ACTRII protein and uses thereof

Abstract

In some aspects, the disclosure relates to compositions and methods for reducing the risk of telangiectasia in patients receiving a therapeutically effective amount of an ActRII polypeptide, particularly dosing regimens that reduce the risk of telangiectasia in patients receiving a therapeutically effective amount of an ActRII polypeptide.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 223,265, filed July 19, 2021, the contents of which are incorporated by reference in their entirety.

[0002] Field The present application describes a method for reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide. The present application also relates to a method for reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30 to 110 of SEQ ID NO:1. [Background technology]

[0003] Telangiectasia refers to a condition in which dilated venules (tiny blood vessels) produce thread-like red lines or patterns on the skin. These patterns, or telangiectasias, are thought to be caused by the release or activation of vasoactive substances under a number of conditions. Although telangiectasias are often benign, they can also be caused by serious diseases such as hereditary hemorrhagic telangiectasia (HHT). In patients with HHT, telangiectasias can develop in vital organs such as the liver. Rupture of these telangiectasias can cause life-threatening hemorrhages.

[0004] Telangiectasia can occur as a side effect upon administration of certain drugs such as ALK-1 receptor fusion protein (dalantercept), endoglin neutralizing antibody (TRC105) and certain ActRII polypeptides (Garber K. Nat Biotechnol. 2016;34(5):458-461).

[0005] There is a high unmet need for effective therapy to reduce the risk of telangiectasia in patients who receive one or more ActRII polypeptides for the treatment of underlying diseases.Therefore, the object of the present disclosure is to provide a method for the risk of telangiectasia in patients who receive ActRII polypeptides. Summary of the Invention

[0006] General In a particular aspect, the disclosure provides a method for reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide, the method comprising: (i) administering a first dose of an ActRII polypeptide to the patient in an amount of 0.3 mg / kg; and (ii) administering a second dose of an ActRII polypeptide to the patient in an amount of 0.7 mg / kg, wherein if the patient exhibits symptoms of telangiectasia or includes risk factors for developing telangiectasia, administration of a third dose is initiated, thereby reducing the risk of telangiectasia. In certain aspects, the disclosure provides a method of reducing the risk of telangiectasia in a patient receiving administration of a therapeutically effective amount of an ActRII polypeptide, wherein the polypeptide comprises an amino acid sequence at least 90% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1, the method comprising administering the ActRII polypeptide in a dosing regimen comprising: (i) administering a first dose of the ActRII polypeptide to the patient in an amount of 0.3 mg / kg; and (ii) administering a second dose of the ActRII polypeptide to the patient in an amount of 0.7 mg / kg, wherein if the patient exhibits symptoms of telangiectasia or comprises risk factors for developing telangiectasia, administration of a third dose is initiated, thereby reducing the risk of telangiectasia. In some embodiments, administration of the third dose comprises administering a dose of the ActRII polypeptide to the patient in an amount of 0.3 mg / kg. In some embodiments, administration of the third dose comprises withholding treatment with the ActRII polypeptide for at least 2-6 weeks. In some embodiments, administration of the third dose comprises withholding treatment with the ActRII polypeptide for at least 3 weeks. In some embodiments, the first dose is administered to the patient for at least 3 weeks. In some embodiments, the second dose is administered for at least 21 weeks. In some embodiments, the second dose is administered for at least 45 weeks.

[0007] In a particular aspect, the disclosure provides a method for reducing the risk of telangiectasia when treating a patient with an ActRII polypeptide, the method comprising: (i) administering an ActRII polypeptide in a dosing regimen comprising administering to the patient one or more first doses of the ActRII polypeptide in an amount of 0.3 mg / kg once every three weeks for 24 weeks, wherein if the patient exhibits one or more symptoms or risk factors for developing telangiectasia, one or more second doses of the ActRII polypeptide are administered to the patient in an amount at least half reduced by the amount of the first dose.

[0008] In certain aspects, the disclosure provides a method for reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide, the method comprising: (i) administering the ActRII polypeptide in a dosing regimen comprising administering the ActRII polypeptide to the patient in an amount of 0.3 mg / kg every three weeks, wherein if the patient exhibits symptoms of telangiectasia or includes risk factors for developing telangiectasia, the ActRII polypeptide is administered to the patient in an amount of 0.3 mg / kg every six weeks, thereby reducing the risk of telangiectasia. In certain aspects, the disclosure provides a method of reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide, wherein the polypeptide comprises an amino acid sequence at least 90% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1, the method comprising (i) administering the ActRII polypeptide in a dosing regimen comprising administering the ActRII polypeptide to the patient in an amount of 0.3 mg / kg every three weeks, wherein if the patient exhibits symptoms of telangiectasia or comprises risk factors for developing telangiectasia, the ActRII polypeptide is administered to the patient in an amount of 0.3 mg / kg every six weeks, thereby reducing the risk of telangiectasia. In some embodiments, the therapeutically effective amount of the ActRII polypeptide is 0.7 mg / kg every three weeks.

[0009] In a particular aspect, the disclosure provides a method for treating pulmonary arterial hypertension, the method comprising administering an ActRII polypeptide to a patient in need thereof in a dosing regimen comprising: (i) administering to the patient a first dose of the ActRII polypeptide in an amount of 0.3 mg / kg; and (ii) administering to the patient a second dose of the ActRII polypeptide in an amount of 0.7 mg / kg once every three weeks for as long as the patient requires treatment, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30 to 110 of SEQ ID NO:1.

[0010] In some embodiments, the symptoms of telangiectasia are selected from the group consisting of pain, itching, red thread marks on the skin, mucocutaneous telangiectasia, gastrointestinal bleeding, lesions on the skin, nosebleeds, bleeding gums, arteriovenous malformations, internal telangiectasia, and red spots on the skin. In some embodiments, the symptoms of telangiectasia include lesions on the skin. In some embodiments, the symptoms of telangiectasia include bleeding gums. In some embodiments, the symptoms of telangiectasia include nosebleeds. In some embodiments, the symptoms of telangiectasia include arteriovenous malformations. In some embodiments, the symptoms of telangiectasia include internal telangiectasia. In some embodiments, the arteriovenous malformations or internal telangiectasia occur in internal organs (e.g., the brain, liver, lungs, spleen, urinary tract, and spine). In some embodiments, the risk factors for developing telangiectasia are selected from the group consisting of low BMP9 levels, low BMP10 levels, low VEGF levels, hereditary hemorrhagic telangiectasia (HHT) and connective tissue disease (CTD).

[0011] In some embodiments, the risk reduction comprises reducing or ameliorating severe telangiectasia. In some embodiments, the risk reduction comprises preventing the progression of telangiectasia. In some embodiments, the third dose comprises withholding treatment with the ActRII polypeptide until the telangiectasia improves to grade 1 or less. In some embodiments, the method improves the severity of telangiectasia from grade 2 to grade 1. In some embodiments, the method improves the severity of telangiectasia from grade 3 to grade 2. In some embodiments, the method improves the severity of telangiectasia from grade 3 to grade 1. In some embodiments, the method prevents the progression of telangiectasia from grade 1 to grade 2. In some embodiments, the method prevents the progression of telangiectasia from grade 2 to grade 3. In some embodiments, the patient is receiving an ActRII polypeptide for the treatment of PH. In some embodiments, the patient is receiving an ActRII polypeptide for the treatment of PAH.

[0012] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1. In some embodiments, the ActRII polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the ActRII polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the ActRII polypeptide is a fusion protein further comprising an immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is an IgG1 immunoglobulin Fc domain. In some embodiments, the Fc fusion protein further comprises a linker domain located between the ActRII polypeptide domain and the immunoglobulin Fc domain. In some embodiments, the linker domain is selected from the group consisting of TGGG (SEQ ID NO:20), TGGGG (SEQ ID NO:18), SGGGG (SEQ ID NO:19), GGGGS (SEQ ID NO:22), GGG (SEQ ID NO:16), GGGG (SEQ ID NO:17) and SGGG (SEQ ID NO:21). In some embodiments, the ActRII polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:23.In some embodiments, the ActRII polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:40.

[0013] In some embodiments, the polypeptide is lyophilized. In some embodiments, the polypeptide is soluble. In some embodiments, the polypeptide is administered to the patient using subcutaneous injection. In some embodiments, the polypeptide is administered to the patient on a schedule selected from the group consisting of weekly, every two weeks, every three weeks, and every four weeks. In some embodiments, the polypeptide is administered to the patient every three weeks. In some embodiments, the polypeptide is administered to the patient every four weeks. In some embodiments, the polypeptide is part of a homodimeric protein complex. In some embodiments, the polypeptide is glycosylated. In some embodiments, the polypeptide has a glycosylation pattern that can be obtained by expression in Chinese hamster ovary cells. In some embodiments, the ActRII polypeptide binds to one or more ligands selected from the group consisting of activin A, activin B, and GDF11. In some embodiments, the ActRII polypeptide binds to activin and / or GDF11. In some embodiments, the ActRII polypeptide further binds to one or more ligands selected from the group consisting of BMP10, GDF8, and BMP6.

[0014] In some embodiments, the method further comprises administering to the patient an additional active agent and / or supportive care, hi some embodiments, the additional active agent and / or supportive care is selected from the group consisting of a beta blocker, angiotensin converting enzyme inhibitor (ACE inhibitor), angiotensin receptor blocker (ARB), a diuretic, a lipid lowering agent, an endothelin blocker, a PDE5 inhibitor, a prostacyclin, or a left ventricular assist device (LVAD). In some embodiments, the additional active agent and / or supportive care is selected from the group consisting of: prostacyclin and its derivatives (e.g., epoprostenol, treprostinil, and iloprost); prostacyclin receptor agonists (e.g., selexipag); endothelin receptor antagonists (e.g., thelin, ambrisentan, macitentan, and bosentan); calcium channel blockers (e.g., amlodipine, diltiazem, and nifedipine); anticoagulants (e.g., warfarin); diuretics; oxygen therapy; atrial septal defect creation; pulmonary endarterectomy; phosphodiesterase type 5 inhibitors (e.g., sildenafil and tadalafil); activators of soluble guanylate cyclase (e.g., cinaciguat and riociguat); ASK-1 inhibitors (e.g., CIIA; SCH79797; GS-4997; MSC2032964A; 3H-naphtho[1,2,3-de]quinyline-2,7-dione, NQDI-1; 2-thioxo-thiazolidine, 5-bromo-3-(4-oxo-2-thioxo-thiazolidine-5-ylidene)-1,3-dihydro-indol-2-one; NF-κB antagonists (e.g., dh404, CDDO-epoxide; 2.2-difluoropropionamide; C 28-imidazole (CDDO-Im);2-Cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO);3-Acetyloleanolic acid;3-Trifluoroacetyloleanolic acid;28-Methyl-3-acetyloleanane;28-Methyl-3-trifluoroacetyloleanane;28-Methyloxyoleanolic acid;SZC014;SCZ015;SZC017;PEGylated derivatives of oleanolic acid;3-O-(beta-D-glucopyranosyl)oleanolic acid;3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl]oleanolic acid;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl]oleanolic acid;3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;3-O-[alpha-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid;3-O-[alpha-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;28-O-β-D-glucopyranosyl-oleanolic acid;3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosiduronic acid (CS1);Oleanolic acid 3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosiduronic acid (CS2);Methyl 3,11-dioxoolean-12-en-28-olate (DIOXOL);ZCVI4-2;Benzyl 3-Dehydroxyoxy-1,2,5-oxadiazolo[3',4':2,3]oleanolate);Left ventricular assist device (LVAD), or lung and / or heart transplant. In some embodiments, the patient is treated with one or more substances selected from the group consisting of phosphodiesterase type 5 inhibitors, soluble guanylate cyclase stimulators, prostacyclin receptor agonists, and endothelin receptor antagonists. In some embodiments, the one or more substances are selected from the group consisting of bosentan, sildenafil, beraprost, macitentan, selexipag, epoprostenol, treprostinil, iloprost, ambrisentan, and tadalafil. In some embodiments, the method further comprises administration of one or more substances selected from the group consisting of phosphodiesterase type 5 inhibitors, soluble guanylate cyclase stimulators, prostacyclin receptor agonists, and endothelin receptor antagonists. In some embodiments, the one or more substances are selected from the group consisting of bosentan, sildenafil, beraprost, macitentan, selexipag, epoprostenol, treprostinil, iloprost, ambrisentan and tadalafil. In some embodiments, the patient has been treated with one or more vasodilators prior to administration of the polypeptide. In some embodiments, the method further comprises administration of one or more vasodilators. In some embodiments, the one or more vasodilators are selected from the group consisting of prostacyclin, epoprostenol and sildenafil. In some embodiments, the vasodilator is prostacyclin. In some embodiments, the patient is undergoing one or more therapies for PAH. In some embodiments, the one or more therapies for PAH are selected from the group consisting of: prostacyclin and its derivatives (e.g., epoprostenol, treprostinil, and iloprost); prostacyclin receptor agonists (e.g., selexipag); endothelin receptor antagonists (e.g., thelin, ambrisentan, macitentan, and bosentan); calcium channel blockers (e.g., amlodipine, diltiazem, and nifedipine); anticoagulants (e.g., warfarin); diuretics; oxygen therapy; atrial septal defect creation; pulmonary endarterectomy;phosphodiesterase type 5 inhibitors (e.g., sildenafil and tadalafil); activators of soluble guanylate cyclase (e.g., cinaciguat and riociguat); ASK-1 inhibitors (e.g., CIIA; SCH79797; GS-4997; MSC2032964A; 3H-naphtho[1,2,3-de]quinyline-2,7-dione, NQDI-1; 2-thioxo-thiazolidine, 5-bromo-3-(4-oxo-2-thioxo-thiazolidine-5-ylidene)-1,3-dihydro-indol-2-one); NF-κB antagonists (e.g., dh404, CDDO-epoxide; 2.2-difluoropropionamide; C28 imidazole (CDDO-Im); 2-cyano-3,12-dioxo- oleanolic acid;3-acetyloleanolic acid;3-trifluoroacetyloleanolic acid;28-methyl-3-acetyloleanan;28-methyl-3-trifluoroacetyloleanan;28-methyloxyoleanolic acid;SZC014;SCZ015;SZC017;PEGylated derivatives of oleanolic acid;3-O-(beta-D-glucopyranosyl)oleanolic acid;3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl]oleanolic acid;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl]oleanolic acid;3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;3-O-[aL-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid;3-O-[alpha-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;28-O-β-D-glucopyranosyl-oleanolic acid;3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosiduronic acid (CS1);Oleanolic acid 3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosiduronic acid (CS2);methyl 3,11-dioxoolean-12-en-28-olate (DIOXOL); ZCVI4-2; benzyl 3-dehydroxyoxy-1,2,5-oxadiazolo[3',4':2,3]oleanolate; left ventricular assist devices (LVADs), or lung and / or heart transplants; [Brief description of the drawings]

[0015] [Figure 1] Figure 1 shows an alignment of the extracellular domain of human ActRIIB (sequence number 31) with the extracellular domain of human ActRIIA (sequence number 2), in which residues predicted in this specification to directly contact the ligand based on a combined analysis of multiple ActRIIB and ActRIIA crystal structures are indicated in boxes. [Diagram 2] FIG. 2 shows a multiple sequence alignment of various vertebrate ActRIIA proteins and human ActRIIA (sequence numbers 6-10 and 36-38). [Diagram 3] Figure 3 shows a multiple sequence alignment of Fc domains from human IgG isotypes using Clustal2.1. The hinge region is shown with a dotted underline. Double underlining indicates examples of positions engineered in IgG1 Fc (SEQ ID NO: 32) to promote asymmetric chain pairing, as well as the corresponding positions for the other isotypes IgG2 (SEQ ID NO: 33), IgG3 (SEQ ID NO: 34) and IgG4 (SEQ ID NO: 35). [Figure 4A] Figures 4A and 4B show the purification of ActRIIA-hFc expressed in CHO cells. The protein is purified as a single, well-defined peak (left lane: molecular weight standard; right lane: ActRIIA-hFc) as visualized by sizing column (Figure 4A) and Coomassie-stained SDS-PAGE (Figure 4B). [Figure 4B]Figures 4A and 4B show the purification of ActRIIA-hFc expressed in CHO cells. The protein is purified as a single, well-defined peak (left lane: molecular weight standard; right lane: ActRIIA-hFc) as visualized by sizing column (Figure 4A) and Coomassie-stained SDS-PAGE (Figure 4B). [Figure 5A] Figures 5A and 5B show binding of ActRIIA-hFc to activin (Figure 5A) and GDF-11 (Figure 5B) as measured by Biacore™ assay. [Figure 5B] Figures 5A and 5B show binding of ActRIIA-hFc to activin (Figure 5A) and GDF-11 (Figure 5B) as measured by Biacore™ assay.

[0016] Detailed Description 1. Overview The present disclosure relates to compositions and methods for reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide described herein, the method comprising (i) administering to the patient a first dose of the ActRII polypeptide in an amount of 0.3 mg / kg, and (ii) administering to the patient a second dose of the ActRII polypeptide in an amount of 0.7 mg / kg, wherein if the patient exhibits symptoms of telangiectasia or includes risk factors for developing telangiectasia, administration of a third dose is initiated, thereby reducing the risk of telangiectasia.

[0017] In certain embodiments, the present disclosure provides a method for reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide for the treatment of pulmonary arterial hypertension (e.g., functional class II or functional class III), wherein the pulmonary arterial hypertension is treated by administering to a patient in need thereof an effective amount of an ActRII polypeptide described herein.

[0018] Pulmonary arterial hypertension [World Health Organization (WHO) Group 1 PH] is a severe, progressive, life-threatening disease of the pulmonary vascular system characterized by abnormal proliferation of smooth muscle cells in the walls of the pulmonary arteries and severe vasoconstriction. Severe constriction of blood vessels in the lungs leads to extremely high pulmonary artery pressures. These high pressures make it difficult for the heart to pump blood to the lungs so that it can be oxygenated. Patients with PAH suffer from extreme shortness of breath because the heart tries to pump against these high pressures. Patients with PAH typically develop a significant increase in PVR and a persistent elevation in mPAP, which ultimately leads to right ventricular failure and death. Patients diagnosed with PAH have a poor prognosis and similarly impaired quality of life, with an average life expectancy of 2-5 years from the time of diagnosis if left untreated.

[0019] PAH can be diagnosed based on a mean pulmonary artery pressure greater than 25 mmHg (or, in updated guidelines, greater than 20 mmHg) at rest and normal pulmonary artery capillary wedge pressure. PAH can cause shortness of breath, dizziness, fainting, and other symptoms, all of which are worsened by exertion. PAH can be a severe disease with markedly reduced exercise tolerance and heart failure. The two main types of PAH include idiopathic PAH (e.g., PAH with no identified predisposition) and hereditary PAH (e.g., PAH associated with mutations in BMPR2, ALK1, ENG, SMAD9, CAV1, KCNK3, or EIF2AK4). In 70% of cases of familial PAH, there is a mutation in the BMPR2 gene. Risk factors for the development of PAH include family history of PAH, drug and toxin use (e.g. methamphetamine or cocaine use), infections (e.g. HIV infection or schistosomiasis), liver cirrhosis, congenital heart anomalies, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis or connective tissue / autoimmune disorders (e.g. scleroderma or lupus).PAH can be associated with long-term responders to calcium channel blockers, pronounced features of venous / capillary (PVOD / PCH) involvement and persistent PH in the newborn syndrome.

[0020] The terms used herein generally have their usual meaning in the art in the context of this disclosure and in the specific context in which each term is used.Specific terms are explained below or elsewhere in this specification in order to provide additional guidance to practitioners in describing the compositions and methods of this disclosure and their preparation and use methods.The scope or meaning of any use of a term will be clear from the specific context in which it is used.

[0021] The term "sequence similarity" in all its grammatical forms means the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

[0022] "Sequence identity (%)" to a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to those in a reference polypeptide (nucleotide) sequence after aligning the sequences and introducing gaps as necessary to obtain the maximum sequence identity (%) without considering any conservative substitutions as part of the sequence identity. Alignment to determine amino acid sequence identity (%) can be achieved by various methods within the skill of the art, for example, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, ALIGN-2, Clustal Omega or Megaalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, amino acid (nucleic acid) sequence identity (%) values ​​are obtained using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.Other algorithms for determining sequence identity or homology include Clustal Omega, LALIGN, FASTA, SIM and EMBOSS Needle. In a preferred embodiment, the algorithm used to determine sequence identity is Clustal Omega.

[0023] "Stimulate" ("agonize"), in all its grammatical forms, refers to the process of activating a protein and / or gene (e.g., by activating or amplifying gene expression of that protein or by inducing an inactive protein to become active) or enhancing the activity of a protein and / or gene.

[0024] "Antagonize" in all its grammatical forms means the process of inhibiting a protein and / or gene (e.g., by inhibiting or reducing gene expression of that protein or by inducing an active protein to become inactive) or the process of reducing the activity of a protein and / or gene.

[0025] The terms "about" and "approximately" used throughout this specification and claims in reference to numerical values ​​indicate a range of accuracy that is well known and acceptable to those skilled in the art. Generally, such a range of accuracy is ±10%. Alternatively, particularly in biological systems, the terms "about" and "approximately" can mean values ​​that are within a single order of magnitude of a given value, preferably within 5 times, more preferably within 2 times, of a given value.

[0026] The numerical ranges disclosed herein are inclusive of the numbers defining the range.

[0027] Singular terms include plural referents unless the context in which the term is used clearly dictates otherwise. Singular terms and the terms "one or more" and "at least one" may be used interchangeably herein. Furthermore, "and / or" as used herein should be interpreted as a specific disclosure of two or more specified features or components, respectively, in the presence or absence of the other. Thus, the term "and / or" as used herein in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" as used in a phrase such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C, A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0028] Throughout this specification, the word "comprise" or variations such as "including" or "including" are understood to imply the inclusion of a stated integer or group of integers and not the exclusion of any other integer or group of integers.

[0029] 2. ActRII Polypeptide In certain aspects, the present disclosure relates to ActRII polypeptides and uses thereof (e.g., reducing the risk of telangiectasia in patients receiving a therapeutically effective amount of the ActRII polypeptide). As used herein, the term "ActRII" refers to the family of type II activin receptors. This family includes activin receptor type IIA (ActRIIA) and activin receptor type IIB (ActRIIB).

[0030] In certain embodiments, the disclosure relates to ActRII polypeptides having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 1, 2, 3, 23, 27, 30, and 40. In other embodiments, the disclosure relates to ActRII polypeptides having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:31. The term "ActRII" as used herein refers to the activin receptor type IIA (ActRIIA) protein family, the activin receptor type IIB (ActRIIB) protein family, or combinations and / or variants thereof. ActRII polypeptides can be derived from any species, and include variants derived from such ActRII proteins by mutagenesis or other modifications. References to ActRII herein are understood to refer to any one of the currently identified forms. ActRII family members are generally transmembrane proteins that are composed of a ligand-binding extracellular domain that includes a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain that has a predicted serine / threonine kinase activity.

[0031] The term ActRII polypeptide includes any naturally occurring polypeptide of an ActRII family member, and any variant thereof (including mutants, fragments, fusions and peptidomimetics) that retains useful activity. Examples of such variant ActRII polypeptides are described throughout this disclosure, as well as in International Patent Application Publication Nos. WO 2006 / 012627, WO 2007 / 062188, WO 2008 / 097541, WO 2010 / 151426 and WO 2011 / 020045, which are incorporated herein by reference in their entirety. The amino acid numbering of all ActRII-related polypeptides described herein is based on the numbering of the human ActRII precursor protein sequence (SEQ ID NO: 1) shown below, unless otherwise indicated.

[0032] The canonical human ActRII precursor protein sequence is as follows:

number

[0033] The signal peptide is single underlined, the extracellular domain is bolded, and potential endogenous N-linked glycosylation sites are double underlined.

[0034] The processed (mature) extracellular human ActRII polypeptide sequence is as follows:

number

[0035] The C-terminal "tail" of the extracellular domain is indicated by a single underline. The sequence with the "tail" deleted (Δ15 sequence) is as follows:

number

[0036] The nucleic acid sequence encoding the human ActRII precursor protein is shown below (SEQ ID NO: 4), according to nucleotides 159-1700 of the Genbank reference sequence NM_001616.4. The signal sequence is underlined.

number

[0037] The nucleic acid sequence encoding the processed soluble (extracellular) human ActRII polypeptide is as follows:

number

[0038] ActRII is well conserved among vertebrates, with large stretches of the extracellular domain being completely conserved. For example, FIG. 2 shows a multiple sequence alignment of the human ActRIIA extracellular domain compared to various ActRIIA orthologues. Many of the ligands that bind to ActRIIA are also highly conserved. Thus, from these alignments, it is possible to predict important amino acid positions in the ligand-binding domain that are important for normal ActRII-ligand binding activity, and also to predict amino acid positions that are likely to tolerate substitutions without significantly altering normal ActRII-ligand binding activity. Thus, active human ActRII mutant polypeptides that are useful in the methods disclosed herein can contain one or more amino acids at the corresponding positions from the sequence of another vertebrate ActRII, or can contain residues similar to those in the sequence of human or other vertebrates.

[0039] An alignment of the amino acid sequences of the human ActRIIA extracellular domain and the human ActRIIB extracellular domain is shown in FIG. 1. This alignment shows the amino acid residues in both receptors that are believed to directly contact the ActRII ligand. For example, the composite ActRII structure showed that the ActRIIA-ligand binding pocket is defined in part by residues F31, N33, N35, K38-T41, E47, Y50, K53-K55, R57, H58, F60, T62, K74, W78-N83, Y85, R87, E92, and K94-F101. Conservative mutations are expected to be tolerated at these positions.

[0040] The following non-limiting examples illustrate this approach to define active ActRII variants. As shown in Figure 2, F13 in the human extracellular domain is Y in Ovis aries (sheep) (SEQ ID NO: 7), Gallus gallus (chicken) (SEQ ID NO: 10), Bos Taurus (cow) (SEQ ID NO: 36), Tyto alba (owl) (SEQ ID NO: 37) and Myotis davidii (bat) ActRIIA, indicating that aromatic residues including F, W and Y are tolerated at this position. Q24 in the human extracellular domain is R in Bos Taurus (cow) ActRIIA, indicating that charged residues including D, R, K, H and E are tolerated at this position. S95 in the human extracellular domain is F in Gallus gallus (chicken) and Tyto alba (owl) ActRIIA, indicating that this site may be tolerant to a wide variety of changes, including polar residues such as E, D, K, R, H, S, T, P, G, Y, and possibly hydrophobic residues such as L, I, or F. E52 in the human extracellular domain is D in Ovis aries (sheep) ActRIIA, indicating that acidic residues, including D and E, are tolerated at this position. P29 in the human extracellular domain is less conserved and appears as S in Ovis aries (sheep) ActRIIA and L in Myotis davidii (bat) ActRIIA, indicating that virtually all amino acids should be tolerated at this position.

[0041] Moreover, as mentioned above, ActRII proteins have been characterized in the art in terms of structural / functional features, particularly with respect to ligand binding [Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1):18-22; Allendorph et al. (2006) PNAS 103(20:7643-7648; Thompson et al. (2003) The EMBO Journal, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2030, 2040, 2050, 2060, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2009 22(7):1555-1566; and U.S. Patent Nos. 7,709,605, 7,612,041, and 7,842,663]. For example, a defining structural motif known as the three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine ​​residues located at various positions within the extracellular domain of each monomeric receptor [Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. In addition to the teachings herein, these references provide detailed guidance on how to produce ActRII mutants that retain one or more desired activities (e.g., ligand binding activity).

[0042] For example, a defining structural motif known as the three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine ​​residues located at various positions within the extracellular domain of each monomeric receptor [Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. Thus, the core ligand-binding domain of human ActRII, bounded by the outermost of these conserved cysteines, corresponds to positions 30-110 of SEQ ID NO:1 (ActRII precursor). Thus, the less structurally ordered amino acids adjacent to the core sequence delimited by these cysteines can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 residues at the N-terminus and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues at the C-terminus without necessarily altering ligand binding. Exemplary ActRII extracellular domain truncations include SEQ ID NOs: 2 and 3.

[0043] Thus, the general formula for an active portion (e.g., ligand binding) of ActRII is a polypeptide that comprises, consists essentially of, or consists of amino acids 30 to 110 of SEQ ID NO: 1. Thus, an ActRII polypeptide can be, for example, an ActRII polypeptide beginning with a residue corresponding to any one of amino acids 21 to 30 of SEQ ID NO: 1 (e.g., beginning with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ending at a position corresponding to any one of amino acids 110 to 135 of SEQ ID NO: 1 (e.g., beginning with amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, In some embodiments, the amino acid sequence of ActRII may comprise, consist essentially of, or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a portion of ActRII (ending in any one of the following amino acids: 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135). Other examples include those starting at a position selected from 21 to 30 of SEQ ID NO:1 (e.g., starting at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), or those starting at a position selected from 22 to 30 (e.g., starting at any one of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30), or those starting at a position selected from 23 to 30 (e.g., starting at any one of amino acids 23, 24, 25, 26, 27, 28, 29, or 30), or those starting at a position selected from 24 to 30. (e.g., starting with any one of amino acids 24, 25, 26, 27, 28, 29, or 30) and ending at a position selected from 111 to 135 of SEQ ID NO: 1 (e.g., ending with any one of amino acids 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), or ending at a position selected from 112 to 135 (e.g., ending with any one of amino acids 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), or ending at a position selected from 113-135 (e.g., ending at any one of amino acids 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), or ending at a position selected from 120-135 or terminating at a position selected from 130-135 (e.g., terminating at any one of amino acids 130, 131, 132, 133, 134, or 135); or terminating at a position selected from 111-134 (e.g., terminating at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135); 1, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 or 134), or terminating at a position selected from 111-133 (e.g., terminating at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133), or terminating at a position selected from 111-132 (e.g., terminating at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133). , 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 or 132), or terminating at a position selected from 111-131 (e.g., terminating at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 or 131).Also contemplated are those that comprise, consist essentially of, or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the corresponding portion of SEQ ID NO: 1. Thus, in some embodiments, an ActRII polypeptide can comprise, consist essentially of, or consist of a polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 30-110 of SEQ ID NO:1. Optionally, the ActRII polypeptide can include a polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 30-110 of SEQ ID NO:1 and contains no more than 1, 2, 5, 10 or 15 conservative amino acid changes within the ligand binding pocket. In some embodiments, the ActRII polypeptide is part of a homodimeric protein complex.

[0044] In certain embodiments, the present disclosure relates to ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof) (including fragments, functional variants, and modified forms thereof) and uses thereof (e.g., reducing the risk of telangiectasia in patients receiving a therapeutically effective amount of an ActRII polypeptide). Preferably, the ActRII polypeptide is soluble (e.g., the extracellular domain of ActRII). In some embodiments, the ActRII polypeptide inhibits one or more GDF / BMP ligands (e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15) (e.g., Smad signaling). In some embodiments, the ActRII polypeptide binds to one or more GDF / BMP ligands (e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15). In some embodiments, an ActRII polypeptide of the disclosure begins with a residue corresponding to amino acids 21-30 of SEQ ID NO:1 (e.g., begins with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ends at a position corresponding to any one of amino acids 110-135 (e.g., begins with any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, , 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a portion of ActRII.In some embodiments, an ActRII polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 30-110 of SEQ ID NO: 1. In certain embodiments, an ActRII polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 21-135 of SEQ ID NO: 1. In some embodiments, the ActRII polypeptide comprises, consists of, or essentially consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 27, 30 and 40.

[0045] In some embodiments, the ActRII polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23. In some alternative embodiments, the ActRII polypeptide (e.g., SEQ ID NO: 23) may lack a C-terminal lysine. In some embodiments, the ActRII polypeptide lacking a C-terminal lysine is SEQ ID NO: 40. In some embodiments, the ActRII polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, an ActRII polypeptide is administered to a patient that comprises, consists of, or consists essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the patient is administered an ActRII polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, a combination of SEQ ID NO: 23 and SEQ ID NO: 40 is administered to the patient.

[0046] In certain aspects, the present disclosure relates to an ActRII polypeptide (e.g., an ActRIIA polypeptide, an ActRIIB polypeptide, or a combination thereof). In some embodiments, the ActRII trap of the present disclosure is a mutant ActRII polypeptide (e.g., an ActRIIA polypeptide, an ActRIIB polypeptide, or a combination thereof) that comprises one or more mutations (e.g., amino acid additions, deletions, substitutions, and combinations thereof) in the extracellular domain (also referred to as the ligand-binding domain) of an ActRIIB polypeptide (e.g., a "wild-type" or unmodified ActRII polypeptide), and the mutant ActRII polypeptide has one or more modified ligand-binding activities compared to the corresponding wild-type ActRII polypeptide. In a preferred embodiment, the mutant ActRII polypeptide of the present disclosure retains at least one activity similar to the corresponding wild-type ActRII polypeptide. For example, a preferred ActRII polypeptide binds to GDF11 and / or GDF8 and inhibits (e.g., antagonizes) their function. In some embodiments, the ActRII polypeptides of the disclosure further bind to and inhibit one or more of the ligands of GDF / BMP (e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15).Thus, the disclosure provides ActRII polypeptides with altered binding specificity for one or more ActRII ligands.

[0047] By way of illustration, one or more mutations can be selected that increase the selectivity of the modified ligand binding domain for GDF11 and / or GDF8 compared to one or more ActRII binding ligands, such as activin (activin A or activin B), particularly activin A. Optionally, the modified ligand binding domain has a K for GDF11 and / or GDF8 binding that is at least 2, 5, 10, 20, 50, 100 or even 1000 times greater than the ratio for the wild-type ligand binding domain. d K for activin binding dOptionally, the variant ligand-binding domain has an IC for inhibiting GDF11 and / or GDF8 that is at least 2, 5, 10, 20, 50, 100, or even 1000 times greater than the wild-type ligand-binding domain. 50 IC for inhibition of activin against 50 Optionally, the modified ligand binding domain has an IC 50 IC at least 2, 5, 10, 20, 50, 100, or even 1000 times lower than 50 and inhibits GDF11 and / or GDF8.

[0048] In certain embodiments, the present disclosure contemplates specific mutations of an ActRII polypeptide (e.g., an ActRIIA polypeptide, an ActRIIB polypeptide, or a combination thereof) to modify the glycosylation of the polypeptide. Such mutations may be selected to introduce or remove one or more glycosylation sites, such as an O-linked or N-linked glycosylation site. An asparagine-linked glycosylation recognition site generally comprises a tripeptide sequence, asparagine-X-threonine or asparagine-X-serine (where "X" is any amino acid), that is specifically recognized by an appropriate cellular glycosylation enzyme. Modifications may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the polypeptide (in the case of an O-linked glycosylation site). Various amino acid substitutions or deletions at either or both of the first or third amino acid positions (and / or amino acid deletions at the second position) of the glycosylation recognition site result in non-glycosylation in the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Depending on the coupling method used, sugars can be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline; (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine. Removal of one or more carbohydrate moieties present on a polypeptide can be accomplished chemically and / or enzymatically. Chemical deglycosylation can include, for example, exposure of the polypeptide to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) while keeping the amino acid sequence intact.Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by using various endoglycosidases and exoglycosidases as described by Thotakura et al. [Meth. Enzymol. (1987) 138:350]. The sequence of the polypeptide can be adjusted as necessary depending on the type of expression system used, since mammalian cells, yeast cells, insect cells and plant cells can all introduce different glycosylation patterns that can be influenced by the amino acid sequence of the peptide. In general, the polypeptides of the present disclosure for use in humans can be expressed in mammalian cell lines that provide appropriate glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are expected to be useful as well.

[0049] The present disclosure further contemplates methods for producing mutants, particularly combinatorial mutant sets and truncation mutants of ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof). A pool of combinatorial mutants is particularly useful for identifying functionally active (e.g., GDF / BMP ligand-binding) ActRII sequences. The purpose of screening such combinatorial libraries may be to produce polypeptide variants with altered properties, e.g., altered pharmacokinetics or altered ligand binding. Various screening assays are provided below, and such assays may be used to evaluate variants. For example, ActRII mutants can be screened for their ability to bind to one or more GDF / BMP ligands [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10 and / or BMP15], to interfere with the binding of GDF / BMP ligands to ActRII polypeptides and their heteromultimers, and / or to interfere with signal transduction triggered by GDF / BMP ligands.

[0050] The activity of ActRII polypeptide (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or combination thereof) or variants thereof can also be tested in cell-based or in vivo assays. For example, the effect of ActRII polypeptide on the expression of genes involved in the pathogenesis of pulmonary arterial hypertension can be evaluated. This can be performed in the presence of one or more recombinant ligand proteins [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15], if necessary, and cells can be transfected to produce ActRII polypeptide and optionally GDF / BMP ligand. Similarly, ActRII polypeptide can be administered to mice or other animals, and the effect on the pathogenesis of pulmonary arterial hypertension can be evaluated using art-recognized methods. Similarly, the activity of ActRII polypeptide or variants thereof can be tested in blood cell progenitor cells for any effect on the proliferation of these cells, which can be performed, for example, by the assays described herein and generally known in the art. SMAD-responsive reporter genes can be used in such cell lines to monitor effects on downstream signaling.

[0051] Combinatorially derived variants can be produced that have increased selectivity or generally increased potency compared to a reference ActRII polypeptide (e.g., an ActRIIA polypeptide, an ActRIIB polypeptide, or a combination thereof). Such variants can be used in gene therapy protocols when expressed from recombinant DNA constructs. Similarly, mutagenesis can generate variants that have dramatically different intracellular half-lives than the corresponding unmodified ActRII polypeptide. For example, modified proteins can be made more stable or less stable to proteolysis or other cellular processes that result in the destruction or inactivation of the unmodified polypeptide. Such variants and the genes that encode them can be used to alter polypeptide complex levels by modulating the half-life of the polypeptide. For example, a shorter half-life can result in a more transient biological effect and, when part of an inducible expression system, can allow for tighter control of recombinant polypeptide complex levels within cells. In Fc fusion proteins, mutations can be made in the linker (if present) and / or the Fc portion to alter the half-life of the ActRII polypeptide.

[0052] Combinatorial libraries can be generated by a degenerate library of genes encoding a library of polypeptides, each of which contains at least a portion of a potential ActRII polypeptide sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into a gene sequence such that a degenerate set of potential ActRII-encoding nucleotide sequences can be expressed as individual polypeptides or as a set of larger fusion proteins (e.g., for phage display).

[0053] There are many ways in which a library of potential homologues can be generated from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automatic DNA synthesizer, and the synthetic genes can then be ligated into a suitable vector for expression. The synthesis of degenerate oligonucleotides is well known in the art [Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, AG Walton, ed., Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; and Ike et al. (1983) Nucleic Acid Res. 11:477]. Such techniques have been used in the directed evolution of other proteins [Scott et al., (1990) Science 249:386-390; Roberts et al. (1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249:404-406; Cwirla et al., (1990) PNAS USA 87:6378-6382; and U.S. Patent Nos. 5,223,409, 5,198,346, and 5,096,815].

[0054] Alternatively, other forms of mutagenesis can be used to generate combinatorial libraries. For example, an ActRII polypeptide of the disclosure (e.g., an ActRIIA polypeptide, an ActRIIB polypeptide, or a combination thereof) can be modified by, for example, alanine scanning mutagenesis [Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601; Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085], linker scanning mutagenesis [Gustin et al. (1993) Virology 193:653-660; and Brown et al. (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al. (1982) Science 232:316], saturation mutagenesis [Meyers et al., (1986) Science 232:613], PCR mutagenesis [Leung et al. (1989) Method Cell Mol Biol 1:11-19], or random mutagenesis, such as chemical mutagenesis [Miller et al. (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al. (1994) Strategies in Mol Biol 7:32-34], and can be isolated from libraries by screening with them. Linker scanning mutagenesis is an attractive method for identifying truncated (bioactive) forms of ActRII polypeptides, especially in a combinatorial setting.

[0055] A wide variety of techniques are known in the art for screening the gene products of combinatorial libraries made by point mutation and truncation, and even for screening cDNA libraries for gene products with specific properties. Such techniques are generally applicable to the rapid screening of gene libraries made by combinatorial mutagenesis of ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof). The most widely used techniques for screening large gene libraries typically include cloning gene libraries into replicable expression vectors, transforming suitable cells with the resulting vector library, and expressing combinatorial genes under conditions where detection of desired activity facilitates relatively easy isolation of the vector encoding the gene of the detection product. Preferred assays include ligand (e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10 and / or BMP15) binding assays and / or ligand-mediated cell signaling assays.

[0056] As will be recognized by those skilled in the art, most of the mutations, variants or modifications described herein can be made at the nucleic acid level, or in some cases by post-translational modification or chemical synthesis. Such techniques are well known in the art, some of which are described herein. In part, this disclosure identifies functionally active parts (fragments) and variants of ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof) that can be used as a guide to make and use other variant ActRII polypeptides within the scope of the disclosure described herein.

[0057] In certain embodiments, functionally active fragments of the ActRII polypeptides of the present disclosure can be obtained by screening recombinantly produced polypeptides from the corresponding fragments of the nucleic acid encoding the ActRII polypeptide.Fragments can also be chemically synthesized using techniques known in the art, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.Fragments can be produced (recombinantly or chemically synthesized) and tested to identify peptidyl fragments that can function as antagonists (inhibitors) of the ActRII receptor and / or one or more ligands [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10 and / or BMP15].

[0058] In certain embodiments, the ActRII polypeptides of the present disclosure (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof) may further include post-translational modifications in addition to any that naturally occur in the ActRII polypeptide. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the ActRII polypeptides may contain non-amino acid elements, such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effect of such non-amino acid elements on the function of the ligand trap polypeptide may be tested as described herein for other ActRII variants. When the polypeptides of the present disclosure are produced in cells by cleaving the nascent form of the polypeptide, post-translational processing may also be important for the proper folding and / or function of the protein. Various cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellular and characteristic mechanisms for such post-translational activities and can be selected to ensure proper modification and processing of ActRII polypeptides.

[0059] In certain aspects, the ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof) of the present disclosure include fusion proteins having at least a portion (domain) of an ActRII polypeptide and one or more heterologous portions (domains). Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP) or human serum albumin. The fusion domains can be selected to confer desired properties. For example, some fusion domains are particularly useful for isolating fusion proteins by affinity chromatography. For affinity purification purposes, glutathione, amylase, and related matrices for affinity chromatography, such as nickel- or cobalt-bound resins, are used. Many such matrices are available in "kit" form, such as the Pharmacia GST purification system and the QIAexpress™ system (Qiagen), which are useful in the presence of (HIS6) (SEQ ID NO: 39) fusion partners. As another example, the fusion domain may be selected to facilitate detection of the ActRII polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are usually short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain contains a protease cleavage site, for example for factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein therefrom. The released protein may then be isolated from the fusion domain by subsequent chromatographic separation.Other types of fusion domains that may be selected include multimerization (e.g., dimerization, tetramerization) domains and functional domains (which impart additional biological function), including, for example, constant domains from immunoglobulins (e.g., Fc domains).

[0060] In certain aspects, the ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof) of the present disclosure include one or more modifications that can "stabilize" the polypeptide. "Stabilize" refers to anything that increases the in vitro half-life, serum half-life, whether this is through reduced destruction of the substance, reduced clearance by the kidney, or other pharmacokinetic effects. For example, such modifications may increase the shelf life of the polypeptide, increase the circulating half-life of the polypeptide, and / or reduce proteolysis of the polypeptide. Such stabilizing modifications include, but are not limited to, fusion proteins (e.g., including fusion proteins comprising an ActRII polypeptide domain and a stabilizing domain), modifications of glycosylation sites (e.g., including the addition of glycosylation sites to the polypeptides of the present disclosure), and modifications of carbohydrate moieties (e.g., including the removal of carbohydrate moieties from the polypeptides of the present disclosure). The term "stabilization domain" as used herein refers not only to fusion domains (e.g., immunoglobulin Fc domains) as in the case of fusion proteins, but also includes non-proteinaceous modifications, such as carbohydrate moieties, or non-proteinaceous moieties, such as polyethylene glycol. In certain preferred embodiments, ActRII polypeptides are fused to heterologous domains that stabilize the polypeptide ("stabilization" domains), preferably heterologous domains that enhance the stability of the polypeptide in vivo. Fusion with constant domains of immunoglobulins (e.g., Fc domains) is known to confer desirable pharmacokinetic properties to a wide range of proteins. Similarly, fusion with human serum albumin may also confer desirable properties.

[0061] An example of a natural amino acid sequence that may be used for the Fc portion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 11). The dotted underline indicates the hinge region and the solid underline indicates the position of the naturally occurring mutation. In part, the present disclosure provides a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 11. Naturally occurring mutations in G1Fc would include E134D and M136L according to the numbering system used in SEQ ID NO: 11. (See Uniprot P01857).

number

[0062] Optionally, the IgG1 Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In certain cases, mutant IgG1 Fc domains having one or more of these mutations (e.g., Asp-265 mutations) have reduced binding ability to Fcγ receptors compared to the wild-type Fc domain. In other cases, mutant Fc domains having one or more of these mutations (e.g., Asn-434 mutations) have enhanced binding ability to MHC class I-associated Fc receptors (FcRN) compared to the wild-type IgG1 Fc domain.

[0063] An example of a naturally occurring amino acid sequence that may be used for the Fc portion of human IgG2 (G2Fc) is shown below (SEQ ID NO:12). The dotted underline indicates the hinge region and the double underline indicates where in the sequence there is a database discrepancy (according to UniProt P01859). In part, the disclosure provides polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:12.

number

[0064] Two examples of amino acid sequences that may be used for the Fc portion of human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be up to four times longer than other Fc chains and contains three identical 15-residue segments preceded by a similar 17-residue segment. The first G3Fc sequence shown below (SEQ ID NO: 13) contains a short hinge region consisting of a single 15-residue segment, while the second G3Fc sequence (SEQ ID NO: 14) contains the full-length hinge region. In both cases, the dotted underlines indicate the hinge region and the solid underlines indicate the positions of naturally occurring mutations based on UniProt P01859. In part, the disclosure provides polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs:13 and 14.

number

[0065] Naturally occurring mutations in G3Fc (see, e.g., Uniprot P01860) include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del, F221Y when converted to the numbering system used in SEQ ID NO: 13, and the present disclosure provides fusion proteins comprising a G3Fc domain containing one or more of these mutations. Also, the human immunoglobulin IgG3 gene (IGHG3) exhibits structural polymorphisms characterized by different hinge lengths [see Uniprot P01859]. In particular, mutant WIS lacks most of the V region and all of the CH1 region. It has an additional interchain disulfide bond at position 7 in addition to position 11, which is normally present in the hinge region. Mutant ZUC lacks most of the V region, all of the CH1 region, and part of the hinge. Mutant OMM may represent an allelic form or another gamma chain subclass. The present disclosure provides additional fusion proteins that include a G3 Fc domain that contains one or more of these variants.

[0066] An example of a naturally occurring amino acid sequence that may be used for the Fc portion of human IgG4 (G4Fc) is shown below (SEQ ID NO:15). The dotted underline indicates the hinge region. In part, the disclosure provides polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:15.

number

[0067] Various engineered mutations in the Fc domain are shown herein for the G1Fc sequence (SEQ ID NO: 11), and similar mutations in G2Fc, G3Fc and G4Fc can be derived from their alignment with G1Fc in Figure 3. Similar Fc positions based on isotype alignment (Figure 3) have different amino acid numbers in SEQ ID NOs: 11, 12, 13, 14 and 15, due to unequal hinge lengths. H 2 and C H A given amino acid position in a three-region immunoglobulin sequence (e.g., SEQ ID NOs: 11, 12, 13, 14 and 15) is numbered based on the entire IgG1 heavy chain constant domain (C H 1, hinge, C H 2 and C H For example, the human G1Fc sequence (SEQ ID NO: 11), the human IgG1 heavy chain constant domain (Uniprot P01857), and selected Cs in the human IgG1 heavy chain are identified by numbers different from the same position in the Uniprot database. H The correspondence between the three positions is as follows:

[0068] [Table 1]

[0069] Various methods are known in the art to increase the desired pairing of Fc-containing fusion polypeptide chains in a single cell line to produce preferred asymmetric fusion proteins in acceptable yields [Klein et al. (2012) mAbs 4:653-663; and Spiess et al. (2015) Molecular Immunology 67(2A):95-106]. Methods for obtaining the desired pairing of Fc-containing chains include, but are not limited to, charge-based pairing (electrostatic steering), "knob-into-hole" steric pairing, SEEDbody pairing, and leucine zipper-based pairing [Ridgway et al. (1996) Protein Eng 9:617-621; Merchant et al. (1998) Nat Biotech 16:677-681; Davis et al. (2010) Protein Eng Des Sel 23:195-202; Gunasekaran et al. (2010); 285:19637-19646; Wranik et al. (2012) J Biol Chem 287:43331-43339; US5932448; WO 1993 / 011162; WO 2009 / 089004 and WO 2011 / 034605].

[0070] It is understood that the different elements of a fusion protein (e.g., an immunoglobulin Fc fusion protein) can be arranged in any manner consistent with the desired function. For example, an ActRII polypeptide domain can be arranged C-terminal to a heterologous domain, or a heterologous domain can be arranged C-terminal to an ActRII polypeptide domain. The ActRII polypeptide domain and the heterologous domain do not have to be adjacent in the fusion protein, and additional domains or amino acid sequences can be included at the C-terminus or N-terminus of either domain or between the domains.

[0071] For example, an ActRII receptor fusion protein can comprise an amino acid sequence of the formula: ABC. The B portion corresponds to an ActRII polypeptide domain (e.g., an ActRIIA polypeptide, an ActRIIB polypeptide, or a combination thereof). The A and C portions can independently be zero, one, or more amino acids, and both the A and C portions, if present, are heterologous to the B portion. The A and / or C portions may be linked to the B portion via a linker. The linker may be rich in glycine residues (e.g., 2-10, 2-5, 2-4, 2-3 glycine residues), or glycine and proline residues, and may contain, for example, a single sequence of threonine / serine and glycine, or a repeating sequence of threonine / serine and / or glycine, such as single or repeat sequences of GGG (SEQ ID NO: 16), GGGG (SEQ ID NO: 17), TGGGG (SEQ ID NO: 18), SGGGG (SEQ ID NO: 19), TGGG (SEQ ID NO: 20), SGGG (SEQ ID NO: 21), or GGGGS (SEQ ID NO: 22). In certain embodiments, the ActRII fusion protein comprises an amino acid sequence of the formula: ABC, where A is a leader (signal) sequence, B consists of an ActRII polypeptide domain, and C is a polypeptide moiety that enhances one or more of in vivo stability, in vivo half-life, uptake / administration, tissue localization or distribution, protein complex formation, and / or purification. In certain embodiments, the ActRII fusion protein comprises an amino acid sequence of the formula: ABC, where A is the TPA leader sequence, B consists of an ActRII receptor polypeptide domain, and C is an immunoglobulin Fc domain. Preferred fusion proteins comprise the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 30, and 40.

[0072] In a preferred embodiment, the ActRII polypeptide used according to the methods described herein is an isolated polypeptide. As used herein, an isolated protein or polypeptide is one that has been separated from the components of its natural environment. In some embodiments, the polypeptide of the present disclosure is purified to greater than 95%, 96%, 97%, 98% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). Methods for assessing purity are well known in the art [see, e.g., Flatman et al. (2007) J. Chromatogr. B 848:79-87]. In some embodiments, the ActRII polypeptide used according to the methods described herein is a recombinant polypeptide.

[0073] The ActRII polypeptides of the present disclosure can be produced by various techniques known in the art. For example, the polypeptides of the present disclosure can be synthesized using standard protein chemistry techniques, such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen / Biosearch 9600). Alternatively, the polypeptides of the present disclosure (including fragments or variants thereof) can be recombinantly produced using various expression systems (e.g., E. coli, Chinese hamster ovary (CHO) cells, COS cells, baculovirus), as known in the art. In another embodiment, modified or unmodified polypeptides of the present disclosure can be produced by digestion of recombinantly produced full-length ActRII polypeptides, for example, by using a protease, such as trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE). Computer analysis (e.g., using commercially available software, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic cleavage sites. Alternatively, such polypeptides can be produced from recombinantly produced full-length ActRII polypeptides using chemical cleavage (e.g., cyanogen bromide, hydroxylamine, etc.).

[0074] 3. Nucleic acids encoding ActRII polypeptides In certain embodiments, the disclosure provides isolated and / or recombinant nucleic acids encoding ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof) (including fragments, functional variants, and fusion proteins thereof).

[0075] As used herein, an isolated nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0076] In certain embodiments, nucleic acids encoding the ActRII polypeptides of the disclosure are understood to include nucleic acids that are variants of any one of SEQ ID NOs: 4, 5, or 28. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions (including allelic variants), and thus include coding sequences that differ from the nucleotide sequences set forth in any one of SEQ ID NOs: 4, 5, or 28.

[0077] In certain embodiments, the ActRII polypeptides of the disclosure are encoded by isolated and / or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 4, 5 or 28. One of skill in the art will appreciate that nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence that is complementary to SEQ ID NO: 4, 5 or 28 and variants thereof are also within the scope of the disclosure. In other embodiments, the nucleic acid sequences of the disclosure can be isolated and / or recombinant and / or fused to a heterologous nucleotide sequence or in a DNA library.

[0078] In other embodiments, the nucleic acid of the present disclosure also includes a nucleotide sequence that hybridizes under highly stringent conditions to the nucleotide sequence shown in SEQ ID NO: 4, 5, or 28, the complementary sequence of SEQ ID NO: 4, 5, or 28, or a fragment thereof. As mentioned above, one skilled in the art will readily understand that suitable stringency conditions that promote DNA hybridization can be varied. One skilled in the art will readily understand that suitable stringency conditions that promote DNA hybridization can be varied. For example, hybridization can be performed at about 45°C in 6.0× sodium chloride / sodium citrate (SSC), followed by washing at 50°C in 2.0×SSC. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0×SSC at 50°C to a high stringency of about 0.2×SSC at 50°C. Also, the temperature in the washing step can be increased from a low stringency condition of room temperature (about 22°C) to a high stringency condition of about 65°C. Both temperature and salt can be varied, or temperature or salt concentration can be held constant and the other variable varied. In one embodiment, the disclosure provides nucleic acids that hybridize under low stringency conditions of 6xSSC at room temperature followed by a wash with 2xSSC at room temperature.

[0079] Also within the scope of this disclosure are isolated nucleic acids that differ from the nucleic acids set forth in SEQ ID NO: 4, 5, or 28 due to the degeneracy of the genetic code. For example, some amino acids are represented by multiple triplets. Multiple codons specifying the same amino acid, i.e., synonyms (e.g., CAU and CAC are synonyms for histidine), may result in "silent" mutations that do not affect the amino acid sequence of the protein. However, DNA sequence polymorphisms that lead to changes in the amino acid sequence of a protein of interest are expected to exist in mammalian cells. One of skill in the art will understand that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of a nucleic acid encoding a particular protein may exist between individuals of a given species due to natural allelic mutations. Any such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure.

[0080] In certain embodiments, the recombinant nucleic acid of the disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequence is generally one that is appropriate for the host cell used for expression. Numerous types of suitable expression vectors and suitable regulatory sequences are known in the art and may be used in a variety of host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and end sequences, translational start and end sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are contemplated by the disclosure. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of multiple promoters. The expression construct may be present in the cell on an episome, such as a plasmid, or the expression construct may be inserted within a chromosome. In some embodiments, the expression vector contains a selection marker gene that allows for the selection of transformed host cells. Selection marker genes are well known in the art and may vary depending on the host cell used.

[0081] In certain embodiments, the nucleic acid of the subject matter disclosed herein is provided in an expression vector comprising a nucleotide sequence encoding an ActRII polypeptide (e.g., an ActRIIA polypeptide, an ActRIIB polypeptide, or a combination thereof) operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct the expression of an ActRII polypeptide. Thus, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of a wide variety of expression regulatory sequences that control the expression of DNA sequences and are operably linked thereto can be used in these vectors to express DNA sequences encoding ActRII polypeptides. Such useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, the adenovirus or cytomegalovirus immediate early promoters, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is driven by the T7 RNA polymerase, the major operator and promoter region of the phage lambda, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase, e.g., Pho5, the promoter of yeast alpha mating factor, the polyhedron promoter of the baculovirus system, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. Additionally, the copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0082] The recombinant nucleic acid of the present disclosure can be produced by ligating the cloned gene or a portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian cells), or both. Expression vehicles for the production of recombinant ActRII polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells such as E. coli.

[0083] Some mammalian expression vectors contain both prokaryotic sequences that facilitate propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences derived from bacterial plasmids such as pBR322 to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the discussion of gene therapy delivery systems below. Various methods used for preparing plasmids and transforming host organisms are well known in the art. For other suitable expression systems and general recombinant methods for both prokaryotic and eukaryotic cells, see, for example, Molecular Cloning A Laboratory Manual, 3rd Ed., edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (e.g., pAcUW1) and pBlueBac-derived vectors (e.g., pBlueBac III containing β-gal).

[0084] In a preferred embodiment, a vector is designed for producing a subject ActRII polypeptide in CHO cells, such as, for example, the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wisc.). As will become apparent, the subject genetic constructs can be used to direct expression of a subject ActRII polypeptide in cells grown in culture, for example, to produce proteins, including fusion or mutant proteins for purification.

[0085] The present disclosure also relates to a host cell transfected with a recombinant gene comprising the coding sequence of one or more subject ActRII polypeptides. The host cell can be any prokaryotic or eukaryotic cell. For example, the ActRII polypeptides of the present disclosure can be expressed in bacterial cells, such as E. coli, insect cells (e.g., using the baculovirus expression system), yeast or mammalian cells (e.g., Chinese hamster ovary (CHO) cell lines). Other suitable host cells are known to those skilled in the art.

[0086] Therefore, the present disclosure further relates to a method for producing a subject ActRII polypeptide. For example, a host cell transfected with an expression vector encoding an ActRII polypeptide can be cultured under suitable conditions that allow expression of the ActRII polypeptide to occur. The polypeptide can be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the ActRII polypeptide can be retained in the cytoplasm or membrane fraction, the cells can be harvested, lysed, and the protein can be isolated. Cell culture includes host cells, medium, and other by-products. Suitable media for cell culture are well known in the art. The subject polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification using an antibody specific for a particular epitope of the ActRII polypeptide, and affinity purification using a substance that binds to a domain fused to the ActRII polypeptide (e.g., a protein A column can be used to purify an ActRII-Fc fusion protein). In some embodiments, the ActRII polypeptide is a fusion protein that contains a domain that facilitates its purification.

[0087] In some embodiments, purification is achieved by a series of column chromatography steps, including, for example, three or more of the following in any order: Protein A chromatography, Q Sepharose chromatography, phenyl sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be completed with virus filtration and buffer exchange. ActRII protein can be purified to a purity of more than 90% (i.e., greater than 90%), more than 95%, more than 96%, more than 98%, or more than 99% as measured by size exclusion chromatography, and to a purity of more than 90%, more than 95%, more than 96%, more than 98%, or more than 99% as measured by SDS PAGE. The target level of purity should be sufficient to achieve the desired results in mammalian systems, particularly non-human primates, rodents (mouse), and humans.

[0088] In another embodiment, a fusion gene encoding a purification leader sequence, such as a poly-(His) / enterokinase cleavage site sequence, at the N-terminus of the desired portion of the recombinant ActRII polypeptide is 2+ This may allow for purification of the expressed fusion protein by affinity chromatography using a metal resin. The purification leader sequence may then be removed by treatment with enterokinase to obtain the purified ActRII polypeptide. See, e.g., Hochuli et al. (1987) J. Chromatography 411:177; and Janknecht et al. (1991) PNAS USA 88:8972.

[0089] The techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments encoding different polypeptide sequences is carried out according to conventional techniques using blunt or staggered ends for ligation, restriction enzyme digestion to obtain suitable ends, filling in of sticky ends where appropriate, alkaline phosphatase treatment to avoid undesired ligation, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including an automated DNA synthesizer. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that generate complementary overhangs between two consecutive gene fragments, which can then be annealed to generate a chimeric gene sequence. See, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992.

[0090] 4.How to use In part, the present disclosure relates to a method for reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide described herein, comprising administering an ActRII polypeptide in a dosage regimen comprising: (i) administering a first dose of the ActRII polypeptide to the patient in an amount of 0.3 mg / kg; and (ii) administering a second dose of the ActRII polypeptide to the patient in an amount of 0.7 mg / kg, wherein if the patient shows symptoms of telangiectasia or includes risk factors for developing telangiectasia, then initiating administration of a third dose, thereby reducing the risk of telangiectasia. In some embodiments, the patient is receiving a therapeutically effective amount of the ActRII polypeptide described herein for the treatment of pulmonary arterial hypertension (PAH).

[0091] These methods are particularly aimed at therapeutic and prophylactic treatment of animals, more particularly humans. The terms "subject", "individual" or "patient" are used interchangeably throughout this specification and refer to either human or non-human animals. These terms include mammals, such as humans, non-human primates, laboratory animals, farm animals (including cows, pigs, camels, etc.), pet animals (e.g., dogs, cats, other farm animals, etc.) and rodents (e.g., mice and rats). In certain embodiments, the patient, subject or individual is a human.

[0092] The terms "treatment", "treat", "alleviate" and the like are generally used herein to mean to obtain a desired pharmacological and / or physiological effect, and may also mean to improve, alleviate and / or reduce the severity of one or more clinical complications of the condition being treated (e.g., reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide). The effect can be preventative in that it completely or partially delays the onset or recurrence of a disease, condition or its complications, and / or can be therapeutic in that it partially or completely cures a disease, condition and / or side effects caused by the disease or condition. As used herein, "treatment" includes any treatment of a disease or condition in a mammal, particularly a human. As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that reduces the occurrence of a disorder or condition in a treated sample compared to an untreated control sample, or delays the onset of a disease or condition compared to an untreated control sample, in a statistical sample.

[0093] In general, treatment or prevention of a disease or condition described in the present disclosure (e.g., reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide) is achieved by administering one or more ActRII polypeptides of the present disclosure in an "effective amount". An effective amount of an agent means an amount effective at the dosage and for the period of time necessary to achieve the desired therapeutic or prophylactic result. The "therapeutically effective amount" of an agent of the present disclosure may vary depending on factors such as the condition, age, sex, and weight of the individual and the ability of the agent to induce a desired response in the individual. A "prophylactically effective amount" means an amount effective at the dosage and for the period of time necessary to achieve the desired prophylactic result.

[0094] In certain aspects, the present disclosure contemplates the use of an ActRII polypeptide in combination with one or more additional active agents or other supportive therapies to treat or prevent a disease or condition (e.g., to reduce the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide). As used herein, "in combination with", "combination of", "combined with" or "combined" administration refers to any form of administration in which the additional active agent or supportive therapy (e.g., second, third, fourth, etc.) is still effective in the body (e.g., multiple compounds are effective in the patient simultaneously over a period of time, which may include synergistic effects of the compounds). Efficacy may not correlate with measurable concentrations of the agents in blood, serum, or plasma. For example, different therapeutic compounds may be administered in the same formulation or in separate formulations, simultaneously or sequentially, on different schedules. Thus, subjects receiving such treatment may benefit from the combined effects of different active agents or therapies. One or more ActRII polypeptides of the present disclosure may be administered simultaneously with, prior to, or subsequent to one or more other additional agents or supportive therapies (e.g., those disclosed herein). In general, each active agent or therapy is administered at a dose and / or time schedule determined for that particular agent. The particular combination to be used in the regimen will take into consideration the compatibility of the ActRII polypeptides of the present disclosure with the additional active agents or therapies and / or the desired effect.

[0095] Overview of the WHO classification The pulmonary arterial hypertension conditions treated by the methods described herein can include any one or more of the conditions recognized according to the World Health Organization (WHO). See, e.g., Simonneau (2019) Eur Respir J:53:1801913.

[0096] [Table 2]

[0097] The clinical purpose of the classification of PAH is to divide clinical conditions associated with PAH into specific subgroups according to their pathophysiological mechanisms, clinical symptoms, hemodynamic characteristics, and treatment strategies. This clinical classification may be updated when new data regarding the above features become available or when additional clinical factors are taken into account.

[0098] The term "pulmonary hemodynamic parameters" as used herein refers to any parameter used to describe or evaluate blood flow through the heart and pulmonary vasculature. Examples of pulmonary hemodynamic parameters include, but are not limited to, mean pulmonary artery pressure (mPAP), diastolic pulmonary artery pressure (dPAP) [also known as pulmonary artery diastolic pressure (PADP)], systolic pulmonary artery pressure (sPAP) [also known as pulmonary artery systolic pressure (PASP)], mean right atrial pressure (mRAP), pulmonary capillary wedge pressure (PCWP) [also known as pulmonary artery wedge pressure (PAWP)], pulmonary vascular resistance (PVR) and cardiac output (CO).

[0099] Many of the above pulmonary hemodynamic parameters are interrelated. For example, PVR is related to mPAP, PCWP and CO according to the following equation: PVR = (mPAP-PCWP) / CO [Wood units]

[0100] PVR assesses the resistance to flow imposed by the pulmonary vasculature in the absence of the influence of left-sided filling pressure. PVR may also be assessed by the following formula: PVR=TPG×80 / CO [unit: dyne-second-cm -5 ] or PVR = (mPAP-PCWP) x 80 / CO [unit: dyne-second-cm -5 ]

[0101] In some embodiments, the total perimeter resistance (TPR) may be evaluated using the following formula: TPR=mPAP / CO

[0102] According to some embodiments, the precapillary pulmonary artery contribution to PH may be reflected by an elevated PVR. In some embodiments, a normal PVR is between 20 and 130 dyne-sec-cm. -5 Or between 0.5 and 1.1 wood units. According to some embodiments, elevated PVR can mean a PVR of more than 2 wood units, more than 2.5 wood units, more than 3 wood units, or more than 3.5 wood units.

[0103] As yet another example, mPAP is related to dPAP and sPAP according to the following equation: mPAP=(2 / 3)dPAP+(1 / 3)sPAP.

[0104] Additionally, using dPAP and sPAP, pulse pressure (mmHg) can be calculated using the following formula: Pulse Pressure = sPAP - dPAP

[0105] Pulse pressure can be used to calculate pulmonary artery compliance using the following equation: Pulmonary artery compliance (mI.mmHg -1 )=stroke volume / pulse pressure

[0106] In some embodiments, the pulmonary hemodynamic parameters are measured directly, for example during right heart catheterization, while in other embodiments, the pulmonary hemodynamic parameters are estimated and / or assessed by other techniques, such as magnetic resonance imaging (MRI) or echocardiography.

[0107] Exemplary pulmonary hemodynamic parameters include mPAP, PAWP and PVR. One or more of the pulmonary hemodynamic parameters can be measured by any suitable method, such as by using right heart catheterization or echocardiography. Various hemodynamic characteristics of PH and PAH are shown in Table 2.

[0108] [Table 3]

[0109] The clinical classification or hemodynamic characteristics and associated diagnostic parameters of PAH described herein may be updated or changed based on the availability of new or existing data sources or when additional clinical factors are taken into account.

[0110] Characteristics of PAHs Pulmonary arterial hypertension (WHO group 1 PH) is a severe, progressive, life-threatening disease of the pulmonary vascular system characterized by abnormal proliferation of smooth muscle cells in the walls of the pulmonary arteries and severe vasoconstriction. Severe constriction of blood vessels in the lungs leads to very high pulmonary artery pressures. These high pressures make it difficult for the heart to pump blood to the lungs so that it can be oxygenated. Patients with PAH suffer from extreme shortness of breath because the heart tries to pump against these high pressures. Patients with PAH typically develop a significant increase in PVR and a persistent elevation in mPAP, which ultimately leads to right ventricular failure and death. Patients diagnosed with PAH have a poor prognosis and similarly impaired quality of life, with an average life expectancy of 2-5 years from the time of diagnosis if left untreated.

[0111] Various factors are involved in the pathogenesis of pulmonary hypertension, including proliferation of pulmonary cells that may contribute to vascular remodeling (i.e., hyperplasia). For example, pulmonary vascular remodeling is primarily caused by proliferation of arterial endothelial cells and smooth muscle cells in patients with pulmonary hypertension. Overexpression of various cytokines is believed to promote pulmonary hypertension. In addition, it has been found that pulmonary hypertension may result from hyperproliferation of pulmonary arterial smooth cells and pulmonary endothelial cells. Furthermore, progressive PAH may be characterized by muscularization of distal pulmonary arterioles, concentric intimal thickening, and obstruction of the vascular lumen by proliferating endothelial cells. Pietra et al., J. Am. Coll. Cardiol., 43:255-325 (2004).

[0112] PAH can be diagnosed based on a mean pulmonary artery pressure greater than 25 mmHg (or, in updated guidelines, greater than 20 mmHg) at rest and normal pulmonary artery capillary wedge pressure. PAH can cause shortness of breath, dizziness, fainting, and other symptoms, all of which are worsened by exertion. PAH can be a severe disease with markedly reduced exercise tolerance and heart failure. The two main types of PAH include idiopathic PAH (e.g., PAH with no identified predisposition) and hereditary PAH (e.g., PAH associated with mutations in BMPR2, ALK1, ENG, SMAD9, CAV1, KCNK3, or EIF2AK4). In 70% of cases of familial PAH, there is a mutation in the BMPR2 gene. Risk factors for the development of PAH include family history of PAH, drug and toxin use (e.g. methamphetamine or cocaine use), infections (e.g. HIV infection or schistosomiasis), liver cirrhosis, congenital heart anomalies, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis or connective tissue / autoimmune disorders (e.g. scleroderma or lupus).PAH can be associated with long-term responders to calcium channel blockers, pronounced features of venous / capillary (PVOD / PCH) involvement and persistent PH in the newborn syndrome.

[0113] Diagnosis of PAH The diagnosis of PAH, including functional groups, may be determined based on symptoms and physical examination, with a review of a comprehensive set of parameters to determine whether hemodynamic and other criteria are met. Some of the criteria that may be considered include the patient's clinical symptoms (e.g., shortness of breath, fatigue, weakness, angina, syncope, dry cough, exercise-induced nausea and vomiting), electrocardiogram (ECG) results, chest radiograph results, pulmonary function tests, arterial blood gases, echocardiography results, ventilation / perfusion lung scan results, high-resolution computed tomography results, contrast-enhanced computed tomography results, pulmonary angiography results, cardiac magnetic resonance imaging, blood tests (e.g., biomarkers such as BNP or NT-proBNP), immunology, abdominal ultrasound scan, right heart catheterization (RHC), vascular reactivity, and genetic testing. See, for example, Galie N. et al., Euro Heart J. (2016) 37, 67-119.

[0114] In some embodiments, biomarkers may be used to aid in the diagnosis of PAH. For example, in some embodiments, the biomarker is a marker of vascular dysfunction (e.g., asymmetric dimethylarginine (ADMA), endothelin-1, angiopoietin, or von Willebrand factor). In some embodiments, the biomarker is a marker of inflammation (C-reactive protein, interleukin 6, chemokines). In some embodiments, the biomarker is a marker of myocardial stress (e.g., atrial natriuretic peptide, brain natriuretic peptide (BNP) / NT-proBNP, or troponin). In some embodiments, the biomarker is a marker of low CO and / or tissue hypoxia (e.g., pCO2, uric acid, growth differentiation factor 15 (GDF15), or osteopontin). In some embodiments, the biomarker is a marker of secondary organ damage (e.g., creatinine or bilirubin). See, e.g., Galie N. et al., Euro Heart J. (2016) 37, 67-119.

[0115] Measurement of pH In certain aspects, the disclosure relates to a method of reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of a therapeutic ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1) for the treatment and / or progression of pulmonary arterial hypertension (PAH). In some embodiments, the method relates to treating a PAH patient with idiopathic PAH. In some embodiments, the method relates to treating a PAH patient with hereditary PAH (e.g., PAH due to one or more mutations in BMPR2, ALK-1, ENG, SMAD9, CAV1, and KCNK3). In some embodiments, the method relates to treating a PAH patient with hereditary PAH due to an unknown mutation. In some embodiments, the method relates to treating a PAH patient with drug- or toxin-induced PAH. In some embodiments, the method relates to treating a PAH patient with PAH associated with a connective tissue disease. In some embodiments, the method relates to treating a PAH patient with PAH associated with HIV infection. In some embodiments, the method relates to treating PAH patients with PAH associated with portal hypertension. In some embodiments, the method relates to treating PAH patients with PAH associated with schistosomiasis. In some embodiments, the method relates to treating PAH patients classified as long-term responders to calcium channel blockers. In some embodiments, the method relates to treating PAH patients with clear features of venous / capillary (PVOD / PCH) involvement. In some embodiments, the method relates to treating PAH patients with persistent pulmonary hypertension (PH) of the newborn syndrome. In some embodiments, the method relates to treating PAH patients with PAH associated with simple congenital systemic-pulmonary shunts at least one year after shunt repair.

[0116] Functional Classes PAH at baseline can be mild, moderate or severe, as assessed, for example, by the World Health Organization (WHO) functional class, a measure of disease severity in patients with pulmonary hypertension. The WHO functional class is an adaptation of the New York Heart Association (NYHA) system and is routinely used to qualitatively assess activity tolerance, for example, in monitoring disease progression and treatment response (Rubin (2004) Chest 126:7-10). In the WHO system, four functional classes are recognized: Functional Class I: Pulmonary hypertension without consequential limitation of physical activity; ordinary physical activity does not cause undue dyspnea or fatigue, chest pain or pre-syncope; Functional Class II: Pulmonary hypertension causing slight limitation of physical activity; Patient is comfortable at rest; ordinary physical activity causes undue dyspnea or fatigue, chest pain or pre-syncope; Functional Class III: Pulmonary hypertension causing significant limitation of physical activity; Patient is comfortable at rest; less than ordinary activity causes undue dyspnea or fatigue, chest pain or pre-syncope; Functional Class IV: Pulmonary hypertension resulting in the inability to perform any physical activity without symptoms; Patient shows signs of right heart failure; dyspnea and / or fatigue can occur even at rest; any physical activity increases discomfort.

[0117] Known Treatments for PAH There is no known cure for PAH. Current treatment methods focus on extending the patient's lifespan and improving the patient's quality of life. This is usually associated with good exercise capacity, good right ventricular function and low risk of death (e.g., leading and / or maintaining the patient in WHO functional class I or functional class II). Current treatment methods for PAH may include administration of vasodilators, such as prostacyclin, epoprostenol and sildenafil; endothelin receptor antagonists, such as bosentan; calcium channel blockers, such as amlodipine, diltiazem and nifedipine; anticoagulants, such as warfarin; and diuretics. PAH has also been treated with oxygen therapy, atrial septal defect creation, pulmonary endarterectomy and lung and / or heart transplantation. However, each of these methods has one or more drawbacks, which may include lack of efficacy, severe side effects, poor patient compliance and high cost. In certain embodiments, the method relates to reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1) in combination with one or more additional active agents and / or supportive therapies for treating PAH (e.g., vasodilators, such as prostacyclin, epoprostenol, and sildenafil; endothelin receptor antagonists, such as bosentan; calcium channel blockers, such as amlodipine, diltiazem, and nifedipine; anticoagulants, such as warfarin; diuretics; oxygen therapy; atrial septal defect creation; pulmonary endarterectomy; and lung and / or heart transplantation); bardoxolone methyl or a derivative thereof; oleanolic acid or a derivative thereof.

[0118] Dosing regimens to reduce the risk of telangiectasia The probability that a patient will suffer from telangiectasia may be higher during initial treatment with an ActRII polypeptide. In certain embodiments, a dosing regimen may be used to prevent, improve or reduce symptoms of telangiectasia. In some embodiments, the ActRII polypeptide of the present disclosure is administered using a dosing regimen. In some embodiments, the method includes administering to a patient a dosing regimen of a therapeutically effective amount of an ActRII polypeptide disclosed herein, the dosing regimen comprising a first dose of 0.1 mg / kg to 1.0 mg / kg of the polypeptide for a first period, and then a second dose of 0.1 mg / kg to 1.0 mg / kg of the polypeptide administered for a second period. In some embodiments, the method comprises administering to the patient a dosing regimen of a therapeutically effective amount of an ActRII polypeptide disclosed herein, the dosing regimen comprising a first dose of 0.1 mg / kg to 1.0 mg / kg of the polypeptide for a first period of time, a second dose of 0.1 mg / kg to 1.0 mg / kg of the polypeptide administered for a second period of time, and then a third dose of 0.1 mg / kg to 1.0 mg / kg of the polypeptide administered for a third period of time. In some embodiments, the method comprises administering to the patient a dosing regimen of a therapeutically effective amount of an ActRII polypeptide disclosed herein, the dosing regimen comprising a first dose of 0.1 mg / kg to 1.0 mg / kg of the polypeptide for a first period of time, a second dose of 0.1 mg / kg to 1.0 mg / kg of the polypeptide administered for a second period of time, and a third dose for a third period of time during which treatment with the ActRII polypeptide is withheld. In some embodiments, the first dose of ActRII polypeptide is administered to the patient in an amount of about 0.2 mg / kg to about 0.4 mg / kg. In some embodiments, the first dose of ActRII polypeptide is administered to the patient in a dose of 0.3 mg / kg. In some embodiments, the second dose of ActRII polypeptide is administered to the patient in an amount of about 0.5 mg / kg to about 0.8 mg / kg. In some embodiments, the second dose of ActRII polypeptide is administered to the patient in a dose of 0.7 mg / kg.In some embodiments, the third dose of the ActRII polypeptide is administered to the patient in an amount of about 0.2 mg / kg to about 0.4 mg / kg. In some embodiments, the third dose of the ActRII polypeptide is administered to the patient at a dose of 0.3 mg / kg. In some embodiments, the third dose comprises withholding treatment with the ActRII polypeptide for a period of time. In some embodiments, treatment with the ActRII polypeptide is withheld for at least 2-6 weeks. In some embodiments, treatment with the ActRII polypeptide is withheld for at least 3 weeks. In some embodiments, treatment with the ActRII polypeptide is withheld for at least 6 weeks. In some embodiments, treatment with the ActRII polypeptide is withheld for at least 9 weeks. In some embodiments, treatment with the ActRII polypeptide is resumed after the period during which treatment with the ActRII polypeptide was withheld.

[0119] In some embodiments, the dosing regimen comprises administering a first dose of ActRII polypeptide to the patient in an amount of 0.3 mg / kg, and then administering a second dose of ActRII polypeptide to the patient in an amount of 0.7 mg / kg. In some embodiments, the dosing regimen comprises administering a first dose of ActRII polypeptide to the patient in an amount of 0.3 mg / kg, administering a second dose of ActRII polypeptide to the patient in an amount of 0.7 mg / kg, and administering a third dose of ActRII polypeptide to the patient in an amount of 0.3 mg / kg. In some embodiments, the dosing regimen comprises administering a first dose of ActRII polypeptide to the patient in an amount of 0.3 mg / kg, administering a second dose of ActRII polypeptide to the patient in an amount of 0.7 mg / kg, and administering a third dose of ActRII polypeptide to the patient, comprising withholding the treatment of ActRII polypeptide for at least 3 weeks. In some embodiments, the dosing regimen comprises administering to the patient a first dose of an ActRII polypeptide in an amount of 0.3 mg / kg, and administering to the patient a second dose of an ActRII polypeptide, comprising withholding treatment with the ActRII polypeptide for at least 3 weeks. In some embodiments, the dosing regimen comprises administering to the patient a first dose of an ActRII polypeptide in an amount of 0.3 mg / kg, administering to the patient a second dose of an ActRII polypeptide in an amount of 0.7 mg / kg, administering to the patient a third dose of an ActRII polypeptide, comprising withholding treatment with the ActRII polypeptide for at least 3 weeks, and administering to the patient a fourth dose of an ActRII polypeptide in an amount of 0.3 mg / kg. In some embodiments, the second dose is greater than the first dose. In some embodiments, the first dose is greater than the second dose. In some embodiments, the third dose is greater than the second dose. In some embodiments, the second dose is greater than the third dose. In some embodiments, the third dose comprises administering to the patient a dose of the ActRII polypeptide in an amount of 0.3 mg / kg. In some embodiments, the third dose comprises withholding treatment with the ActRII polypeptide for at least 2-6 weeks.In some embodiments, the second dose comprises withholding treatment with the ActRII polypeptide for at least 2-6 weeks. In some embodiments, the third dose comprises withholding treatment with the ActRII polypeptide for at least 3 weeks. In some embodiments, the second dose comprises withholding treatment with the ActRII polypeptide for at least 3 weeks. In some embodiments, the first period is at least 3 weeks. In some embodiments, the second period is at least 3 weeks. In some embodiments, the third period is at least 3 weeks. In some embodiments, the second period is at least 21 weeks. In some embodiments, the second period is at least 45 weeks. In some embodiments, the second period is longer than the first period. In some embodiments, the third period is longer than the first period. In some embodiments, the third period is longer than the second period. In some embodiments, the first dose is administered to the patient for at least 3 weeks. In some embodiments, the second dose is administered for at least 21 weeks. In some embodiments, the second dose is administered for at least 45 weeks.

[0120] In some embodiments, the dose change between the first dose and the second dose is determined by the attending physician, taking into account various factors (e.g., symptoms and / or risk factors of telangiectasia). In some embodiments, the dose change between the second dose and the third dose is determined by the attending physician, taking into account various factors (e.g., symptoms and / or risk factors of telangiectasia). In some embodiments, the dose change between the third dose and the fourth dose is determined by the attending physician, taking into account various factors (e.g., symptoms and / or risk factors of telangiectasia).

[0121] In some embodiments, the various factors include, but are not limited to, the patient's risk factors for developing telangiectasia. In some embodiments, the risk factors for developing telangiectasia are selected from the group consisting of low BMP9 levels, low BMP10 levels, low VEGF levels, hereditary hemorrhagic telangiectasia (HHT) and connective tissue disease (CTD). In some embodiments, the patient includes one or more of these risk factors before treatment with ActRII polypeptide. In some embodiments, the patient includes one or more of these risk factors during treatment with ActRII polypeptide. In some embodiments, the risk factors for developing telangiectasia are the patient includes one or more mutations associated with hereditary hemorrhagic telangiectasia (HHT) and / or connective tissue disease (CTD). In some embodiments, the one or more mutations associated with HHT are mutations in genes selected from the group consisting of ENG, ACVRL1, EPHB4, SMAD4, GDF2, BMPR9 and RASA1. In some embodiments, the one or more mutations associated with the CTD are selected from the group consisting of ABCC6, ACTA2, ADAMTS2, ADAMTS10, ADAMTSL2, ALDH18A1, ATP6V0A2, ATP7A, B3GALT6, B4GALT7, BGN, C1R, C1S, CBS, CHST14, COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL9A1, COL9A2, COL9A3, COL11A1, COL11A2, DSE, EFEMP2(FBLN4), ELN, FBLN5, FBN1, FBN2, FBN3, FBN4, FBN5, FBN6, FBN7, FBN8, FBN9, FBN10, FBN11, FBN12, FBN13, FBN14, FBN15, FBN16, FBN17, FBN18, FBN19, FBN20, FBN21, FBN22, FBN23, FBN24, FBN25, FBN26, FBN27, FBN28, FBN29, FBN21, FBN21, FBN22, FBN26, FBN27, FBN28, FBN29, FBN20, FBN21, FBN22, FBN23, FBN24, FBN25 ... , FBN2, FKBP14, FLCN, FLNA, FOXE3, GORAB, LOX, LTBP4, MED12, MFAP5, MYH11, MYLK, NOTCH1, NOTCH2, PKD1, PKD2, PLOD1, PRDM5, PRKG1, PTDSS1, PYCR1, RIN2, SKI, SLC2A10, SLC39A13, SMAD2, SMAD3, SMAD4, SMAD6, TAB2, TGFB2, TGFB3, TGFBR1, TGFBR2, TNXB and ZNF469.In some embodiments, if one or more of the patient's symptoms or risk factors are abnormal before or during treatment, the patient's dose of an ActRII polypeptide disclosed herein is maintained (e.g., maintained at 0.3 mg / kg or 0.7 mg / kg).

[0122] In some embodiments, the dosing regimen prevents, ameliorates or reduces side effects of the ActRII polypeptide. In some embodiments, administering an ActRII polypeptide according to the dosing regimen provided herein reduces the risk of telangiectasia. In some embodiments, administering an ActRII polypeptide according to the dosing regimen provided herein reduces the risk of telangiectasia during a second period. In some embodiments, administering an ActRII polypeptide according to the dosing regimen provided herein reduces the risk of telangiectasia during a third period. In some embodiments, administering an ActRII polypeptide according to the dosing regimen provided herein reduces the risk of telangiectasia during a fourth period. In some embodiments, administering an ActRII polypeptide according to the dosing regimen provided herein reduces the risk of telangiectasia after the first 21 weeks of treatment. In some embodiments, administering an ActRII polypeptide according to the dosing regimen provided herein reduces the risk of telangiectasia after the first 45 weeks of treatment.

[0123] Symptoms and Risk Factors for Telangiectasia Telangiectasias are also known as spider veins, hyphen webs, thread veins, sunburst veins, stellate veins and venous flares. Telangiectasias involve dilated venules (tiny blood vessels) that produce thread-like red lines or patterns on the skin. These patterns, or telangiectasias, are thought to be caused by the release or activation of vasoactive substances under a number of conditions. Symptoms of telangiectasias include pain, itching, thread-like red marks on the skin, mucocutaneous telangiectasias, gastrointestinal bleeding, lesions on the skin, nosebleeds, bleeding gums, arteriovenous malformations, internal telangiectasias and red spots on the skin.

[0124] Although telangiectasias are often benign, they can also be caused by serious diseases such as hereditary hemorrhagic telangiectasia (HHT). In people with HHT, telangiectasias can develop in vital organs such as the liver. Rupture of these telangiectasias can cause life-threatening bleeding.

[0125] In certain aspects, the disclosure relates to a method of reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1), the method comprising administering the ActRII polypeptide according to a dosing regimen when the patient exhibits symptoms of telangiectasia or includes risk factors for developing telangiectasia, thereby reducing the risk of telangiectasia. In some embodiments, the symptoms of telangiectasia are selected from the group consisting of pain, itching, thread-like red marks on the skin, mucocutaneous telangiectasia, gastrointestinal bleeding, lesions on the skin, nosebleeds, bleeding gums, arteriovenous malformations, internal telangiectasia, and red spots on the skin. In some embodiments, the symptoms of telangiectasia are lesions on the skin. In some embodiments, the symptoms of telangiectasia are bleeding gums. In some embodiments, the symptoms of telangiectasia are nosebleeds. In some embodiments, the telangiectasia symptom is arteriovenous malformation. In some embodiments, the telangiectasia symptom is internal telangiectasia. In some embodiments, the arteriovenous malformation or internal telangiectasia occurs in internal organs (e.g., brain, liver, lung, spleen, urinary tract and spine). In some embodiments, the risk factor for developing telangiectasia is selected from the group consisting of low BMP9 level, low BMP10 level, low VEGF level, hereditary hemorrhagic telangiectasia (HHT) and connective tissue disease (CTD).

[0126] In certain embodiments, the present disclosure provides a method for managing a patient being treated with or a candidate for treatment with one or more ActRII polypeptides of the present disclosure (e.g., an amino acid sequence at least 90% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1) by measuring one or more symptoms or risk factors of telangiectasia in the patient. The one or more symptoms and / or risk factors of telangiectasia can be used to assess appropriate dosing for a patient who is a candidate for treatment with one or more ActRII polypeptides of the present disclosure, and / or to monitor symptoms and / or risk factors of telangiectasia during treatment, and / or to assess whether to adjust dosage during treatment with one or more ActRII polypeptides of the present disclosure, and / or to assess an appropriate maintenance dose of one or more ActRII polypeptides of the present disclosure. If one or more of the symptoms and / or risk factors of telangiectasia are abnormal, administration of one or more ActRII polypeptides can be reduced, delayed, or discontinued.

[0127] In one embodiment, if one or more symptoms and / or risk factors of telangiectasia are abnormal in a patient who is a candidate for treatment with one or more ActRII polypeptides, initiation of administration of one or more ActRII polypeptides of the present disclosure is delayed until the symptoms and / or risk factors of telangiectasia return to normal or acceptable levels, either naturally or through therapeutic intervention.

[0128] In certain embodiments, if one or more symptoms and / or risk factors of telangiectasia are abnormal in a patient who is a candidate for treatment with one or more ActRII polypeptides, the start of administration may not be delayed.However, the dosage or frequency of administration of one or more ActRII polypeptides of the present disclosure may be set to an amount that reduces the risk of telangiectasia occurring upon administration of one or more ActRII polypeptides of the present disclosure.Alternatively, a treatment regimen that combines a therapeutic agent that addresses the risk of telangiectasia with one or more ActRII polypeptides may be developed for the patient.

[0129] The severity of telangiectasia in the patient Telangiectasia and its severity level can be described and evaluated using various classifications. For example, the Clinical-Etiology-Anatomy-Pathophysiology (CEAP) classification of chronic venous disorders grades venous conditions in order of increasing severity. CEAP classifies telangiectasia into the mildest category (C1), which describes telangiectasia as a confluence of dilated intradermal venules less than 1 mm in internal diameter. See, for example, Table 3 below.

[0130] [Table 4]

[0131] Telangiectasia may be further classified into specific grades of severity. For example, telangiectasia may be classified into three grades according to the degree and extent of venous tortuosity and protuberance. See, e.g., Ruckley CV et al., European Journal of Vascular and Endovascular Surgery. 2008;36(6):719-724.

[0132] In certain aspects, the disclosure relates to methods of reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1), where the patient has C1 telangiectasia as classified by Clinical-Etiology-Anatomy-Pathophysiology (CEAP). In some embodiments, the method improves telangiectasia from C1 to C0 as classified by CEAP.

[0133] In certain aspects, the disclosure relates to a method of reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1), wherein the patient has grade 1, grade 2, or grade 3 telangiectasia. In some embodiments, the patient has grade 1 telangiectasia. In some embodiments, the patient has grade 2 telangiectasia. In some embodiments, the patient has grade 3 telangiectasia. In some embodiments, the risk of telangiectasia is reduced by reducing or improving the severity of telangiectasia. In some embodiments, the risk of telangiectasia is reduced by improving the grade of telangiectasia. In some embodiments, the method improves telangiectasia from grade 2 to grade 1. In some embodiments, the method improves telangiectasia from grade 3 to grade 2. In some embodiments, the method improves telangiectasia from grade 3 to grade 1. In some embodiments, the risk of telangiectasia is reduced by preventing the progression of telangiectasia. In some embodiments, the method prevents the progression of telangiectasia from grade 1 to grade 2. In some embodiments, the method prevents the progression of telangiectasia from grade 2 to grade 3.

[0134] In certain aspects, the disclosure relates to a method of reducing the risk of telangiectasia in a patient receiving a therapeutically effective amount of an ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1), the method comprising: (i) administering to the patient a first dose of the ActRII polypeptide in an amount of 0.3 mg / kg; and (ii) administering to the patient a second dose of the ActRII polypeptide in an amount of 0.7 mg / kg, wherein if the patient exhibits symptoms of telangiectasia or includes risk factors for developing telangiectasia, administration of a third dose is initiated, thereby reducing the risk of telangiectasia. In some embodiments, the third dose comprises withholding treatment with the ActRII polypeptide until telangiectasia improves to grade 1 or less. In some embodiments, the third dose comprises withholding treatment with the ActRII polypeptide until telangiectasia improves to grade 2 or less. The third dose involves withholding treatment with the ActRII polypeptide until telangiectasia improves to Grade 3 or less.

[0135] 5. Pharmaceutical Compositions and Methods of Administration In certain embodiments, the therapeutic method of the present disclosure comprises administering the composition systemically or locally as an implant or device.The therapeutic composition for use in the present disclosure is in a substantially pyrogen-free or pyrogen-free physiologically acceptable form when administered.The therapeutically useful substance other than ActRII polypeptide, which may be optionally included in the composition, may be administered simultaneously or sequentially with the subject compound in the method disclosed herein.

[0136] Typically, the protein therapeutics disclosed herein are administered parenterally, particularly intravenously or subcutaneously. Pharmaceutical compositions suitable for parenteral administration can include one or more ActRII polypeptides together with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The formulations may be presented in unit-dose or multi-dose sealed containers, for example ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid vehicle for injection, for example water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind described herein.

[0137] The compositions and formulations may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0138] Furthermore, the composition may be injected or encapsulated in a form for delivery to a target tissue site. In certain embodiments, the composition of the present invention may include a matrix that can deliver one or more therapeutic compounds (e.g., ActRII polypeptides) to a target tissue site to provide structure to developing tissue, and optimally can be resorbed in the body. For example, the matrix may provide sustained release of the ActRII polypeptide. Such a matrix may be composed of materials currently used for other implantation medical applications.

[0139] The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, aesthetic appearance and interface properties. The particular application of the subject composition will determine the appropriate formulation. Potential matrices for the composition can be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Other matrices are composed of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates or other ceramics. Matrices can be composed of any combination of the above types of materials, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can be modified in composition, such as in calcium-aluminate-phosphate, and can be treated to modify pore size, particle size, particle shape and biodegradability.

[0140] In certain embodiments, the methods of the invention can be orally administered, for example, in the form of a capsule, cachet, pill, tablet, lozenge (using a flavored base, usually sucrose and acacia or tragacanth), powder, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a lozenge (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and / or mouthwash, each containing a predetermined amount of the agent as an active ingredient. The agent can also be administered as a bolus, electuary, or paste.

[0141] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more therapeutic compounds of the present invention may be mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) bulking agents or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glutaraldehyde, glyceryl stearate, sorbitol, glyceryl stearate, and / or sorbitol; (4) glyceryl stearate, sorbitol ... glycerol;(4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate;(5) solution retarders such as paraffin;(6) absorption accelerators such as quaternary ammonium compounds;(7) wetting agents such as cetyl alcohol and glycerol monostearate;(8) absorbents such as kaolin and bentonite clay;(9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents.Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols, and the like.

[0142] Liquid dosage forms for oral administration include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions (suspensions), syrups and elixirs.In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.In addition to the inert diluent, oral compositions may also contain auxiliary agents, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, coloring agents, excipients and preservatives.

[0143] Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0144] The compositions of the present invention may also contain auxiliary agents, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action may be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. Prolonged absorption of the injectable pharmaceutical form may also be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0145] It is understood that the administration regimen is determined by the attending physician, taking into consideration various factors that modify the action of the subject compounds (e.g., ActRII polypeptides) of the present disclosure. Various factors include, but are not limited to, the age, sex, and diet of the patient, the severity of the disease, the administration time, and other clinical factors. If desired, the administration amount may vary depending on the type of matrix used for reconstitution and the type of compound in the composition. In some embodiments, the patient's hematological parameters may be monitored by periodic evaluation to determine whether the patient has higher than normal red blood cell levels and / or hemoglobin levels (e.g., hemoglobin levels above 16.0 g / dL or hemoglobin levels above 18.0 g / dL). In some embodiments, patients with higher than normal red blood cell levels and / or hemoglobin levels may receive delayed or reduced administration until their levels return to normal or acceptable levels.

[0146] In some embodiments, the ActRII polypeptide of the disclosure is administered at a dose range of 0.1 mg / kg to 2.0 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 0.1 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 0.2 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 0.3 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 0.4 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 0.5 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 0.6 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 0.7 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 0.8 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 0.9 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 1.0 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 1.1 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 1.2 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 1.3 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 1.4 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 1.5 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 1.6 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 1.7 mg / kg. In some embodiments, an ActRII polypeptide of the disclosure is administered at 1.8 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 1.9 mg / kg. In some embodiments, the ActRII polypeptide of the disclosure is administered at 2.0 mg / kg.

[0147] In certain embodiments, the ActRII polypeptide of the present disclosure is administered once a day. In certain embodiments, the ActRII polypeptide of the present disclosure is administered twice a day. In certain embodiments, the ActRII polypeptide of the present disclosure is administered once a week. In certain embodiments, the ActRII polypeptide of the present disclosure is administered twice a week. In certain embodiments, the ActRII polypeptide of the present disclosure is administered three times a week. In certain embodiments, the ActRII polypeptide of the present disclosure is administered every two weeks. In certain embodiments, the ActRII polypeptide of the present disclosure is administered every three weeks. In certain embodiments, the ActRII polypeptide of the present disclosure is administered every four weeks. In certain embodiments, the ActRII polypeptide of the present disclosure is administered monthly.

[0148] In certain embodiments, the present invention also provides gene therapy for in vivo production of ActRII polypeptide. Such therapy will achieve its therapeutic effect by introducing the polynucleotide sequence of ActRII polypeptide into cells or tissues with the above-mentioned disorder. The delivery of the polynucleotide sequence of ActRII polypeptide can be achieved using recombinant expression vectors, such as chimeric viruses or colloidal dispersion systems. For therapeutic delivery of the polynucleotide sequence of ActRII polypeptide, the use of targeted liposomes is preferred.

[0149] Various viral vectors that can be utilized in the gene therapy taught herein include adenovirus, herpes virus, vaccinia virus, or preferably RNA viruses, such as retroviruses. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), and Rous sarcoma virus (RSV). Multiple genes can be incorporated into many additional retroviral vectors. All of these vectors can introduce and incorporate genes for selectable markers so that transduced cells can be identified and generated. Retroviral vectors can be made target specific, for example, by binding to sugars, glycolipids, or proteins. Preferred targeting is achieved by using antibodies. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to the viral envelope to enable target-specific delivery of retroviral vectors containing ActRII polypeptides. In a preferred embodiment, the vector is targeted to bone or cartilage.

[0150] Alternatively, tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol, and env by conventional calcium phosphate transfection. These cells are then transfected with a vector plasmid containing the gene of interest. The resulting cells release the retroviral vector into the culture medium.

[0151] Another targeted delivery system for polynucleotides of ActRII polypeptides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated within the aqueous interior space and delivered to cells in a biologically active form (see, for example, Fraley et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using liposomal vehicles are known in the art. See, for example, Mannino et al., Biotechniques, 6:682, 1988. The composition of liposomes is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0152] Examples of lipids useful for preparing liposome include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides.Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.Targeting of liposome can also be based on, for example, organ specificity, cell specificity and organelle specificity, and is known in the art.

[0153] The disclosure provides formulations that can be modified to include acids and bases to adjust the pH as well as buffers to maintain the pH within narrow ranges.

[0154] 6. Working Example The foregoing disclosure will be more readily understood by reference to the following examples, which are provided merely for illustrative purposes of certain embodiments of the invention and are not intended to be limiting.

[0155] Example 1: ActRIIA-Fc fusion protein Soluble ActRIIA fusion proteins were constructed that contained the extracellular domain of human ActRIIa fused to the human or mouse Fc domain with a minimal linker between them, and these constructs are designated ActRIIA-hFc and ActRIIA-mFc, respectively.

[0156] ActRIIA-hFc purified from a CHO cell line is shown below (SEQ ID NO: 23).

number

[0157] An additional ActRIIA-hFc purified from a CHO cell line and lacking the C-terminal lysine is shown below (SEQ ID NO: 40).

number

[0158] ActRIIA-hFc and ActRIIA-mFc proteins were expressed in a CHO cell line. Three different leader sequences were considered. (i) Honeybee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 24); (ii) tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 25); (iii) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 26).

[0159] The form selected uses the TPA leader and has the following unprocessed amino acid sequence:

number

[0160] This polypeptide is encoded by the following nucleic acid sequence:

number

[0161] Both ActRIIA-hFc and ActRIIA-mFc were highly suitable for recombinant expression. As shown in Figures 4A and 4B, the protein was purified as a single, well-defined protein peak. N-terminal sequencing showed a single sequence of -ILGRSETQE (SEQ ID NO: 29). Purification could be achieved by a series of column chromatography steps, including, for example, three or more of the following in any order: Protein A chromatography, Q Sepharose chromatography, Phenyl Sepharose chromatography, Size exclusion chromatography, and Cation exchange chromatography. Purification could be completed with viral filtration and buffer exchange. ActRII protein was purified to greater than 98% (i.e., greater than 98%) purity as measured by size exclusion chromatography and greater than 95% purity as measured by SDS PAGE.

[0162] ActRIIA-hFc and ActRIIA-mFc showed high affinity for the ligand. GDF11 or activin A was immobilized on a Biacore™ CM5 chip using standard amine coupling methods. ActRIIA-hFc and ActRIIA-mFc proteins were loaded into the system and binding was measured. ActRIIA-hFc was 5×10 -12 Dissociation constant (K D ) binds to activin and is 9.96 × 10 -9 K DActRIIA-hFc bound to GDF11 at 100-fold increase in affinity for activin B, GDF8, BMP6 and BMP10. Using similar binding assays, ActRIIA-hFc was determined to have high to moderate affinity for other TGF-beta superfamily ligands, including activin B, GDF8, BMP6 and BMP10. ActRIIA-mFc also showed similar properties.

[0163] ActRIIA-hFc was very stable in pharmacokinetic studies. Rats were administered 1 mg / kg, 3 mg / kg, or 10 mg / kg of ActRIIA-hFc protein, and plasma levels of the protein were measured at 24, 48, 72, 144, and 168 hours. In another study, rats were administered 1 mg / kg, 10 mg / kg, or 30 mg / kg. In rats, ActRIIA-hFc had a serum half-life of 11-14 days, and circulating levels of the drug were very high after 2 weeks (11 μg / ml, 110 μg / ml, or 304 μg / ml, respectively, for initial doses of 1 mg / kg, 10 mg / kg, or 30 mg / kg). In cynomolgus monkeys, the plasma half-life was substantially longer than 14 days and circulating levels of the drug were 25 μg / ml, 304 μg / ml or 1440 μg / ml following initial doses of 1 mg / kg, 10 mg / kg or 30 mg / kg, respectively.

[0164] Example 2: Characterization of ActRIIA-hFc Protein ActRIIA-hFc fusion protein was expressed in stably transfected CHO-DUKX B11 cells from pAID4 vector (SV40 ori / enhancer, CMV promoter) using the tissue plasminogen leader sequence of SEQ ID NO: 25. The protein, purified as described in Example 1 above, had the sequence of SEQ ID NO: 23. The Fc portion is the human IgG1 Fc sequence shown in SEQ ID NO: 23. Protein analysis showed that the ActRIIA-hFc fusion protein was formed as a homodimer with disulfide bonds.

[0165] The material expressed in CHO cells has a higher affinity for activin B ligand than that reported for an ActRIIA-hFc fusion protein expressed in human 293 cells [see del Re et al. (2004) J Biol Chem. 279(51):53126-53135]. The use of the TPA leader sequence also resulted in higher production than other leader sequences and provided a highly pure N-terminal sequence, unlike ActRIIA-Fc expressed with the native leader. The use of the native leader sequence produced two major species of ActRIIA-Fc, each with a different N-terminal sequence.

[0166] Example 3: Alternative ActRIIA-Fc Proteins Various ActRIIA mutants that can be used according to the methods described herein are described in the international patent application published as WO2006 / 012627, which is incorporated by reference in its entirety (see, e.g., pages 55-58). Another construct can have a deletion of the C-terminal tail (the last 15 amino acids of the extracellular domain of ActRIIA). The sequence of such a construct is shown below (the Fc portion is underlined) (SEQ ID NO: 30).

number

Claims

1. A pharmaceutical composition comprising an ActRII fusion protein for use in reducing the risk of telangiectasia in a patient, wherein the ActRII fusion protein (i) administering a first dose of the ActRII fusion protein to the patient in an amount of 0.3 mg / kg; (ii) administering a second dose of the ActRII fusion protein to the patient in an amount of 0.7 mg / kg once every three weeks, three weeks after the first dose, as long as the patient requires treatment; (iii) delaying the administration of the second dose for at least six weeks until one or more symptoms or risk factors for the development of telangiectasia improve, if the patient exhibits one or more symptoms or risk factors for telangiectasia; and (iv) restarting the administration of one or more doses of the ActRII fusion protein to the patient in an amount of 0.7 mg / kg once every three weeks, as long as the patient requires treatment The pharmaceutical composition, comprising being administered according to a dosing regimen comprising.

2. A pharmaceutical composition comprising an ActRII fusion protein for use in reducing the risk of telangiectasia in a patient being treated with an ActRII fusion protein, wherein the ActRII fusion protein (i) administering a first dose of the ActRII fusion protein to the patient in an amount of 0.3 mg / kg; (ii) administering one or more second doses of the ActRII fusion protein to the patient in an amount of 0.7 mg / kg once every three weeks, three weeks after the first dose, as long as the patient requires treatment; and (iii) reducing the second dose to an amount of 0.3 mg / kg once every three weeks until one or more symptoms or risk factors for the development of telangiectasia improve, if the patient exhibits one or more symptoms or risk factors for telangiectasia The pharmaceutical composition, comprising being administered according to a dosing regimen comprising.

3. The pharmaceutical composition according to claim 1 or 2, wherein the one or more symptoms of telangiectasia are selected from the group consisting of pain, itching, thread-like red marks on the skin, cutaneous mucosal telangiectasia, gastrointestinal bleeding, skin lesions, nosebleeds, gum bleeding, arteriovenous malformations, internal telangiectasia, and red spots on the skin.

4. The pharmaceutical composition according to claim 1 or 2, wherein the use alleviates or improves the severity of telangiectasia in the patient.

5. The pharmaceutical composition according to claim 1 or 2, wherein the use prevents the progression of telangiectasia in the patient.

6. The pharmaceutical composition according to claim 1 or 2, comprising an ActRII polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 and an ActRII fusion protein.

7. The pharmaceutical composition according to claim 1 or 2, comprising an ActRII polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 and an ActRII fusion protein.

8. The pharmaceutical composition according to claim 1 or 2, wherein the ActRII fusion protein further comprises an Fc domain of an immunoglobulin.

9. The pharmaceutical composition according to claim 8, wherein the Fc domain of the immunoglobulin is the Fc domain of IgG1 immunoglobulin.

10. The pharmaceutical composition according to claim 1 or 2, wherein the ActRII fusion protein further comprises a linker domain located between the ActRII polypeptide and the Fc domain of the immunoglobulin.

11. The pharmaceutical composition according to claim 10, wherein the linker domain is selected from the group consisting of TGGG (SEQ ID NO: 20), TGGGG (SEQ ID NO: 18), SGGGG (SEQ ID NO: 19), GGGGS (SEQ ID NO: 22), GGG (SEQ ID NO: 16), GGGG (SEQ ID NO: 17), and SGGG (SEQ ID NO: 21).

12. The pharmaceutical composition according to claim 1 or 2, comprising an ActRII polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 23 and an ActRII fusion protein.

13. The pharmaceutical composition according to claim 1 or 2, comprising an ActRII polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 23 and an ActRII fusion protein.

14. The pharmaceutical composition according to claim 1 or 2, comprising an ActRII polypeptide comprising the amino acid sequence of SEQ ID NO: 23 and an ActRII fusion protein.

15. The pharmaceutical composition according to claim 1 or 2, comprising an ActRII polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40 and an ActRII fusion protein.

16. The pharmaceutical composition according to claim 1 or 2, comprising an ActRII polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 40 and an ActRII fusion protein.

17. The pharmaceutical composition according to claim 1 or 2, comprising an ActRII polypeptide comprising the amino acid sequence of SEQ ID NO: 40 and an ActRII fusion protein.

18. The pharmaceutical composition according to claim 1 or 2, wherein the fusion protein is reconstituted after lyophilization.

19. The pharmaceutical composition according to claim 1 or 2, wherein the fusion protein is soluble.

20. The pharmaceutical composition according to claim 1 or 2, wherein the fusion protein is administered to a patient using subcutaneous injection.

21. The pharmaceutical composition according to claim 1 or 2, wherein the fusion protein is part of a homodimeric protein complex.

22. The pharmaceutical composition according to claim 1 or 2, wherein the patient is receiving one or more therapies for PAH.

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