEG moiety of TPGS has a molecular weight of about 1000 Da, and the TPGS molecule is referred to as D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS 1000). As used herein, the term TPGS includes TPGS 1000. TPGS 1000 has an HLB value of 13.2. See Wu and Hopkins. (1999). Characteristics of D-alpha-tocopheryl PEG 1000 succinate for applications as an absorption enhancer in drug delivery systems. Pharm Tech. 23:52-60. TPGS has a melting point of about 37 °C to about 41 °C or about 38 °C, is stable at a pH of about 4.5 to about 7.5, and has a solubility in water of about 20% at 20 °C. TPGS is a highly stable form of vitamin E. See PMCIsochem. (2015). Vitamin E TPGS: NF and Food Grade. Available at pmcisochem.fr / page / info-center. TPGS is generally recognized as safe (GRAS) and has been approved by the Federal Drug Administration (FDA) as an inactive ingredient for oral and topical formulations. TPGS 1000 has a CMC of 0.02% (w / w) at 37 °C. See Wu and Hopkins. (1999). Characteristics of D-alpha-tocopheryl PEG 1000 succinate for applications as an absorption enhancer in drug delivery systems. Pharm Tech. 23:52-60.

[0295] TPGS formulations generally contain a mixture of monomers and dimers, where the monomers include a single vitamin E molecule covalently linked to a water-soluble moiety such as polyethylene glycol (PEG) via a linker, where the water-soluble moiety, e.g., the PEG, has a free, unreacted terminal reactive group, e.g., a free terminal hydroxyl group. The dimers include two vitamin E molecules covalently linked to a water-soluble moiety such as polyethylene glycol (PEG) via one or more linkers, where both terminals of the water-soluble moiety, e.g., both terminal hydroxyl groups of the PEG moiety, have reacted with the linker attached to the vitamin E molecule, such that there is no free terminal reactive group, e.g., hydroxyl group. The monomers and dimers are formed during esterification reactions. In some embodiments, the TPGS formulation comprises at least about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, or about 90% TPGS monomers and less than about 30%, about 25%, about 20%, about 15%, about 14%, about 13%, about 12%, about 11%, or about 10% TPGS dimers. In some embodiments, the TPGS formulation comprises at least about 85% TPGS monomers and less than about 15% TPGS dimers.

[0296] In further embodiments, the lipase-resistant surfactant is selected from the group consisting of P188, TPGS, Kolliphor HS15, Kolliphor EL, Kolliphor RH40, PEG 300, PEG 400, Brij 58, and Brij 35.

[0297] In some embodiments of any aspect of the disclosure, the concentration of the lipase-resistant surfactant is 0.001% (w / v) to 1% (w / v), optionally 0.001% (w / v) to 0.5% (w / v). In some embodiments, the concentration of the lipase-resistant surfactant is 0.005% (w / v) to 1% (w / v), optionally 0.005% (w / v) to 0.2% (w / v). In some embodiments, the concentration of the lipase-resistant surfactant is 0.01% (w / v) to 0.15% (w / v). In some embodiments, the concentration of the lipase-resistant surfactant is 0.01% (w / v) to 0.1% (w / v). In particular embodiments, the concentration of the lipase-resistant surfactant is 0.02% (w / v) to 0.06% (w / v), optionally 0.02% (w / v), 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), or 0.06% (w / v). In particular embodiments, the concentration of the lipase-resistant surfactant is 0.04% (w / v).

[0298] In some embodiments, the lipase-resistant surfactant is poloxamer 188 (P188). Thus, in some embodiments, the formulations of the present disclosure comprise about 0.01% (w / v) to about 0.1% (w / v) of poloxamer 188 (P188). In some embodiments, the formulations of the present disclosure comprise about 0.02% (w / v) to about 0.06% (w / v) of poloxamer 188 (P188). In some embodiments, the formulations of the present disclosure comprise about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), or about 0.06% (w / v) of poloxamer 188 (P188). In particular embodiments, the formulations of the present disclosure comprise about 0.04% (w / v) of poloxamer 188 (P188).

[0299] Buffer and pH

[0300] During the development and optimization of the pharmaceutical formulations of the present disclosure comprising a heterodimeric fusion as described herein (e.g., HFUS1), the inventors have identified that proteases can be the cause of HFUS1 fragmentation and clipping. This enzymatic activity is more likely to occur at lower pH, where proteases are most effective at cleaving molecules. Thus, the inventors identified that the pH of the formulation can be important for minimizing chemical degradation impacts, such as fragmentation and amino acid (AA) clipping of the molecule, as demonstrated in the pH optimization studies (see Example 2).

[0301] In some embodiments of any aspect of the present disclosure, the formulation comprises a buffer having a pH of about 3 to about 10, optionally about 5 to about 8. In some embodiments, the formulation comprises a buffer having a pH of about 5.5 to about 7.5. At lower pH values, the beneficial effects of ionic excipients (e.g., arginine-HCl) and lipase-resistant surfactants (e.g., P188) can still be obtained, although protease activity is higher.

[0302] In some embodiments, the formulation has a pH in the range of about 6 to about 7. In some embodiments, the formulation has a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In some embodiments, the formulation has a pH of about 6. In some embodiments, the formulation has a pH of about 7. In particular embodiments, the formulation has a pH of about 6.5.

[0303] As used herein, “buffer” refers to an acid-base conjugate component that resists changes in pH as known in the art.

[0304] In some embodiments of any aspect of the disclosure, the concentration of the buffering agent is about 0.1 mM to about 100 mM, optionally about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of the buffering agent is about 1 mM to about 50 mM, about 10 mM to about 50 mM, or about 10 mM to about 30 mM. In particular embodiments, the concentration of the buffering agent is about 10 mM to about 30 mM.

[0305] In some embodiments, the concentration of the buffering agent is about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 75 mM, or about 100 mM. In some embodiments, the concentration of the buffering agent is about 10 mM. In some embodiments, the concentration of the buffering agent is about 15 mM. In some embodiments, the concentration of the buffering agent is about 25 mM. In some embodiments, the concentration of the buffering agent is about 30 mM. In particular embodiments, the concentration of the buffering agent is about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM. In particular embodiments, the concentration of the buffering agent is about 20 mM.

[0306] In some embodiments, the buffering agent is selected from the group consisting of acetate, acetic acid, succinate, succinic acid, phosphate, phosphoric acid, ascorbate, ascorbic acid, lactate, lactic acid, tartaric acid, maleic acid, glycine, gluconate, citrate, histidine, imidazole, bicarbonate and carbonic acid, sodium benzoate, benzoic acid, edetate, malate, tris, glycinamide, and mixtures thereof. In particular embodiments, the buffering agent is selected from the group consisting of histidine, citrate, acetate, phosphate, tris, succinate, and mixtures thereof. In particular embodiments, the buffering agent is selected from the group consisting of citrate buffer and histidine buffer. In some embodiments, the buffering agent is a citrate buffer. In some embodiments, the buffering agent is a histidine buffer.

[0307] In particular embodiments, the buffering agent is histidine, histidine hydrochloride, or a histidine / histidine hydrochloride buffer. In particular embodiments, the buffering agent is a histidine / histidine hydrochloride buffer (i.e., a combination of histidine and histidine hydrochloride). In one embodiment, the buffering agent is L-histidine / L-histidine hydrochloride monohydrate.

[0308] In some embodiments, the formulation of the present disclosure comprises about 10 mM to about 50 mM of a histidine / histidine hydrochloride buffer. In some embodiments, the formulation of the present disclosure comprises about 10 mM to about 30 mM of a histidine / histidine hydrochloride buffer. In some embodiments, the formulation of the present disclosure comprises about 15 mM to about 25 mM of a histidine / histidine hydrochloride buffer. In some embodiments, the formulation of the present disclosure comprises about 17 mM to about 23 mM of a histidine / histidine hydrochloride buffer. In some embodiments, the formulation of the present disclosure comprises about 10 mM of a histidine / histidine hydrochloride buffer. In some embodiments, the formulation of the present disclosure comprises about 15 mM of a histidine / histidine hydrochloride buffer. In some embodiments, the formulation of the present disclosure comprises about 25 mM of a histidine / histidine hydrochloride buffer. In some embodiments, the formulation of the present disclosure comprises about 30 mM of a histidine / histidine hydrochloride buffer. In particular embodiments, the formulation of the present disclosure comprises about 20 mM of a histidine / histidine hydrochloride buffer.

[0309] Excipients

[0310] During the development and optimization of the pharmaceutical formulations of the present disclosure comprising a heterodimeric fusion (e.g., HFUS1) as described herein, the inventors have demonstrated that the heterodimeric fusion (e.g., HFUS1) tends to self-associate (see Example 1). High levels of self-association of the molecule can lead to the formation of soluble aggregates, which can become precursors of insoluble large size aggregates, and eventually particles, significantly impacting the stability profile of the molecule. The optimization of the pH, buffering agent, and excipients used in the pharmaceutical formulations of the present disclosure helps to reduce aggregation. Overall, the histidine-arginine HC1 system was identified to provide the highest colloidal and conformational stability to the heterodimeric fusion (e.g., HFUS1) (see Example 2).

[0311] Thus, in some embodiments of any aspect of the present disclosure, the formulation additionally comprises an excipient to reduce protein aggregation.

[0312] The pharmaceutical formulations of the present disclosure can comprise one or more excipients. Pharmaceutically acceptable excipients are known in the art, see, e.g., Remington's Pharmaceutical Sciences (Joseph P. Remington, ed., 18th ed., Mack Publishing Co., Easton, PA), which is incorporated herein in its entirety.

[0313] As used herein, the term "pharmaceutically acceptable" 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.

[0314] In some embodiments, the concentration of the excipient is about 10 mM to about 500 mM, optionally 50 mM to 500 mM. In some embodiments, the concentration of the excipient is 100 mM to 300 mM. In some embodiments, the concentration of the excipient is 100 mM to 150 mM, 150 mM to 200 mM, 200 mM to 250 mM, or 250 mM to 300 mM. In particular embodiments, the concentration of the excipient is 140 mM to 240 mM. In some embodiments, the concentration of the excipient is 140 mM. In some embodiments, the concentration of the excipient is 150 mM. In some embodiments, the concentration of the excipient is 160 mM. In some embodiments, the concentration of the excipient is 170 mM. In some embodiments, the concentration of the excipient is 180 mM. In some embodiments, the concentration of the excipient is 190 mM. In some embodiments, the concentration of the excipient is 200 mM. In some embodiments, the concentration of the excipient is 210 mM. In some embodiments, the concentration of the excipient is 220 mM. In some embodiments, the concentration of the excipient is 230 mM. In some embodiments, the concentration of the excipient is 240 mM. In particular embodiments, the concentration of the excipient is 190 mM.

[0315] In one aspect, the pharmaceutical formulation comprises about 1 mg / ml to about 50 mg / ml, about 5 mg / ml to about 25 mg / ml, about 10 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 10 mg / ml of at least one amino acid as an excipient. In one aspect, the pharmaceutical formulation comprises about 1 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, or about 25 mg / ml to about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 50 mg / ml of at least one amino acid as an excipient. In one aspect, the pharmaceutical formulation comprises about 1 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 50 mg / ml of at least one amino acid as an excipient.

[0316] In some embodiments, the excipient is an ionic excipient. In some embodiments, the ionic excipient is an amino acid salt. By "amino acid salt" is meant an amino acid in cationic or anionic form in combination with a counterion of opposite charge. In some embodiments, the amino acid salt is a pharmacologically acceptable salt. In some embodiments, the amino acid salt is an inorganic salt. In some embodiments, the amino acid salt is an organic salt. In some embodiments, the amino acid salt includes a sodium salt, a potassium salt, a calcium salt, a magnesium salt, an ammonium salt, a hydrochloride salt, a sulfate salt, a nitrate salt, or a phosphate salt. In some embodiments, the amino acid salt includes an organic acid salt, such as an acetate salt, a citrate salt, a maleate salt, a malate salt, or an oxalate salt. In some embodiments, the formulation comprises an amino acid salt in the form of a salt selected from arginine, cysteine, glycine, lysine, ornithine, proline, alanine, glutamine, glutamic acid, histidine, valine, or a combination thereof. In some embodiments, the amino acid salt includes arginine, lysine, or histidine. In some embodiments, the amino acid salt is selected from an arginine salt or a lysine salt. In some embodiments, the ionic excipient is selected from arginine HC1 or lysine HC1. In particular embodiments, the ionic excipient is arginine HC1.

[0317] In some embodiments, the formulation of the present disclosure comprises about 100 mM to about 300 mM arginine HC1. In specific embodiments, the formulation of the present disclosure comprises about 140 mM to about 240 mM arginine HC1. In some embodiments, the formulation of the present disclosure comprises about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, or about 240 mM arginine HC1. In specific embodiments, the formulation of the present disclosure comprises about 180 mM to about 200 mM arginine HC1. In specific embodiments, the formulation of the present disclosure comprises about 190 mM arginine HC1.

[0318] Additional components

[0319] In some embodiments of any aspect of the present disclosure, the formulation further comprises a sugar. The presence of a sugar can improve the tonicity of the formulation. In specific embodiments, the concentration of the sugar is sufficient to make the formulation isotonic or near isotonic. In some embodiments, the sugar is selected from the group consisting of sucrose, trehalose, fructose, glucose, mannose, melibiose, melezitose, raffinose, manninotriose, stachyose, sorbose, xylose, lactose, maltose, maltulose, isomaltulose, lactulose, amylopectin, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble dextran, polyols such as trihydric or higher sugar alcohols (e.g., glycerol, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol), and mixtures thereof. In specific embodiments, the sugar is sucrose.

[0320] In some embodiments, the concentration of the sugar is 1 mg / ml to about 300 mg / ml, about 10 mg / ml to about 200 mg / ml, about 50 mg / ml to about 100 mg / ml, or about 80 mg / ml to about 90 mg / ml. In some embodiments, the concentration of the sugar is about 30 mg / ml to about 90 mg / ml.

[0321] Concentration of heterodimeric fusions

[0322] The present inventors evaluated the effect of heterodimer fusion protein (e.g., HFUS1) concentration on the stability of the pharmaceutical formulation. Heterodimer fusion protein (e.g., HFUS1) showed good stability in formulations with different heterodimer fusion protein (e.g., HFUS1) concentrations (see Examples 4 to 6).

[0323] mg / mL, 1.1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42, mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, or 50 mg / mL. In particular embodiments, the concentration of the heterodimeric fusion protein (e.g., HFUS1) in the formulation is 0.25 mg / mL, 1 mg / mL, 5 mg / mL, or 50 mg / mL. In some embodiments, the concentration of the heterodimeric fusion protein (e.g., HFUS1) in the formulation is 0.25 mg / mL. In some embodiments, the concentration of the heterodimeric fusion protein (e.g., HFUS1) in the formulation is 1 mg / mL. In some embodiments, the concentration of the heterodimeric fusion protein (e.g., HFUS1) in the formulation is 1.1 mg / mL. In some embodiments, the concentration of the heterodimeric fusion protein (e.g., HFUS1) in the formulation is 5 mg / mL. In some embodiments, the concentration of the heterodimeric fusion protein (e.g., HFUS1) in the formulation is 30 mg / mL. In some embodiments, the concentration of the heterodimeric fusion protein (e.g., HFUS1) in the formulation is 33 mg / mL. In some embodiments, the concentration of the heterodimeric fusion protein (e.g., HFUS1) in the formulation is 50 mg / mL.

[0324] In some example embodiments, the formulation comprises 0.2 mg / mL to 50 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0325] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0326] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.0.

[0327] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 7.0.

[0328] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 140 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0329] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 240 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0330] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.02% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0331] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.06% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0332] In some embodiments, the formulation comprises 5 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0333] In some embodiments, the formulation comprises 1.1 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0334] In some embodiments, the formulation comprises 1 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0335] In some embodiments, the formulation comprises 1 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 140 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 7.0.

[0336] In some embodiments, the formulation comprises 0.25 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0337] In some embodiments, the formulation comprises 0.25 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 140 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 7.0.

[0338] In some embodiments, the formulation comprises 33 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.0.

[0339] In some embodiments, the formulation comprises 33 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0340] In some embodiments, the formulation comprises 33 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 7.0.

[0341] In some embodiments, the formulation comprises 33 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 150 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0342] In some embodiments, the formulation comprises 33 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 230 mM Arginine HC1, 0.04% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0343] In some embodiments, the formulation comprises 33 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.02% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0344] In some embodiments, the formulation comprises 33 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM Histidine / Histidine hydrochloride buffer, 190 mM Arginine HC1, 0.06% (w / v) Poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0345] In some embodiments, the formulation comprises 30 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HC1, 0.04% (w / v) poloxamer 188 (P188), and wherein the formulation has a pH of 6.0.

[0346] In some embodiments, the formulation comprises 30 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HC1, 0.04% (w / v) poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0347] In some embodiments, the formulation comprises 30 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HC1, 0.04% (w / v) poloxamer 188 (P188), and wherein the formulation has a pH of 7.0.

[0348] In some embodiments, the formulation comprises 30 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 150 mM arginine HC1, 0.04% (w / v) poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0349] In some embodiments, the formulation comprises 30 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 230 mM arginine HC1, 0.04% (w / v) poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0350] In some embodiments, the formulation comprises 30 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HC1, 0.02% (w / v) poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0351] In some embodiments, the formulation comprises 30 mg / mL of a heterodimeric fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HC1, 0.06% (w / v) poloxamer 188 (P188), and wherein the formulation has a pH of 6.5.

[0352] Stability

[0353] The present inventors have described some deliberately designed studies to identify the root cause of the instability of using a heterodimeric fusion (e.g., HFUS1) in the formulations of the present disclosure (see Example 1), as well as formulation development and optimization work to identify stable liquid formulations of the molecule to meet its drug product shelf-life requirements (see Examples 2-6).

[0354] A “stable” formulation is one in which the heterodimeric fusion (e.g., HFUS1) retains its physical stability, chemical stability, or biological activity during storage. “Chemical stability” can be assessed by detecting and quantifying chemically altered forms of the heterodimeric fusion (e.g., HFUS1), including, for example, deamidation, including, for example, asparagine (Asn) deamidation; isomerization, including, for example, aspartic acid (Asp) isomerization; oxidation, including, for example, methionine (Met) oxidation; clipping / hydrolysis / fragmentation, including, for example, antibody hinge region fragmentation; succinimide formation; racemization; beta-elimination; glycation; adduct formation; disulfide scrambling; N-terminal extensions; C-terminal processing; and glycosylation differences. “Physical stability” can be assessed by detecting and quantifying physically altered forms of the heterodimeric fusion (e.g., HFUS1), including, but not limited to, those resulting from denaturation, aggregation, precipitation, or particle formation, and surface adsorption.

[0355] Stability can be evaluated qualitatively and / or quantitatively in a number of different ways, including, but not limited to, evaluation of aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino- or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE; peptide mapping; or by evaluating biological activity, e.g., via in vitro, in vivo, or in situ assays that are indicative of heterodimeric fusion (e.g., HFUS1) activity.

[0356] A heterodimeric fusion (e.g., HFUS1) is “stable” in a pharmaceutical formulation if its physical stability, chemical stability, or biological activity at a given time is within about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, or about 25% (within error standards) of the physical stability, chemical stability, or biological activity exhibited by the heterodimeric fusion (e.g., HFUS1) at an initial time point, e.g., at the time of preparation of the pharmaceutical formulation.

[0357] Generally, stability is determined relative to a selected temperature and a selected time period. In some embodiments, the formulation is stable at a temperature of about 5°C ± 10°C, about 5°C ± 5°C for at least about 2 weeks, about 1 month, about 2 months, or about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the formulation is stable at a temperature of about 25°C ± 10°C, about 25°C ± 5°C for at least about 2 weeks, about 1 month, about 2 months, or about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the formulation is stable at a temperature of about 40°C ± 10°C, about 40°C ± 5°C for at least about 2 weeks, about 1 month, about 2 months, or about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months.

[0358] In some embodiments, the formulation is stable at a temperature of about 2°C to about 8°C, or at about 2°C, about 4°C, about 5°C, about 6°C, or about 8°C, for at least about 2 weeks, about 1 month, about 3 months, or about 6 months, and for up to about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the formulation is stable at a temperature of about 22°C to about 28°C, or at about 22°C, about 24°C, about 25°C, about 26°C, or about 28°C, for at least about 2 weeks, about 1 month, about 3 months, or about 6 months, and for up to about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the formulation is stable at a temperature of about 37°C to about 43°C, or at about 37°C, about 39°C, about 40°C, about 41°C, or about 43°C, for at least about 2 weeks, about 1 month, about 3 months, or about 6 months, and for up to about 9 months, about 12 months, about 24 months, or about 36 months.

[0359] In some embodiments, the formulation is stable at 40°C for up to about 3 months. In some embodiments, the formulation is stable at 40°C for up to about 6 months. In some embodiments, the formulation is stable at 25°C for up to about 6 months. In some embodiments, the formulation is stable at 25°C for up to about 12 months. In some embodiments, the formulation is stable at 25°C for up to about 24 months. In some embodiments, the formulation is stable at 5°C for up to about 6 months. In some embodiments, the formulation is stable at 5°C for up to about 12 months. In some embodiments, the formulation is stable at 5°C for up to about 24 months. In some embodiments, the formulation is stable at 5°C for up to about 36 months.

[0360] In some embodiments, the heterodimeric fusion (e.g., HFUS1) in the pharmaceutical formulation is chemically stable. In some embodiments, the increase in chemically altered forms of the heterodimeric fusion (e.g., HFUS1) in the pharmaceutical formulation is less than about 25%, about 20%, about 15%, about 10%, or about 5% when stored at a temperature of about 40 °C, about 25 °C, about 2 °C to about 8 °C, or about 5 °C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the increase in one or more chemically altered forms of the heterodimeric fusion (e.g., HFUS1) is less than about 25%, about 20%, about 15%, about 10%, or about 5%, including, for example, chemically altered forms resulting from deamidation, including, for example, asparagine (Asn) deamidation; oxidation, including, for example, methionine, cysteine, histidine, tyrosine, tryptophan, or phenylalanine oxidation; intra- and inter-residue cyclization (asparagine and glutamic acid, glutamine, N-terminal dipeptidyl motifs); clipping / hydrolysis / fragmentation; beta-elimination; glycation; and disulfide scrambling.

[0361] In some embodiments, the heterodimeric fusion (e.g., HFUS1) in the pharmaceutical formulation is physically stable. In some embodiments, the increase in physically altered forms of the heterodimeric fusion (e.g., HFUS1) in the pharmaceutical formulation is less than about 25%, about 20%, about 15%, about 10%, or about 5% when stored at a temperature of about 40 °C, about 25 °C, about 2 °C to about 8 °C, or about 5 °C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, or about 6 months and up to about 9 months, about 12 months, about 24 months, or about 36 months. In one aspect, the increase in one or more physically altered forms of the heterodimeric fusion (e.g., HFUS1) is less than about 25%, about 20%, about 15%, about 10%, or about 5%, including, for example, physically altered forms resulting from denaturation, aggregation, precipitation, or microparticle formation.

[0362] In some embodiments, the heterodimeric fusion (e.g., HFUS1) in the pharmaceutical formulation is biologically stable. In some embodiments, the decrease in biological activity of the heterodimeric fusion (e.g., HFUS1) in the pharmaceutical formulation is less than about 25%, about 20%, about 15%, about 10%, or about 5% when stored at a temperature of about 40 °C, about 25 °C, about 2 °C to about 8 °C, or about 5 °C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, or about 6 months and up to about 9 months, about 12 months, about 24 months, or about 36 months.

[0363] In some embodiments, the concentration of the lipase-resistant surfactant in the pharmaceutical formulation remains stable over the storage period. In some embodiments, the concentration of the lipase-resistant surfactant in the pharmaceutical formulation decreases by less than about 25%, about 20%, about 15%, about 10%, or about 5% when stored at a temperature of about 40 °C, about 25 °C, about 2 °C to about 8 °C, or about 5 °C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months.

[0364] In some embodiments, the stability of the pharmaceutical formulation is evaluated by measuring the amount of particulate impurities. Particulate impurities can include visible, subvisible, and submicron impurities. Visible impurities have a diameter greater than about 100 pm or about 150 pm and can be detected by visual inspection. Subvisible particles typically range in size from about 1 pm to about 100 pm or about 150 pm. Submicron particles have a diameter less than about 1 pm. Subvisible particles pose the greatest risk when present in a pharmaceutical formulation, particularly when present in a pharmaceutical formulation for parenteral administration, including subcutaneous, intravenous, or intramuscular administration, in some cases due to the ability of subvisible particles to elicit an adverse immunogenic response. Subvisible particles having a diameter greater than or equal to about 10 pm or greater than or equal to about 5 pm can block blood vessels in the lungs following vascular infusion. Methods for detecting and quantifying particulate impurities are known and include dynamic light scattering (DLS) or static light scattering (SLS), nanoparticle tracking analysis (NTA), optical microscopy, electrical sensing zone (ESZ), flow imaging technology, resonance mass measurement, electron microscopy, Fourier transform infrared (FTIR) microscopy, and Raman microscopy. In some embodiments, subvisible particles are detected using microflow imaging (MFI). In some embodiments, subvisible particles are detected using light obstruction methods, such as a high precision product (HIAC) system, such as a HIAC system model 9703 equipped with a HRLD150 sensor.

[0365] In some embodiments of any aspect of the disclosure, the pharmaceutical formulation comprises less than about 10,000, about 6000, about 5,000, about 1,000, about 750, about 600, about 500, about 250, about 150, about 100, or about 50 particles per mL of greater than about 2 pm, about 5 pm, about 10 pm, about 15 pm, about 20 pm, or about 25 pm diameter. In some embodiments, the pharmaceutical formulation comprises less than about 10,000, about 6000, about 5,000, about 1,000, about 750, about 600, about 500, about 250, about 150, about 100, or about 50 particles per mL of greater than 2 pm, 5 pm, or 10 pm diameter. In some embodiments, the pharmaceutical formulation comprises less than about 10,000, about 6000, about 5,000, about 1,000, about 750, about 600, about 500, about 250, about 150, about 100, or about 50 particles per mL of greater than 2 pm diameter. In some embodiments, the pharmaceutical formulation comprises less than about 10,000, about 6000, about 5,000, about 1,000, about 750, about 600, about 500, about 250, about 150, about 100, or about 50 particles per mL of greater than 10 pm diameter.

[0366] The pharmaceutical formulation can comprise a sub-visible particle count within the USP limits (no more than 6000 and 600 for particle sizes of >10 pm and >25 pm, respectively; Pharmacopeia US. 2014. USP 787 and 788). Thus, in particular embodiments, the pharmaceutical formulation comprises less than about 6000 particles per mL of greater than about 10 pm diameter. In particular embodiments, the pharmaceutical formulation comprises less than about 600 particles per mL of greater than about 25 pm diameter.

[0367] In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 40°C ± 10°C, about 30°C ± 10°C, about 25°C ± 10°C, about 20°C ± 10°C, or about 5°C ± 3°C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 40°C ± 10°C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 40°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 30°C ± 10°C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 30°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 25°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 25°C ± 5°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 20°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 20°C ± 5°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 24 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 36 months.

[0368] In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 40 °C ± 10 °C, about 30 °C ± 10 °C, about 25 °C ± 10 °C, about 20 °C ± 10 °C, or about 5 °C ± 3 °C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 40 °C ± 10 °C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 40 °C ± 10 °C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 30 °C ± 10 °C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 30 °C ± 10 °C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 25 °C ± 10 °C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 25 °C ± 5 °C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 20 °C ± 10 °C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 20 °C ± 5 °C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 5 °C ± 3 °C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 5 °C ± 3 °C for up to about 24 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 2 pm in diameter when stored at a temperature of about 5 °C ± 3 °C for up to about 36 months.

[0369] In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 40°C ± 10°C, about 30°C ± 10°C, about 25°C ± 10°C, about 20°C ± 10°C, or about 5°C ± 3°C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 40°C ± 10°C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 40°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 30°C ± 10°C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 30°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 25°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 25°C ± 5°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 20°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 20°C ± 5°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 24 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 36 months.

[0370] In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 40 °C ± 10 °C, about 30 °C ± 10 °C, about 25 °C ± 10 °C, about 20 °C ± 10 °C, or about 5 °C ± 3 °C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 40 °C ± 10 °C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 40 °C ± 10 °C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 30 °C ± 10 °C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 30 °C ± 10 °C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 25 °C ± 10 °C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 25 °C ± 5 °C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 20 °C ± 10 °C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 20 °C ± 5 °C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 5 °C ± 3 °C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 5 °C ± 3 °C for up to about 24 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 5 pm in diameter when stored at a temperature of about 5 °C ± 3 °C for up to about 36 months.

[0371] In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 40°C ± 10°C, about 30°C ± 10°C, about 25°C ± 10°C, about 20°C ± 10°C, or about 5°C ± 3°C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 40°C ± 10°C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 40°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 30°C ± 10°C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 30°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 25°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 25°C ± 5°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 20°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 20°C ± 5°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 24 months. In some embodiments, the pharmaceutical formulation comprises less than about 6,000 or 5,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 36 months.

[0372] In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 40°C ± 10°C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 40°C ± 10°C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 40°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 30°C ± 10°C for up to about 3 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 30°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 25°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 25°C ± 5°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 20°C ± 10°C for up to about 6 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 20°C ± 5°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 12 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 24 months. In some embodiments, the pharmaceutical formulation comprises less than about 1,000 particles per mL greater than about 10 pm in diameter when stored at a temperature of about 5°C ± 3°C for up to about 36 months.

[0373] Methods of treatment

[0374] The present disclosure encompasses therapies involving administration of a pharmaceutical formulation of the present disclosure to an animal, particularly a mammal (e.g., a human) to prevent, treat, or ameliorate symptoms associated with a disease, disorder, or infection.

[0375] Accordingly, the pharmaceutical formulations of the present disclosure are useful in therapy, e.g., for treating a disease or disorder. Also provided is a method of treating a disease or disorder, the method comprising administering to a subject or patient in need thereof a therapeutically effective amount of a fusion polypeptide as described herein (e.g., HFUS1). The use or the method can comprise administering a therapeutically effective regimen having a lower frequency of doses of the fusion polypeptide as described herein (e.g., HFUS1) than a therapeutically effective dosing regimen of a wild-type relaxin molecule.

[0376] The heterodimeric fusions described herein have been described as useful for treating various disorders, including heart failure and heart failure with pulmonary arterial hypertension (see WO 2023 / 111112, which is incorporated by reference herein). Accordingly, it is understood that the pharmaceutical formulations of the present disclosure are useful for treating cardiovascular diseases, e.g., for treating heart failure, and more particularly for treating heart failure with pulmonary arterial hypertension.

[0377] The pharmaceutical formulations of the present disclosure are also useful for treating renal, pulmonary, and fibrotic disorders, e.g., fibrotic disorders of the kidney, heart, lung, and liver, and for wound healing (Sherwood OD (2004) Endocrine Reviews 25(2):205-234). The fusion polypeptides as described herein (e.g., HFUS1) are also useful for reversing insulin resistance in diabetic patients (Bonner JS et al. (2013) Diabetes 62(9):3251-3260). The pharmaceutical formulations of the present disclosure are also useful for various forms of pulmonary arterial hypertension. The pharmaceutical formulations of the present disclosure are also useful for disorders caused by arteriosclerosis, reduced arterial elasticity, reduced arterial compliance and distensibility, including hypertension, renal disease, peripheral arterial disease, carotid and cerebrovascular disease (i.e., stroke and dementia), diabetes, microvascular disease leading to end-organ damage, coronary heart disease, and heart failure.

[0378] The present disclosure encompasses methods of treating a subject having heart failure, particularly heart failure with pulmonary arterial hypertension, by administering a pharmaceutical formulation as described herein, and the use of the pharmaceutical formulation for said methods. In particular, the subject can be an animal, particularly a mammal, more particularly a human.

[0379] The use or the method can comprise administering a therapeutically effective regimen having a lower frequency of doses of the heterodimeric fusion / fusion polypeptide as described herein (e.g., HFUS1) than a therapeutically effective dosing regimen of a wild-type relaxin molecule.

[0380] 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” can also include more specific diagnoses such as heart failure with preserved ejection fraction (HFpEF), heart failure with mid-range ejection fraction, or heart failure with reduced ejection fraction (HFrEF). Heart failure caused by hypertrophic cardiomyopathy or dilated cardiomyopathy can also be included.

[0381] As used herein, the term“pulmonary arterial hypertension” can be defined as a mean pulmonary arterial pressure of about 20 mmHg or greater, optionally 25 mmHg or greater, in a subject typically when the subject is at rest. It can also be defined as a mean pulmonary arterial pressure of about 30 mmHg or greater, typically when the subject is exercising or has been exercising recently. Thus, the mean pulmonary arterial pressure of a subject can range from about 20 mmHg to about 30 mmHg, optionally from about 25 mmHg to about 30 mmHg or greater. Alternatively or additionally, a subject can have:

[0382] a. a right ventricular systolic pressure of about 40 mmHg or greater;

[0383] b. a pulmonary arterial wedge pressure (PAWP) of greater than 15 mmHg; and / or

[0384] c. a pulmonary vascular resistance of:

[0385] i. less than 3.0 wood units; or

[0386] ii. 3.0 or greater wood units.

[0387] Thus, in some cases, the pulmonary arterial hypertension can be classified as type 2 pulmonary arterial hypertension according to the World Health Organization’s definition. This can also be referred to as“heart failure with preserved ejection fraction due to left heart disease with pulmonary hypertension.” In other cases, the pulmonary arterial hypertension can be classified as type 1 pulmonary arterial hypertension according to the World Health Organization’s definition (see Ryan et al., 2012, Pulm. Circ. 2(1): 107-121).

[0388] The parameters of pulmonary arterial hypertension and heart failure can be measured or estimated using techniques known in the art. For example, these techniques include echocardiography, pulmonary artery catheterization, and implantable monitoring devices. In certain embodiments, the subject can have an installed blood pressure monitoring device, optionally a pulmonary arterial pressure monitoring device as known in the art. In particular embodiments, the pulmonary arterial pressure monitoring device is a CardioMEMS pressure monitoring device. Typically, the device is installed prior to treatment with the heterodimeric fusion described herein (e.g., HFUS1). Alternatively, the subject has the device installed during or after the treatment period.

[0389] As used herein, the term“heart failure with pulmonary hypertension” refers to the subset of heart failure subjects (HF+PH subjects) who also have pulmonary hypertension.

[0390] “Treatment” refers to amelioration and / or elimination of one or more symptoms or causes of a target disease. In some embodiments, this can involve modulating the level of one or more biomarkers or functions to a non-diseased range (as compared to a healthy cohort). For example, a pharmaceutical preparation of the present disclosure can reduce pulmonary vascular resistance (PVR) in a subject. For example, 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 after treatment as compared to baseline PVR (prior to administration of a heterodimeric fusion (e.g., HFUS1) as described herein to a subject). Thus, a pharmaceutical preparation of the present disclosure 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 as compared to baseline PVR (prior to administration of a pharmaceutical preparation of the present disclosure to a subject). Additionally or alternatively, a pharmaceutical preparation of the present disclosure can reduce mean pulmonary arterial pressure (mPAP) in a subject. For example, mPAP can be reduced by at least 1 mmHg to 15 mmHg or more. Thus, a pharmaceutical preparation of the present disclosure can reduce mean pulmonary arterial pressure in a subject by at least 1 mmHg, at least 2 mmHg, at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 11 mmHg, at least 12 mmHg, at least 13 mmHg, at least 14 mmHg, or at least 15 mmHg or more. Likewise, a pharmaceutical preparation of the present disclosure can reduce estimated pulmonary arterial diastolic pressure (ePAD) in a subject. For example, ePAD can be reduced by at least 1 mmHg to 15 mmHg or more. Thus, a pharmaceutical preparation of the present disclosure can reduce estimated pulmonary arterial diastolic pressure in a subject by at least 1 mmHg, at least 2 mmHg, at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 11 mmHg, at least 12 mmHg, at least 13 mmHg, at least 14 mmHg, or at least 15 mmHg or more. Additionally or alternatively, a pharmaceutical preparation of the present disclosure can increase the percent ejection fraction (EF%) in a subject as a measure of cardiac output. For example, EF% can be increased by at least 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, or 1% to 50% or more. Thus, a pharmaceutical preparation of the present disclosure can increase the percent ejection fraction (EF%) in a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more. Additionally or alternatively, a pharmaceutical preparation of the present disclosure can:

[0391] (a) increase the stroke volume (SV) of the heart;

[0392] (b) reducing systemic vascular resistance (SVR) and / or increasing estimated glomerular filtration rate (eGFR);

[0393] (c) increasing ejection fraction; and / or

[0394] (d) increasing cardiac output;

[0395] The combination of a reduction in SVR and an increase in eGFR is indicative of improved organ perfusion.

[0396] Accordingly, the pharmaceutical formulation of the present disclosure can:

[0397] (a) reduce PVR;

[0398] (b) reduce mPAP;

[0399] (c) reduce ePAD;

[0400] (d) increase stroke volume (SV) of the heart;

[0401] (e) reduce systemic vascular resistance (SVR) and / or increase estimated glomerular filtration rate (eGFR);

[0402] (f) increase ejection fraction; and / or

[0403] (g) increase cardiac output;

[0404] The combination of a reduction in SVR and an increase in eGFR is indicative of improved organ perfusion. Changes in one or more or all of these parameters can each occur after 1 to 24 weeks of treatment. In some embodiments, changes in one or more or all of these parameters occur after 24 weeks of treatment.

[0405] In particular embodiments, the reduction in mPAP as described herein can result in an improvement in dyspnea as described in Solomonica A et al. (2013) Circ Heart Fail. 6:53-60.

[0406] The pharmaceutical formulation of the present disclosure is suitable for parenteral administration to a subject or patient. In some embodiments, the subject or patient is a mammal, especially a human.

[0407] Wild-type human relaxin-2 has a half-life of several minutes in vivo. Thus, it must be administered by continuous intravenous infusion in hospitalized patients and presents serious side effects, including a drop in blood pressure. In contrast, it is understood that embodiments of the pharmaceutical formulations of the present disclosure can be administered to a subject or patient by injection, such as by intravenous, subcutaneous, or intramuscular injection. In some embodiments, the pharmaceutical formulation is administered by subcutaneous injection. Administration by injection, such as by subcutaneous injection, provides the advantage of greater comfort for the subject or patient, and provides the opportunity for administration to the subject or patient outside of a hospital setting. In some embodiments, the pharmaceutical formulation is administered by self-administration.

[0408] In some embodiments, the fusion polypeptides used in the formulations of the present disclosure, thus including heterodimeric fusions, e.g., HFUS1, have increased half-life compared to wild-type relaxin, which allows for lower total exposure on a molar concentration basis. For example, the fusion polypeptides used in the formulations of the present disclosure, thus including heterodimeric fusions, e.g., HFUS1, can be administered less frequently than wild-type relaxin, thus providing a more convenient dosing regimen.

[0409] Articles of manufacture

[0410] In one aspect, an article of manufacture is provided. In some embodiments, the article of manufacture includes a device or container containing a pharmaceutical preparation comprising a heterodimeric fusion having relaxin activity (e.g., HFUS1) as defined herein and a lipase-resistant surfactant as described herein. In one embodiment, the article of manufacture includes a device or container containing a pharmaceutical preparation comprising a heterodimeric fusion having relaxin activity (e.g., HFUS1) as defined herein and a lipase-resistant surfactant selected from poloxamer 188 (P188) and D-a-tocopheryl polyethylene glycol succinate (TPGS). In one embodiment, the article of manufacture includes a device or container containing a pharmaceutical preparation comprising a heterodimeric fusion having relaxin activity (e.g., HFUS1) as defined herein and poloxamer 188 (P188). In some embodiments, the container or the device is a syringe, e.g., a pre-filled syringe; an auto-injector; a bottle; a vial; or a test tube. In particular embodiments, the container or the device is a syringe, optionally a pre-filled syringe. Thus, in particular embodiments, the article of manufacture includes a syringe, e.g., a pre-filled syringe, containing a pharmaceutical preparation comprising a heterodimeric fusion having relaxin activity (e.g., HFUS1) as defined herein and poloxamer 188 (P188). In some embodiments, the article of manufacture includes a device or container containing a pharmaceutical preparation and a label providing instructions for use on or associated with the device or the container. In some embodiments, the article of manufacture further includes other materials that are desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, or package inserts with instructions for use.

[0411] In one aspect, a kit comprising a pharmaceutical preparation of the disclosure is provided. The kit can include a package containing a pharmaceutical preparation of the disclosure and instructions for administering the preparation. In some embodiments, the kit includes at least one device or container containing a pharmaceutical preparation comprising a heterodimeric fusion having relaxin activity (e.g., HFUS1) as defined herein and a lipase-resistant surfactant as described herein. In some embodiments, the at least one device or container is selected from a syringe, e.g., a pre-filled syringe; an auto-injector; a bottle; a vial; or a test tube. In particular embodiments, the at least one device or container is a syringe, e.g., a pre-filled syringe.

[0412] The present disclosure provides a kit comprising a pharmaceutical preparation of the disclosure. The kit can include a package containing a pharmaceutical preparation of the disclosure and instructions for administering the preparation.

[0413] In some embodiments, the kit comprises at least one device or container containing a pharmaceutical formulation of the disclosure and an injection device. In some embodiments, the injection device is suitable for intravenous, intramuscular, or subcutaneous administration. In one embodiment, the injection device is suitable for subcutaneous administration. In a particular embodiment, the injection device is a syringe, e.g., a pre-filled syringe, containing a pharmaceutical formulation comprising a heterodimeric fusion having relaxin activity, e.g., HFUS1, as defined herein, and a fatase-resistant surfactant, e.g., poloxamer 188 (P188), as defined herein.

[0414] In some embodiments, the pharmaceutical formulation of the disclosure is formulated in a single-dose vial or container closure system, e.g., a pre-filled syringe. Optionally, associated with such a container can be a label in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which reflects approval by the agency that the pharmaceutical or biological product was manufactured, used, or sold for human administration. In some embodiments, the kit includes instructions.

[0415] The above embodiments are to be understood as illustrative examples. Additional embodiments are contemplated. It should be understood that any features described in relation to any one embodiment can be used individually and in combination with other features described, and also in combination with one or more features of any other embodiment, or in combination with any combination of any other embodiment. Furthermore, equivalents and modifications not described above can also be employed without departing from the scope of the disclosure defined in the appended claims.

[0416] Other embodiments and variations are within the scope of the disclosure, as described in the appended claims. All documents cited herein are each fully incorporated by reference herein, including all data, tables, graphs, and textual content present in the cited documents.

[0417] Sequences

[0418] Table 3: Sequence Listing. Upper hinge region is italicized, relaxin A is underlined, relaxin B is double underlined, FC region is bolded Example .

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430] Materials and methods

[0431] Materials

[0432] Methods

[0433] All materials used for the study were USP or multi-drug compendia grade. All solutions and buffers were prepared using USP or HPLC water and filtered through 0.2 pm PVDF filters (Millipore, Millex GV, SLG033RB) before further use. HFUS1 samples for formulation screening and stability studies were prepared under aseptic conditions in a biological safety cabinet (BSC). Bulk material was stored at 5 °C. All formulations were filtered through 0.22 pm PVDF filter units and filled into 2R glass vials or pre-filled syringes (PFS) and stored in incubators set to 5 °C, 25 °C and 40 °C for stability time points.

[0434] Protein concentration determination

[0435] Purity determination by size exclusion chromatography

[0436] HFUS1 protein concentration was determined by measuring absorbance at 280 nm with a Trinean HT-A280 using a procedure adapted from SOP DV-050465. An extinction coefficient of 1.47 (mg / mL) -1 cm -1 Protein concentration was calculated.

[0437] Isoelectric point determination

[0438] In an Agilent Technologies 1200 / 1100 HPLC system, using a temperature controlled autosampler at 8 °C, a TSKgel G3000SWXL, 5 pm, 300 A, 7.8 x 300 mm column (Tosoh Bioscience) was used. Size exclusion chromatography was performed on a TSKgel column (7.8 x 300 mm) and guard column of 7.8 mm TSKgel from Tosoh Bioscience and Agilent ChemStation software. The UV lamp was turned on 30 minutes prior to analysis and the system was primed with mobile phase (0.1 M disodium hydrogen phosphate anhydrous, 0.1 M sodium sulphate, pH 6.8) at 1.0 ml / min for at least 30 minutes, monitoring the pressure to a maximum of 130 bar. Samples were diluted to 10 mg / mL in PBS and filtered using an Ultrafree-MC centrifugal filter unit (Merck) with a PVDF membrane and 0.45 pm pore size. All samples were run at 280 nm, the column flow rate was 1.0 ml / min and the stop time was 20 minutes, the injection volume was 25 pl and the column temperature was 20 °C.

[0439] Visual appearance evaluation

[0440] Capillary isoelectric focusing (cIEF) was used using ProteinSimple's Maurice, following the manufacturer's protocol The isoelectric point of HFUS1 was determined.

[0441] Subvisible particle analysis

[0442] All samples and standards were equilibrated at room temperature prior to performing visual testing of the formulation. The exterior of the glass vials were wiped with alcohol to remove any particles or residue on the glass surface. Visual inspection was performed manually in a white and black background light cabinet (Apollo II Liquid Viewer, Adelphi Manufacturing Co Ltd.) with a Lux range of 3000-3400. The cabinet light was turned on 20 minutes prior to use to ensure the appropriate light intensity.

[0443] The determination of visible particles was performed by comparing the sample to a particle standard with increasing concentrations of barium sulphate. The particle standard was numbered from 0 to 7, with 7 being the highest particle number and was used as a guide when determining visible particles in the sample. The sample was vortexed 10-20 times to ensure homogeneity and compared to the standard under a black background approximately half way down the panel and directly under the top light of the panel.

[0444] Differential scanning calorimetry (DSC) measurement of unfolding temperature

[0445] Sub-visible particle counting was performed using MFI or HIAC as indicated herein.

[0446] For MFI, a MFI 5200 series model with 100 pm 1.6 mm silane-coated flow cell (Protein simple, United States) was used. Prior to use, the system was cleaned with approximately 10 mL of non-ionized ultrapure water followed by 1 mL of 10% Decon and rinsed with 10 mL of non-ionized ultrapure water using the System Flush Preview mode. The cell was allowed to dry to check that the silane coating was intact and to ensure that no particles were attached to its surface. If the cell became dirty, the cleaning procedure was performed again. To assess the system cleanliness, a water background run was performed by priming the cell with 250 pi of water at 1 ml / min and analyzing approximately 800 pi of the water sample under optimized illumination. The real-time image was monitored to check for large particles or air bubbles entering the system.

[0447] For HIAC, a High Precision Product (HIAC) system model 9703 equipped with a HRLD150 sensor was used. Samples were diluted to 25 mg / mL prior to analysis and degassed for 30 minutes prior to measurement. For each sample, a representative buffer was taken for particle count to assess the contribution of the dilution buffer to the total particle count. Four measurements were taken for each sample.

[0448] Dynamic light scattering (DLS) measurement

[0449] DSC experiments were performed in an automated MicroCal VP-Capillary DSC system from Malvern Panalytical (UK). Samples were diluted to 5 mg / ml using the respective formulation buffer and filtered with a 0.22 pm PVD centrifugal filter unit. At the beginning and end of each run, lysozyme (3 mg / ml) was used as a standard to assess system suitability. Scans were run from 25 °C to 100 °C at a scan rate of 95 °C / hour, with a filter period of 24 seconds and a clean period between protein sample sets. For each sample, a matching formulation buffer was used as a reference and analyzed under the same conditions. Data analysis was performed with Origin v 7.0552 software. Buffer blank scans were subtracted from the thermograms and normalized to the appropriate protein concentration. Data were fitted using a non-2-state model with cursor initialization for each transition to determine the thermodynamic parameters.

[0450] Amino acid clipping measurement by reverse phase high performance liquid chromatography (RP-HPLC)

[0451] DLS measurements were performed using a Wyatt DynaPro PlateReader II (Wyatt, Santa Barbara, CA) at a laser wavelength of 820.17 nm. Three independent samples were prepared for each sample and 30 μΐ were loaded into wells on a 384 well black untreated polystyrene plate (Thermo Scientific Nunc, UK) with a protein concentration range of 4 mg / ml - 20 mg / ml. For each well, 10 DLS measurements of 5 seconds each were taken at 20 °C and if the percent polydispersity > 15% the data was discarded. Cumulant analysis was performed using Wyatt Dynamics software to directly measure the protein self-diffusion coefficient (D) in triplicate for each condition and then the diffusion coefficients were averaged. Since D scales with protein concentration (c) according to the equation D = Do(1 + k D. c), where Do is the protein diffusion coefficient at infinite dilution, the protein-protein interaction parameter k D can be determined from a plot of D versus c.

[0452] Potency assay to assess HFUS1 binding to RXFP1 receptor

[0453] Product main peak and b-chain clipping were measured using reverse phase high performance liquid chromatography whereby separation was achieved by differential affinity to the hydrophobic chains of the packing material in the column. Test sample concentrations were adjusted and mixed with denaturing buffer in the presence of a reducing agent and incubated at a specified temperature for a specific amount of time. The samples were then injected into a reverse phase column and eluted with an increasing organic solvent gradient. Peaks eluted according to their hydrophobicity.

[0454] More specifically, an Agilent 1260 Infinity series or equivalent instrument was used for reverse phase high performance liquid chromatography. The column used was a Phenomenex, Aeris WIDEPORE 3.6 μιη XB-C8 HPLC column, 4.6 x 150 mm. Each 100 μg sample was denatured (8 M Guanidine, 130 mM Tris(hydroxymethyl)aminomethane, 1 mM EDTA pH 7.6) in the presence of a reducing agent (1 M DTT) then mixed and incubated at 37 °C for 45 minutes ± 5 minutes. The final concentration of each sample was 0.5 mg / mL. For each sample, 10 μΐ^was injected onto the column and eluted with an increasing organic solvent gradient (mobile phase A: 95% water, 5% acetonitrile, 0.1% TFA and mobile phase B: 80% acetonitrile: 20% water, 0.1% TFA) over 40 minutes. Peaks eluted according to their hydrophobicity, detection was at 220 nm.

[0455] Example 1 : Preliminary stability evaluation of HFUS1

[0456] THP1 cells endogenously expressing the RXFP1 receptor were engineered to express CRE-NanoLuc upon activation of the RXFP1 receptor. In this assay, HFUS1 binds to the RXFP1 receptor on the surface of THP1-CRE NanoLuc cells. This results in the production of cyclic adenosine monophosphate (cAMP), which signals through the cAMP response element (CRE) to drive expression of nanoluciferase. Luciferase expression is then measured by the addition of a chemiluminescent substrate. EC50 values representing the concentration of HFUS1 at which half-maximum luciferase expression is observed were generated using four-parameter semi-log curves fit to HFUS1 reference standard (HFUS1 formulated in 20 mM Histidine / Histidine HCL, 190 mM Arginine HCl, 0.02% (w / v) PS80, pH 6.5) and test samples. The relative potency of each HFUS1 sample was assessed by dividing the EC50 value of the reference standard by the EC50 value of each sample and multiplying by 100%.

[0457] Figure 2

[0458] The liquid stability of HFUS1 was first evaluated in a histidine buffer-based formulation containing sucrose and polysorbate (PS) 80 at concentrations of 10 mg / mL and 50 mg / mL. The loss of purity of the molecule was measured by HPSEC during storage at 5°C, 25°C, and 40°C and is shown in Figure 1. The rate of loss of % of the product main peak (MMP) at different temperatures was taken from the slope of the linear regression of the following data points: a) 50 mg / mL, -0.2% at 5°C, -0.5% at 25°C, and -2.0% at 40°C; and b) 10 mg / mL, -0.1% at 5°C, -0.4% at 25°C, and -2.3% at 40°C.

[0459] The conformational stability of the molecule was evaluated by measuring the unfolding temperature (T m ) of the molecule in the histidine / sucrose formulation buffer. The DSC trace is shown in Figure 3 . HFUS1 has two unfolding events. The T m onset is about 57°C, with the first unfolding at 63°C and the second unfolding at 82°C.

[0460] The protein-protein interaction parameter (k D ) of HFUS1 was evaluated by DLS using the methods described in the Methods section. Empirically, a negative k D value indicates a higher attractive tendency between HFUS1 molecules, while a positive value indicates an repulsive protein-protein interaction. The self-diffusion coefficient and the hydrodynamic radius of HFUS1 are shown in Example 2: Phase 1 formulation optimization and development . The k DCalculated as -16 mL / g, indicating a tendency for the molecule to self-associate.

[0461] At the same time, no phase separation or change in visual appearance was observed for HFUS1 upon storage. In general, HFUS1 showed an acceptable stability profile during the evaluation period. However, while the rate of degradation at 25°C and 40°C was low, from the limited data time points (up to 3 months), the molecule showed a relatively high rate of aggregation at 5°C, i.e., -0.21% per month at 50 mg / mL, as determined by HPSEC. If this rate continues, it translates to about 2.5% aggregation or monomer loss per year, making it difficult for the molecule to meet shelf-life requirements. This high rate of aggregation at 5°C can be due to the tendency of the molecule to self-associate in this formulation, as reflected by the negative k D If the molecule is prone to self-association, as the association continues, the molecule can form soluble aggregates (typically detectable by HPSEC), which can become precursors of insoluble large size aggregates, and eventually particles, significantly impacting the stability profile of the molecule. This data indicates the need for formulation optimization to reduce this tendency of self-association and aggregation.

[0462] Table 4: Summary of DLS, DSC, HPSEC, and visual data for HFUS1 formulations

[0463] Optimization of the HFUS1 formulation was performed. This focused on screening of pH and buffer species as well as alternative excipients to sucrose as stabilizers. The pH range of 4.5 to 6.5 was evaluated and citrate or histidine buffer systems were used to cover this range. Arginine HC1 or lysine HC1 were also used in place of sucrose in the formulation. PS80 was still used as a surfactant. HFUS1 was used at a concentration of 50 mg / mL. The interaction parameter k D , DSC was used to measure the onset of unfolding temperature, HPSEC was used to measure purity, and visual assessment was used to measure the level of particle formation. The results are summarized in Table 4. Results from the preliminary study of the histidine-sucrose formulation were included for comparison purposes. In addition, given the new HFUS1 material used in this optimization study, the histidine-sucrose formulation was also repeated in this study to serve as a control.

[0464] Figure 6 .

[0465]

[0466]

[0467] Abbreviations: mon - monomer, agg - aggregation, frag - fragmentation, opal - opalescent a acceptable within target threshold; i near maximum tolerated threshold; e exceeds allowable threshold

[0468] In the repeat study (histidine-sucrose formulation, control), HFUS1 showed similar K D However, it had a lower T 起始 and higher aggregation at 40°C. This difference can be caused by variability in the material used for this study. In the developability study, material from transient expression was used, whereas in the optimization study Chinese hamster ovary (CHO) cells were used. This indicates that process variability can impact the product quality of the molecule and that robust formulations are needed to meet the stability requirements.

[0469] As shown in Table 4, at the low pH end (pH 4.5 or pH 5.0), significant purity loss was observed by HPSEC after 1 month of storage under stress conditions (40°C). Formulation 2 showed high levels of aggregation and fragmentation, while formulation 1 produced severe phase separation and precipitation. The conformational stability of the molecule was also lower at lower pH, as lower unfolding onset temperatures were observed for formulations 1, 2, 3, and 4. The buffer species and excipients seemed to have an impact on the stability of the molecule. Several variables were investigated at the same pH. At pH 5.5, there were formulations containing citrate or histidine buffer systems, as well as formulations containing arginine HC1, lysine HC1, and sucrose. Sucrose and lysine HC1 seemed to provide slightly higher T 起始 , which means better conformational stability, while the histidine system provided better colloidal stability to the molecule, as indicated by higher K D values: i.e. formulation 4 (histidine) had a higher K D than formulation 3 (citrate). Arginine HC1 did not show improved K D at pH 5.5 than sucrose, e.g. the K D of formulation 3 (arginine HC1) was -11.5, which is similar to -12.6 for formulation 7 (sucrose). However, at pH 6.0, arginine HC1 (formulation 5) showed some improvement compared to sucrose (formulation 9), with higher K D and lower aggregation after storage at 40°C. Overall, formulation 6 showed the best stability profile, with the highest K D and lowest purity loss. This can be a combined effect of the pH, buffer system, and use of arginine HC1.

[0470] The chemical stability of the molecules in the formulations was further evaluated by mass spectrometry (MS), with a focus on the histidine-arginine HC1 formulation system, to assess the impact of pH on the molecules. The histidine-sucrose formulation was included as a control for comparison. The attributes evaluated were: 1) cleavage of the amino acid (AA) at the C-terminus of the relaxin B chain; 2) change in % of the tri-sulfide; and 3) methionine 271 (M271) oxidation at the relaxin B chain. The results are summarized in Figure 4.

[0471] No significant change in % of the tri-sulfide was observed across the pH tested. However, pH 5.5 showed the highest cleavage compared to pH 6.0 and pH 6.5, which performed the best. pH 5.5 also showed higher oxidation compared to the higher pH, but the difference was less pronounced compared to cleavage. To further evaluate the histidine-arginine HC1 formulation system, a pH optimization study was performed. Histidine-arginine HC1 formulations were prepared at pH 5.5, 6.0, 6.5, 6.8, and 7.0. The focus was on the HFUS1 purity loss on HPSEC and the data are shown in Figure 5.

[0472] Fragmentation of HFUS1 appeared to be high at low pH. At both 25 °C and 40 °C, pH 5.5 showed the highest molecular fragmentation, followed by pH 6.0. At both temperatures, fragmentation dropped to 0 from pH 6.5. No fragmentation was observed at 5 °C at all of the pH tested. As for aggregation, different profiles were observed at different temperatures. At 40 °C, the highest aggregation was observed at pH 5.5. As the pH increased, a V-shape was observed, with pH 6.5 showing the slowest aggregation rate, while at 5 °C, the aggregation rate increased as the pH increased, with a clear trend observed, although both were at low levels. The 5 °C condition most represents real-time storage of the drug product, and at the histidine-arginine HC1 system, all formulations provided improved stability compared to the histidine-sucrose. Even at pH 7.0, which had the highest aggregation rate, a monthly rate of 0.05% represented a drop of about 0.6% per year. The cleavage of the molecules was also evaluated by mass spectrometry within this pH range, with the data summarized in Figure 6. Similar to the previous finding, pH 5.5 showed the highest cleavage compared to the higher pH formulations. pH 6.8 and pH 7.0 showed low cleavage, and this finding was similar to the fragmentation of the molecules measured by HPSEC. Figure 7

[0473] ​It was hypothesized that the high fragmentation or clipping observed at low pH (5.5 and 6.0) could be due to protease activity. The molecule can be susceptible to certain enzymes co-eluting during purification, thus leading to this chemical degradation of the molecule. To validate this hypothesis, a protease inhibition study was performed. The formulation with histidine-arginine HC1 at pH 5.5 was chosen as it produced the highest fragmentation and clipping. The HFUS1 material was split into four portions. The first portion was evaluated as is. The second portion was spiked with a small amount of protease inhibitor (PI) cocktail. The protease inhibitor cocktail was dissolved in DMSO, so the third portion was spiked in DMSO as a control. The last portion was spiked with EDTA. The samples were stored at 40 °C for 4 weeks, then the purity of the material was evaluated by HPSEC and AA clipping by MS. The data is summarized in Figure 8 and Example 3: Particle formation challenges for HFUS1 and mitigation by surfactant screening

[0474] The samples spiked with PI or EDTA showed a clear reduction in fragmentation and AA clipping, while the control and DMSO spiked samples showed no clear change and both had high levels of fragmentation and clipping. This data resolves the hypothesis that proteases can be the cause of the molecule fragmentation and clipping. This enzymatic activity can be higher at lower pH, where proteases are most effective at cleaving molecules. Therefore, it is important to maintain the pH of the formulation in a higher range, as demonstrated in the pH optimization study. In this formulation optimization study, the histidine-arginine HC1 system was identified to provide the highest colloidal and conformational stability to HFUS1. In this formulation, the self-association tendency of the molecule is reduced and lower aggregation is observed. The optimal pH range was also confirmed to minimize the impact on the molecule’s chemical degradation, such as fragmentation and AA clipping.

[0475] Figure 10

[0476] During long-term stability storage at 5 °C, the formulations from the optimization study started to show visible particles. The onset of particles varied depending on the conditions. The formulations at pH 6.5 and 6.8 started to show particles at 6 months, and eventually all other formulations showed particles after longer storage, as shown in Figure 9. Considering that these formulations have different pH, buffer species, and excipients, it is unlikely that any of these are the root cause of particle formation. Therefore, it is suspected that this particle formation can be related to the presence of PS80, which is a common factor in all formulations. At the same time, a particle study was performed by using Fourier-transform infrared spectroscopy (FTIR) to identify the nature of the particles. The FTIR trace is shown in Table 5: Polysorbate 80 and Poloxamer 188 water levels in HFUS1 formulations post storage detected by HPLC-ELSD. Limit of quantitation (LOQ) is 0.004% The particles showed characteristics similar to the CH region of a protein at 3000 cm -1 -2800 cm -1 and at about 1740 cm -1 ​The particles were shown to be characteristic of an ester linkage region similar to that of PS80. Although this data is not conclusive, it is clear that the particles are protein in nature. It is noteworthy that similar particle formation was observed for the histidine-arginine HC1 formulation in another stability study using a new batch of HFUS1 material (for a GLP toxicology study using a lead clone). This indicates that the particles are not specific to a certain batch of HFUS1 material and that they occur in all HFUS1 material tested to date.

[0477] Host cell protein (HCP) levels were measured for the HFUS1 drug substance material. HCPs are process-related protein impurities that co-purify with the HFUS1 molecule and cannot be removed by the purification process. The HCP levels for the HFUS1 material were about 100 ng / mg to 650 ng / mg, which is a high level. The identity of the HCPs was analyzed by a mass spectrometry-based proteomics identification study. The identified HCPs included lipoprotein lipase (LPL), which is known to degrade polysorbate esters, as well as proteases or peptidases that can degrade proteins or cleave terminal amino acids. Without wishing to be bound by theory, this leads to several hypotheses for the cause of particle formation: 1) LPL degrades polysorbate 80 in the formulation, producing impurities such as free fatty acids (FFAs). Since free fatty acids are not soluble in water, these free fatty acids precipitate out as particles; 2) the free fatty acid impurities can trigger HFUS1 protein aggregation as nuclei, producing particles and at the same time, due to degradation, the level of PS80 decreases, thus losing its protective / surfactant effect against particle formation during storage; 3) proteases can degrade HFUS1 and the degraded HFUS1 has exposed patches that are prone to interact, leading to aggregation; and 4) some proteases or HCPs can also interact with HFUS1, leading to aggregation to occur.

[0478] Given that the particles identified by FTIR are essentially proteinaceous, it is unlikely that they are solely insoluble FFA. On the other hand, HFUS1 in the optimized histidine-arginine-HCl formulation (e.g. formulation 6 of Table 4) did not show abnormal fragmentation or AA clipping, so it is also unlikely that the particles are caused by protease degradation of HFUS1. In this case, hypothesis 2 and hypothesis 4 are the most likely mechanisms of HFUS1 particle formation. To confirm whether the presence of PS80 plays a role in particle formation, a new stability study was set up with alternative surfactants such as poloxamer 188 (P188) and D-a-tocopheryl polyethylene glycol succinate (TPGS) replacing PS80 in the histidine-arginine HC1 formulation. Two different concentrations of HFUS1 were tested, i.e. 50 mg / mL and 5 mg / mL. The formulations were stored at 5°C in vials and pre-filled syringes (PFS) and checked for particle formation at different time points (Figures 11 and 12). After 6 months of storage, the 50 mg / mL formulation containing PS80 started to show a higher milky trace of visual particles. The particle level increased after 9 months and at 12 months, a high level of visible particles was observed. Although the 5 mg / mL PS80 formulation did not show particle formation until the 9-month time point, visible particles were observed at 12 months. In contrast, the formulations containing TPGS and P188 did not show any visible particles after 12 months of storage.

[0479] MFI was also used to check for sub-visible particles in HFUS1 formulations. As shown in Figure 13, the particle count / mL of the 50 mg / mL formulation containing PS80 increased significantly after 6 months, while the 50 mg / mL formulation containing TPGS and P188 did not show any significant increase.

[0480] The PS80 and P188 levels in the HFUS1 formulations after storage were also tested by HPLC equipped with an evaporative light scattering detector (ELSD). The data are summarized in Table 5. ELSD data for the PS80 formulations were obtained after 5 months, while P188 data were obtained after 11 months. The PS80 levels dropped below the limit of quantitation for the 50 mg / mL HFUS1 formulation, while the 5 mg / mL formulation also showed a decrease in PS80 levels. This indicates that PS80 has degraded in the formulation, and without wishing to be bound by theory, this can be caused by the presence of LPL, which cleaves the ester bond of PS80 via enzymatic hydrolysis. This also explains why formulations with lower HFUS1 concentrations exhibit slower PS80 degradation over time. This can be due to lower levels of co-purified LPL in the formulation. In contrast, formulations containing P188 instead of PS80 did not show any changes in surfactant levels. P188 is a class of water-soluble non-ionic triblock copolymers formed from polyethylene oxide (PEO) and polypropylene oxide (PPO) blocks. The PEO and PPO blocks are linked together by ether bonds, which are not susceptible to enzymatic hydrolysis by LPL.

[0481] Figure 14 Figure 16 .

[0482]

[0483] The effect of LPL on PS80 degradation was further validated by LC-MS studies. A histidine-arginine HC1 formulation containing 50 mg / mL HFUS1 and PS80 (e.g., formulation 6 of Table 4) was split into 3 parts. One part was taken as an initial control sample, another was kept at 40 °C for 2 weeks, and the last was spiked with lipase and kept at 40 °C for 2 weeks. The LC-MS chromatograms of the samples are shown in Example 4: Robustness of HFUS1 formulations The heat-stressed HFUS1 sample exhibited hydrolytic degradation and accumulation of polyoxyethylene sorbitan monooleate, polyoxyethylene isosorbide monooleate, as well as di- and tri-substituted species. This profile was similar to the lipase-spiked sample, which showed more pronounced hydrolytic degradation.

[0484] This data and study indicate that the polysorbate 80 degradation hypothesis is the cause of the particle formation observed with HFUS1 formulations. Using alternative, lipase- resistant surfactants such as P188 or TPGS concurrently can mitigate the particle formation challenge.

[0485] In addition to visible particle inspection and sub-visible particle analysis, HPSEC and cIEF were also used to evaluate the purity and chemical degradation profile of HFUS1 formulations with different surfactants. The HPSEC data are shown in Figure 15 and the cIEF data are shown in Table 6: Formulation summary in robustness studyThe formulation containing P188 and PS80 showed similar degradation profiles and, in addition, no significant differences were observed in samples stored in vials or pre-filled syringes (PFS). Therefore, HFUS1 is stable in histidine-arginine HC1 formulation with P188 with no significant changes in physical and chemical stability after 18 months of storage.

[0486] Example 5: Formulation stability at lower HFUS1 concentrations

[0487] After formulation optimization, which focused on buffer species, pH and excipients, and surfactant screening, an optimized formulation in histidine buffer with arginine-HCl and poloxamer 188 at pH 6.5 was developed. This formulation provides long-term protection of HFUS1 with low chemical and physical degradation. In this study, the robustness of the formulation system was evaluated. This was investigated by varying the concentration of arginine-HCl and surfactant, as well as the pH. Different formulations were stored at 5°C, 25°C and 40°C to evaluate their stability profiles. The summary of different conditions tested is given in Table 6. The formulations were contained in 2R glass vials or PFS.

[0488] Table 7: HFUS1 formulation conditions

[0489]

[0490] The formulations were tested by HPSEC, CGE, cIEF and MFI and the data are summarized in Figures 17, 18, 19 and 20. HPSEC was used to evaluate the purity and aggregation of HFUS1 during storage. At the same time, CGE was used to detect any fragmentation of the molecule and cIEF to detect chemical instability. Sub-visible particles were assessed by MFI and visual inspection of the formulations was also performed (data not shown).

[0491] No particle formation issues were observed by MFI and visual inspection after 3 months storage at 40°C and over 12 months storage at 5°C. Also, as a comparison, control formulations containing PS80 instead of P188 showed high levels of sub-visible and visible particles. This data further confirms that the use of P188 mitigates the particle issues observed with PS80 systems. These formulations showed no significant changes in the main product peak (MPP%) detected by HPSEC after over 12 months storage at 5°C and over 6 months storage at 25°C, indicating good physical stability of these formulations. The rate of MPP% decrease at 40°C showed small differences between the different formulation conditions, however, this rate remained very low (<1% decrease per month). The concentration of HFUS1 had an impact on the aggregation of the molecule, with the 5 mg / mL sample showing a much lower MPP% decrease per month (Figure 17). The chemical stability of the formulations was evaluated by CGE and cIEF. For CGE, the MPP% decrease after storage was minimal or negligible for these formulations. This indicates low levels of fragmentation of the molecule. At the same time, the cIEF data also showed negligible changes in MPP% after 12 months storage at 5°C, indicating low chemical instability of the molecule. The MPP% decrease at 25°C and 40°C was more pronounced, but remained at acceptable levels in general. Formulations based on histidine-arginine HC1 showed excellent stability and robustness in general. Formulation F1 / F1P were selected as overall favorable formulations.

[0492] Example 6: Formulation stability at HFUS1 concentration of 33 mg / mL

[0493] The stability of HFUS1 formulations at lower protein concentrations was also investigated. Formulations were filled in 1 mL PFS and stored at 5°C, 25°C and 40°C for testing at multiple time points. The summary of formulation conditions is shown in Table 7. The concentrations evaluated were 0.25 mg / mL and 1 mg / mL HFUS1, and they were formulated in the target formulations (P1 and P3), and also in formulations that can generate less favorable case stability (P2 and P4), which have lower arginine HC1 concentration and higher pH. In the robustness study described in Example 4, higher aggregation was observed at lower arginine HC1 levels, and the pH 7.0 samples showed higher degradation rates as determined by cIEF. This study in Example 5 had to allow generation of formulation data at lower HFUS1 concentrations to support the applicability of the formulations at a wide range of HFUS1 concentrations. The formulations were tested by visual inspection (data not shown), MFI, HPSEC, cIEF and CGE, and the data is summarized in Figures 21-24.

[0494] Table 8: Formulation compositions for robustness stability study arms conducted in 1 mL PFS

[0495]

[0496] HPSEC data indicated that HFUS1 remained stable with no monomer % loss at all temperatures tested after 6 months storage at 5°C and 25°C, and 3 months storage at 40°C. No significant aggregation was observed. CGE was used to monitor any fragmentation of the molecule during storage and no loss of purity was observed at 5°C and 25°C, and only minimal loss was observed at 40°C. cIEF was used to detect any chemical degradation, as shown by changes in the main peak. After storage at 40°C, approximately 18% loss of the main peak was observed per month. This was lower than the rate observed at higher concentration formulations (e.g. 50 mg / mL and 5 mg / mL). While the degradation at 25°C was slightly higher than the 50 mg / mL and 5 mg / mL formulations. These rates of degradation at 25°C and 40°C were at an acceptable level, and importantly, the rate of degradation at 5°C was minimal. Little to no change was observed after 6 months of storage. Particle formation trends were also evaluated by visual inspection (visible particles) and MFI (sub-visible particles). The particle levels of HFUS1 in the formulation remained low during storage. Generally, at lower protein concentration formulations, the risk of aggregation and particle formation is lower, while the risk of chemical degradation is higher. This data set at low concentration indicates that HFUS1 has no chemical or physical stability issues at the concentrations studied.

[0497] Conclusions

[0498] After developing the optimized formulation, a single variable robustness stability study was performed at 33 mg / mL in 1 mL PFS. PFS was chosen as the intended long-term storage container for this molecule.

[0499] The concentration of 33 mg / mL corresponds to the highest concentration within ±10% of the 30 mg / mL formulation. Stability risks associated with sub-visible particle generation are considered highest at the highest protein concentration.

[0500] In this study, the robustness of the formulation was evaluated at the lower and upper limits of the excipient, surfactant, and pH specifications. This was investigated by varying the concentration of arginine-HCl and surfactant, and the pH. Different formulations were stored at 5°C, 25°C, and 40°C and tested at multiple time points to evaluate their stability profile. A summary of the different conditions tested is given in Table 8.

[0501] ​

[0502]

[0503] The formulations were tested by MFI, HIAC, HPSEC, cIEF, potency, and RP-HPLC, and the data is summarized in Figures 26-31.

[0504] Subvisible particles were assessed by MFI and HIAC and visual inspection of the formulations were also performed (data not shown). HIAC is a compendial method for subvisible particle counting and MFI is used as an orthogonal method. HPSEC was used to evaluate the purity of HFUS1 during storage. Meanwhile, cIEF was used to detect any chemical degradation as indicated by changes in the main peak. Potency assay was used to assess the binding of HFUS1 to RXFP1 receptor over time. RP-HPSEC was used to monitor the amino acid (AA) cleavage of the C-terminal of relaxin B chain.

[0505] No trend in particle formation was observed by MFI or visual inspection after 3 months storage at 40°C, 6 months at 25°C or 9 months at 5°C (Figure 26). HIAC data after the same duration of storage did not show a trend in subvisible particle count across all conditions and the subvisible particle count was well within the USP limits for all tested conditions (no more than 6000 and 600 particles for particle size >10 pm and >25 pm, respectively; Pharmacopeia US. 2014. USP 787 and 788) (Figure 27). This data further confirms that the use of P188 mitigated the particle issues observed with PS80 system.

[0506] HPSEC data indicated that all HFUS1 formulations remained stable with no monomer loss after 9 months storage at 5°C. Higher monomer loss was observed at pH 7 compared to pH 6 after 6 months storage at 25°C (-0.11% per month vs -0.05% per month) and 3 months storage at 40°C (-0.71% per month vs -0.36% per month). These data indicate good physical stability of these formulations with no monomer loss at the expected long-term storage temperature of -5°C and <1% per month for the other two temperatures (Figure 28).

[0507] cIEF data also showed negligible changes in the main peak after 6 months storage at 5°C, indicating low chemical instability of the molecule. The decrease in the main peak was more pronounced at 25°C and 40°C, with pH 7 showing the highest rate of change compared to the other formulation conditions (Figure 29). Changes in the cIEF profile are expected under these stress and accelerated conditions to which the molecule will not be exposed during its shelf life, and the degradation rate of all tested formulations was minimal at 5°C.

[0508] Potency results indicated that there was no change in the molecule activity after 6 months storage at 5°C for all formulation compositions tested. The change in potency was similar for all formulations at the other two temperatures, 25°C and 40°C (Figure 30).

[0509] RP-HPLC results at 5°C showed that the product main peak of all formulations did not change over 9 months. At 25°C and 40°C, the pH 6 formulations showed higher B-chain cleavage compared to the higher pH 7 formulations (Figure 31).

[0510] ​

[0511] During formulation development of HFUS1, several challenges were encountered. In some formulations, the molecule had a tendency to self-associate, leading to a risk of aggregation. The molecule also showed a tendency for AA cleavage. Cleavage occurred on the B-chain of the relaxin peptide, which is responsible for the binding of the molecule to its target. In addition, the molecule had high levels of process-related HCPs, and some species of HCPs present caused PS80 degradation, which led to high levels of particle formation of the molecule. This particle formation was not significantly affected by pH, buffer species, and stabilizing excipient type, but was primarily driven by PS80 degradation. Extensive formulation development and optimization studies were performed. Arginine HC1 was selected for its effectiveness in reducing the molecule's tendency to self-associate, and an optimized pH range was identified that effectively prevented AA cleavage and fragmentation of the molecule. Detailed research efforts were undertaken to understand the cause of the particle formation issue and identified PS80 as the root cause. This led to the optimization of the surfactant in the formulation system, and alternative, lipase-resistant surfactants such as P188 and TPGS mitigated the particle formation issue. P188 was selected as the lead surfactant for the formulation. Comprehensive formulation stability studies were performed to evaluate the robustness of the formulation system. The histidine-arginine HC1 formulation showed an excellent stability profile and robustness. The effect of HFUS1 concentration on stability was also evaluated, and HFUS1 showed good stability in both low concentration formulations (e.g., 0.25 mg / mL, 1 mg / mL, and 5 mg / mL) and higher concentration formulations (e.g., 33 mg / mL and 50 mg / mL).

Claims

1. A pharmaceutical formulation comprising a heterodimeric fusion and a lipase-resistant surfactant, wherein the heterodimeric fusion comprises: (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 heterodimeric fusion has relaxin activity.

2. The pharmaceutical formulation of claim 1, wherein the relaxin A chain polypeptide and the relaxin B chain polypeptide are covalently bound by at least one interchain disulfide bond.

3. The pharmaceutical formulation of claim 1 or 2, wherein the relaxin A chain and the relaxin B chain are not covalently linked to each other via an amino acid linker.

4. The pharmaceutical formulation of any one of the preceding claims, wherein the relaxin A chain is relaxin-2A chain and the relaxin B chain is relaxin-2B chain.

5. The pharmaceutical formulation of any one of the preceding claims, wherein the relaxin A chain is linked to the first heterodimerization domain via a linker, and the relaxin B chain is linked to the second heterodimerization domain via a linker, optionally wherein one or both linkers are polypeptides. 6 . The pharmaceutical formulation according to claim 5 , wherein one or both of the linkers have a length of 6 to 40 amino acids, for example one or both linkers have a length of 21 amino acids.

7. The pharmaceutical formulation of any one of the preceding claims, wherein the first and second heterodimerization domains are derived from immunoglobulin Fc regions ("first Fc region" and "second Fc region", respectively), optionally wherein the first and second Fc regions comprise constant domains CH2 and CH3.

8. The pharmaceutical formulation 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 pharmaceutical formulation according to claim 7 or 8, wherein the first Fc region and the second Fc region comprise amino acid mutations and / or modifications that promote heterodimerization, optionally wherein the amino acid mutations that promote heterodimerization are "Fc knob" and "Fc hole" mutations, such as "Fc knob" and "Fc hole" mutations present in the CH3 domain.

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

11. The pharmaceutical preparation according to claim 10, wherein the amino acid mutations that promote heterodimerization include: a. "Fc hole" mutations Y349C, T366S, L368A, and Y407V in one CH3 domain; and b. "Fc knob" mutations S354C and T366W in another CH3 domain, where amino acid numbering is according to the EU index as in Kabat.

12. The pharmaceutical preparation according to claim 11, wherein: a. the first Fc region comprises the "Fc knob" mutation, and the second Fc region comprises the "Fc hole" mutation; or b. the second Fc region comprises the “Fc knob” mutation, and the first Fc region comprises the “Fc hole” mutation.

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

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

15. The pharmaceutical formulation of claim 14, wherein the relaxin-2A chain polypeptide comprises the amino acid mutation K9H, K17M, or K17I.

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

17. The pharmaceutical formulation of claim 1, wherein the heterodimer fusion comprises: (i) FcX-con-A fusion polypeptide; and (ii) Fcγ-con-B fusion polypeptide, in: A is relaxin A chain or a variant thereof, such as relaxin-2A chain or a variant thereof; B is relaxin B chain or a variant thereof, such as relaxin-2 B chain or a variant thereof; Fcγ is the Fc region comprising the constant domains CH2 and CH3 of a human IgG1 immunoglobulin, and comprises an "Fc hole" amino acid mutation and / or modification, optionally the amino acid mutations Y349C:T366S:L368A:Y407V; FcX is an Fc region having "Fc knob" amino acid mutations and / or modifications, optionally comprising the constant domains CH2 and CH3 of a human IgG1 immunoglobulin, and comprising "Fc knob" amino acid mutations and / or modifications, optionally the amino acid mutation S354C:T366W; and con is a linker polypeptide, optionally having the sequence GGGGSGGGGSGGGGSGGGGGS [SEQ ID NO: 5], wherein amino acid numbering is according to the EU index as in Kabat, and wherein FcX and FcY heterodimerize.

18. The pharmaceutical formulation of any one 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 pharmaceutical formulation of 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 pharmaceutical formulation of any one of claims 8 to 19, wherein the heterodimer 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. Fc region.

21. The pharmaceutical formulation of claim 1, wherein the heterodimer fusion comprises: (i) FcX-BLA and FcY, optionally FcY-BLA; or (ii) FcY-BLA and FcX, optionally FcX-BLA; in: Fcγ is an immunoglobulin Fc region having an "Fc hole" amino acid mutation and / or modification, optionally comprising a CH3 domain having the amino acid mutations Y349C:T366S:L368A:Y407V; FcX is an immunoglobulin Fc region with "Fc knob" amino acid mutations and / or modifications, optionally comprising a CH3 domain with amino acid mutations S354C:T366W; B is relaxin B chain or a variant thereof, such as relaxin 2B chain or a variant thereof; A is relaxin A chain or a variant thereof, such as relaxin 2A chain or a variant thereof; and L is a linker polypeptide, optionally having the amino acid sequence GGGSGGGSGG [SEQ ID NO: 60], wherein amino acid numbering is according to the EU index as in Kabat, and wherein FcX and FcY heterodimerize.

22. The pharmaceutical formulation of claim 21, wherein 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, such as 21 amino acids in length.

23. The pharmaceutical formulation of any one of the preceding claims, wherein the formulation comprises less than about 10,000, about 6000, about 5,000, about 1,000, about 750, about 600, about 500, about 250, about 150, about 100, or about 50 particles / mL having a diameter greater than 2 μm, 5 μm, 10 μm, 15 μm, 20 μm, or 25 μm.

24. The pharmaceutical formulation according to any one of the preceding claims, wherein the concentration of the lipase-resistant surfactant is 0.001% (w / v) to 1% (w / v).

25. The pharmaceutical formulation according to any one of the preceding claims, wherein the concentration of the lipase-resistant surfactant is 0.02% (w / v) to 0.06% (w / v), optionally 0.04% (w / v).

26. The pharmaceutical formulation according to any one of the preceding claims, wherein the lipase-resistant surfactant cannot be enzymatically hydrolyzed by a lipase, optionally wherein the lipase is selected from the group consisting of lipoprotein lipase, lipase 9, phospholipase 2 and phospholipase 2A.

27. The pharmaceutical formulation according to any one of the preceding claims, wherein the lipase-resistant surfactant does not comprise an ester bond capable of being enzymatically hydrolyzed by a lipase, optionally wherein the lipase is selected from the group consisting of lipoprotein lipase, lipase 9, phospholipase 2 and phospholipase 2A.

28. The pharmaceutical formulation according to any one of the preceding claims, wherein the lipase-resistant surfactant is a water-soluble nonionic triblock copolymer formed from polyethylene oxide (PEO) and polypropylene oxide (PPO) blocks, optionally wherein the water-soluble nonionic triblock copolymer is poloxamer 188 (P188).

29. The pharmaceutical formulation of any one of claims 1 to 27, wherein the lipase-resistant surfactant is selected from P188, D-α-tocopheryl polyethylene glycol succinate (TPGS), Kolliphor HS15, Kolliphor EL, Kolliphor RH40, PEG 300, PEG 400, Brij 58, and Brij 35; optionally wherein the lipase-resistant surfactant is TPGS.

30. The pharmaceutical formulation of any one of the preceding claims, wherein the formulation further comprises a buffer having a pH of about 3 to about 10, optionally about 5.5 to about 7.

5.

31. The pharmaceutical formulation of claim 30, wherein the formulation has a pH in the range of 6 to 7.

32. The pharmaceutical formulation of claim 31 , wherein the formulation has a pH of 6.

5.

33. The pharmaceutical formulation of any one of claims 30 to 32, wherein the concentration of the buffer is 0.1 mM to 100 mM, optionally 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM.

34. The pharmaceutical formulation of claim 33, wherein the concentration of the buffer is 10 mM to 30 mM, optionally 20 mM.

35. The pharmaceutical formulation of any one of claims 30 to 34, wherein the buffer is selected from the group consisting of acetate, acetic acid, succinate, succinic acid, phosphate, phosphoric acid, ascorbate, ascorbic acid, lactate, lactic acid, tartaric acid, maleic acid, glycine, gluconate, citrate, histidine, imidazole, bicarbonate and carbonic acid, sodium benzoate, benzoic acid, edetate, malate, tris, glycylglycine, and mixtures thereof, optionally wherein the buffer is selected from the group consisting of citrate buffer and histidine buffer.

36. The pharmaceutical formulation according to any one of claims 30 to 35, wherein the buffer is histidine, histidine hydrochloride or histidine / histidine hydrochloride buffer, optionally histidine / histidine hydrochloride buffer.

37. The pharmaceutical formulation according to any one of the preceding claims, wherein the formulation further comprises an excipient, optionally wherein the excipient is an ionic excipient.

38. The pharmaceutical formulation of claim 37, wherein the concentration of the excipient is 10 mM to 500 mM.

39. The pharmaceutical formulation of claim 38, wherein the concentration of the excipient is 140 mM to 240 mM, optionally 190 mM.

40. The pharmaceutical formulation according to any one of claims 37 to 39, wherein the excipient is an ionic excipient selected from arginine salts or lysine salts.

41. The pharmaceutical formulation according to claim 40, wherein the ionic excipient is selected from arginine HCl or lysine HCl, optionally arginine HCl.

42. The pharmaceutical formulation of any preceding claim, wherein the formulation further comprises a sugar, optionally wherein the sugar is sucrose.

43. The pharmaceutical formulation of any one of the preceding claims, wherein the concentration of the heterodimeric fusion is 0.1 mg / mL to 100 mg / mL, optionally 0.2 mg / mL to 50 mg / mL.

44. The pharmaceutical formulation of claim 43, wherein the formulation comprises 0.2 mg / mL-50 mg / mL of the heterodimeric fusion, 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, 0.04% (w / v) poloxamer 188 (P188), and wherein the formulation has a pH of 6.

5.

45. The pharmaceutical formulation of claim 44, wherein the heterodimeric fusion consists of 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.

46. ​​The pharmaceutical formulation of any one of the preceding claims, wherein the formulation comprises 50 mg / mL of the heterodimeric fusion.

47. The pharmaceutical formulation of any one of claims 1 to 45, wherein the formulation comprises 30 mg / mL of the heterodimeric fusion.

48. The pharmaceutical formulation of any one of claims 1 to 45, wherein the formulation comprises 5 mg / mL or 1.1 mg / mL or 1 mg / mL of the heterodimeric fusion.

49. A pharmaceutical formulation according to any preceding claim for use in therapy.

50. The pharmaceutical formulation of any one of claims 1 to 48 for use in the treatment of a subject suffering from heart failure with pulmonary hypertension.

51. The pharmaceutical formulation for use according to claim 49 or 50, wherein the pharmaceutical formulation is administered to the subject by subcutaneous injection.

52. The pharmaceutical formulation for use according to any one of claims 49 to 51, wherein the fusion polypeptide or pharmaceutical formulation is administered by self-administration.

53. The pharmaceutical formulation for use according to any one of claims 50 to 52, wherein the heart failure is heart failure with reduced ejection fraction, heart failure with intermediate ejection fraction or heart failure with preserved ejection fraction.

54. The pharmaceutical formulation for use according to any one of claims 50 to 53, wherein the subject has a mean pulmonary artery pressure of about 25 mmHg or higher, a pulmonary artery wedge pressure (PAWP) above 15 mmHg and / or a right ventricular systolic pressure of about 40 mmHg or higher.

55. The pharmaceutical formulation for use according to any one of claims 50 to 54, wherein the subject has a pulmonary vascular resistance of less than 3.0 Wood units.

56. The pharmaceutical formulation for use according to any one of claims 50 to 54, wherein the subject has a pulmonary vascular resistance of 3.0 or greater Wood units.

57. A pharmaceutical formulation for use according to any one of claims 50 to 56, wherein the subject has fitted a blood pressure monitoring device, optionally a pulmonary artery pressure monitoring device.

58. The pharmaceutical preparation for use according to claim 57, wherein the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device.

59. The pharmaceutical formulation for use according to any one of claims 49 to 58, wherein administration of the pharmaceutical formulation results in one or more of the following compared to baseline levels prior to administration: a) decreased PVR; (b) decreased mPAP; (c) ePAD reduction; (d) Increased cardiac stroke volume (SV); (e) decreased systemic vascular resistance (SVR) and / or increased estimated glomerular filtration rate (eGFR); (f) increased ejection fraction; and / or (g) Increased cardiac output.

60. A kit comprising the pharmaceutical formulation according to any one of claims 1 to 48.

61. A method of treating a subject having a disease or condition, the method comprising administering to the subject a pharmaceutical formulation according to any one of claims 1 to 48.

62. A method of treating a subject suffering from heart failure with pulmonary hypertension, the method comprising administering to the subject a pharmaceutical formulation according to any one of claims 1 to 48.

63. The method of claim 61 or 62, wherein the heterodimeric fusion or pharmaceutical formulation is administered to the subject by subcutaneous injection.

64. The method of any one of claims 61 to 63, wherein the heterodimeric fusion or pharmaceutical formulation is administered by self-administration.

65. The method of any one of claims 62 to 64, wherein the heart failure is heart failure with reduced ejection fraction, heart failure with intermediate ejection fraction, or heart failure with preserved ejection fraction.

66. The method of any one of claims 62 to 65, wherein the subject has a mean pulmonary artery pressure of about 25 mmHg or greater, a pulmonary artery wedge pressure (PAWP) greater than 15 mmHg, and / or a right ventricular systolic pressure of about 40 mmHg or greater.

67. The method of any one of claims 62 to 66, wherein the subject has a pulmonary vascular resistance of less than 3.0 Wood units.

68. The method of any one of claims 62 to 66, wherein the subject has a pulmonary vascular resistance of 3.0 or greater Wood units.

69. The method of any one of claims 62 to 68, wherein the subject has been fitted with a blood pressure monitoring device, optionally a pulmonary artery pressure monitoring device.

70. The method of any one of claims 62 to 69, wherein the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device.

71. The method of any one of claims 61 to 70, wherein administration of the heterodimeric fusion or pharmaceutical formulation results in one or more of the following, compared to baseline levels prior to administration: a) decreased PVR; (b) decreased mPAP; (c) ePAD reduction; (d) Increased cardiac stroke volume (SV); (e) decreased systemic vascular resistance (SVR) and / or increased estimated glomerular filtration rate (eGFR); (f) increased ejection fraction; and / or (g) Increased cardiac output.

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