Compositions and methods for treating pulmonary hypertension

Abstract

Methods of treating, preventing, or reducing the progression rate and / or severity of pulmonary hypertension (PH), particularly treating, preventing, or reducing the progression rate and / or severity of one or more PH-related complications, are provided.SOLUTION: In some aspects, the disclosure relates to GDF / BMP antagonists and methods of using GDF / BMP antagonists to treat, prevent, or reduce the progression rate and / or severity of PH. The disclosure also provides methods of using GDF / BMP antagonists to treat, prevent, or reduce the progression rate and / or severity of a variety of conditions including, but not limited to, pulmonary vascular remodeling, pulmonary fibrosis, and right ventricular hypertrophy. The present disclosure further provides methods of using a GDF / BMP antagonist to reduce right ventricular systolic pressure in a subject in need thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 362,955, filed July 15, 2016. No. 62 / 453,888 filed February 2, 2017, and No. 62 / 453,888 filed May 2, 2017. The application claims the benefit of priority from application Ser. No. 62 / 510,403, filed on 24th January 2016. The disclosures of the aforementioned applications are hereby incorporated by reference in their entireties. [Background technology]

[0002] Background of the Invention Pulmonary hypertension (PH) is a condition characterized by high blood pressure in the pulmonary vasculature, including the pulmonary arteries, veins, and capillaries. PH is a condition characterized by low mean pulmonary artery (PA) pressure. Defined as ≥25 mmHg at rest or ≥30 mmHg during exercise [Hill et al. Respiratory Care, Vol. 54(No. 7): pp. 958-68 (2009)] The main symptom of PH is shortness of breath or difficulty breathing, and other symptoms include fatigue, dizziness, fainting, and endurance. Peripheral edema (swelling of the feet, legs, or ankles), bluish lips and skin, chest pain, angina, exercise Symptoms include dizziness, dry cough, rapid pulse, and palpitations. PH is a condition that affects people with pulmonary hypertension. It can be a severe disease that leads to heart failure, one of the most common causes of death in the United States. Hypertension presents challenges that make many types of surgery or procedures difficult and are associated with high mortality rates. obtain.

[0003] PH shares similarities in pathophysiological mechanisms, clinical findings, and therapeutic approaches. These can be classified based on the different symptoms of the disease [Simonneau et al., JACC, Vol. 54 (No. 1): pp. S44-54 (2009)]. The clinical classification of PH was first proposed in 1973. A recently updated clinical classification was proposed by the World Health Organization (WHO) in 2008. According to the updated PH clinical classification, there are five major groups of PH: Pulmonary arterial hypertension (PAH) characterized by a PA wedge pressure ≤ 15 mmHg; PA wedge pressure > PH (pulmonary venous hypertension or pulmonary venous hypertension) due to left heart disease characterized by a blood pressure of 15 mmHg also known as congestive heart failure); PH due to pulmonary disease and / or hypoxia; chronic thrombosis Embolic PH; and PH with unknown or multifactorial etiology [Simonneau et al., JA CC, vol. 54(1):S44-54 (2009); Hill et al., Respirat Ory Care, Vol. 54 (No. 7): pp. 958-68 (2009)]. PAH is a Idiopathic PAH (IPAH), a sporadic disease with no family history of PAH or identified risk factors; Hereditary PAH; drug- and toxin-induced PAH; connective tissue disease, HIV infection, hives PAH associated with pulmonary arterial hypertension, congenital heart disease, schistosomiasis, and chronic hemolytic anemia; and neonatal persistent PH [Simonneau et al., JACC, Vol. 54 ( 1, pp. S44-54 (2009)]. The diagnosis of various types of PH involves a series of tests. Need.

[0004] Generally, treatment of PH depends on the cause or classification of the PH. When it is caused by a medical condition, it is known as secondary PH, and its treatment is usually Treatment of pulmonary venous hypertension generally involves diuretics, basal by administering ACE inhibitors, ATP blockers, or mitral or aortic valve replacement therapy. PAH treatment involves optimizing left ventricular function by repairing or replacing the aortic valve. Medications include pulmonary vasodilators, digoxin, diuretics, anticoagulants, and oxygen therapy. Tonicity agents include those administered via the prostacyclin route (e.g., intravenous epoprostenol, subcutaneous or intravenous prostacyclins, including intravenous treprostinil and inhaled iloprost), nitric oxide pathways (e.g., phosphodiesterase-5 inhibitors, including sildenafil and tadalafil) agents), and the endothelin-1 pathway (e.g., oral bosentan and oral ambricenta targeting different pathways, including endothelin receptor antagonists (Humb ert, M., Am. J. Respir. Crit. Care Med., 1 79:650-6 (2009); Hill et al., Respiratory Care , Vol. 54 (No. 7): pp. 958-68 (2009)]. However, current treatments for PH are and do not provide a cure, and they may contribute to the underlying vascular limping observed in many PH patients. Dering and muscular arterialization of blood vessels are not directly treated. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hill et al., Respiratory Care, 54(7):958-68 (2009) [Non-patent document 2] Simonneau et al., JACC, Vol. 54(1):S44-54 (2009) [Non-patent document 3] Hill et al., Respiratory Care, 54(7):958-68 (2009) [Non-patent document 4] Simonneau et al., JACC, Vol. 54(1):S44-54 (2009) [Non-patent document 5] Humbert, M., Am. J. Respir. Crit. Care Med., 179:650-6 (2009); Hill et al., Respiratory Care, 54(7):958-68 (2009) Summary of the Invention [Means for solving the problem]

[0006] Thus, there is a high unmet need for effective therapies to treat pulmonary hypertension. Therefore, it is an object of the present disclosure to treat, prevent, or slow the progression and progression of PH. and / or reduce the severity, particularly to treat or prevent one or more PH-related complications. The present invention provides a method for treating or reducing the rate of progression and / or severity of the disease.

[0007] Summary of the Invention In part, the data presented herein demonstrate that GDF / BMP antagonists (inhibitors) For example, the soluble ActRIIA polypeptide can be used to treat pulmonary hypertension. When the peptide and the ALK4:ActRIIB heterodimer were used individually, Reduces blood pressure, cardiac hypertrophy, and lung weight in a talin-induced pulmonary arterial hypertension (PAH) model Similar positive effects were observed in the Sugen hypoxia PAH model. Histological analysis revealed that ActRIIA polypeptides were expressed in the cytoplasm of ActRIA. IA polypeptide inhibits monocrotaline-induced and sugen hypoxia PAH models The surprising effect on vascular remodeling and reduction of muscular arterialization in both Furthermore, it was revealed that ActRIIA polypeptide and Both ALK4 and ALK4:ActRIIB heterodimers surprisingly demonstrated efficacy in the treatment of PAH. Improvement of various complications of PAH compared with sildenafil, a drug approved for PAH Thus, the present disclosure provides a and ActRIIB) signaling pathway antagonists to improve the severity of pulmonary hypertension To establish that soluble ActRIIa polypeptide and ALK4 can reduce the severity of ALK4-related inflammatory bowel disease. : ActRIIB heteromultimers may have mechanisms other than antagonism of ActRIIA / B ligands. Although pulmonary hypertension can be affected through a variety of factors, the present disclosure nonetheless provides a desirable treatment. This study demonstrates that agents can be selected based on their ActRII signaling antagonist activity. Thus, in some embodiments, the present disclosure provides a method for treating hypertension, particularly pulmonary hypertension. For example, one or more ActRIIA / B ligands [e.g., activin ( Activin A, Activin B, Activin AB, Activin C, Activin AC, Activin activin BC, activin E, activin AE, and / or activin BE), GDF8 , GDF11, GDF3, BMP6, BMP15, and BMP10]. agonists; one or more type I and / or type II receptors (e.g., ActRIIA , ActRIIB, ALK4, ALK7, and ALK5); and one or more downstream signaling components (e.g., Smad2 and 3, etc. Various ActRII signaling antagonists, including those that inhibit Smad proteins, As used herein, such antagonists are used in methods of treating inflammatory bowel disease. These signaling antagonists are collectively referred to as "GDF / BMP antagonists" or " Thus, the present disclosure relates, in part, to the treatment of pulmonary hypertension (e.g., , PAH), particularly for treating one or more complications of pulmonary hypertension (e.g., blood pressure GDF / GGF for treating cardiac hypertrophy, vascular remodeling, and muscular arterialization of blood vessels BMP antagonist compositions and methods are provided. GDF / BMP antagonists that may be used include, for example, ligand traps (e.g., soluble ActRIIA polypeptide, ActRIIB polypeptide, ALK4:ActRIIB heterodimer, follistatin polypeptide, and FLRG polypeptide), antibody antagonists, small molecule antagonists, and nucleotide antagonists. Optionally, the GDF / BMP antagonist is administered in combination with one or more It may be used in combination with multiple supportive therapies and / or additional active agents.

[0008] In certain aspects, the present disclosure provides a method for treating pulmonary arterial hypertension, comprising administering to a subject in need thereof a The present invention relates to a method for treating a patient suffering from atopic dermatitis, the method comprising the step of administering an effective amount of an ActRIIA polypeptide to a patient suffering from atopic dermatitis. In some embodiments, the ActRIIA polypeptide comprises amino acids 21, 22 of SEQ ID NO:9. , 23, 24, 25, 26, 27, 28, 29, or 30. and amino acids 110, 111, 112, 113, 114, 115, and 116 of SEQ ID NO: 9. , 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 , 127, 128, 129, 130, 131, 132, 133, 134, or 135 The amino acid sequence ending with one of these has at least 70% (e.g., at least 7 0%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) In some embodiments, the ActRIIA polypeptide comprises an amino acid sequence that is identical to the sequence The amino acid sequence of sequence number 10 has at least 70% (e.g., at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical amino acids In some embodiments, the ActRIIA polypeptide comprises the amino acid sequence of SEQ ID NO: 11. The amino acid sequence has at least 70% (e.g., 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 the first amino acid sequence. In some embodiments, the ActRIIA polypeptide comprises an ActRIIA domain and an A A fusion protein comprising one or more polypeptide domains heterologous to ctRIIA. In some embodiments, the ActRIIA polypeptide is an immunoglobulin protein. In some embodiments, the fusion protein comprises an Fc domain of an immunoglobulin. In some embodiments, the domain is an Fc domain of an IgG1 immunoglobulin. The ActRIIA fusion protein comprises an ActRIIA polypeptide domain and one or more a linker domain located between the heterologous domain (e.g., Fc immunoglobulin domain) of the In some embodiments, the linker domain further comprises TGGG (SEQ ID NO: 23 ), TGGGG (SEQ ID NO: 21), SGGGG (SEQ ID NO: 22), GGGGS (SEQ ID NO: 25), GGG (SEQ ID NO: 19), GGGG (SEQ ID NO: 20), and SGGG (SEQ ID NO: No. 24. In some embodiments, the ActRIIA polypeptide is selected from the group consisting of is at least 70%, 75%, 80%, 85%, 86%, or 90% similar to the amino acid sequence of SEQ ID NO: 32. %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 97%, 98%, 99%, or 100% identical amino acid sequence. In embodiments, the ActRIIA polypeptide comprises the amino acid sequence of SEQ ID NO: 32. In one embodiment, the ActRIIA polypeptide consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIA polypeptide is a member of a homodimeric protein complex. In some embodiments, the ActRIIA polypeptide is glycosylated. In some embodiments, the ActRIIA polypeptide is derived from Chinese hamster egg The glycosylation pattern can be obtained by expression in the foci. In embodiments, administration of an ActRIIA polypeptide reduces pulmonary artery pressure in a patient. In some embodiments, administration of an ActRIIA polypeptide reduces pulmonary artery pressure in a patient. At least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40% , 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% In some embodiments, administration of an ActRIIA polypeptide reduces the In some embodiments, administration of an ActRIIA polypeptide reduces ventricular hypertrophy caused by: Reduce ventricular hypertrophy in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% reduction. Administration of ActRII reduces smooth muscle hypertrophy in a patient. Administration of a polypeptide of formula A induces smooth muscle hypertrophy in a patient by at least 10% (e.g., 10% , 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, or at least 80%. Administration of ActRIIA polypeptide reduces pulmonary arteriolar muscularization in patients. In some embodiments, administration of an ActRIIA polypeptide is inducible by the administration of an ActRIIA polypeptide to a subject with pulmonary arteriolar muscularization. at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80 In some embodiments, administration of an ActRIIA polypeptide reduces the incidence of leukemia in a patient by 100%. In some embodiments, administration of an ActRIIA polypeptide reduces pulmonary vascular resistance. The goal is to reduce the pulmonary vascular resistance in patients by at least 10% (e.g., 10%, 15%, 20%, 2 5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 7 In some embodiments, the ActRIIA polynucleotide is reduced by 5%, or by at least 80%. Administration of the peptide reduces pulmonary vascular resistance in patients by at least 25-30%. In one embodiment, the patient has pulmonary arterial hypertension and is classified as having a pulmonary hypertension syndrome according to the World Health Organization Functional Classification System. In some embodiments, the patient has pulmonary hypertension of functional class II or III according to the pulmonary hypertension stem cell line. , patients have idiopathic or hereditary pulmonary arterial hypertension, drug- and / or toxin-induced pulmonary hypertension pulmonary hypertension associated with connective tissue disease, and congenital pulmonary hypertension at least 1 year after shunt repair pulmonary hypertension associated with left-to-right shunt In some embodiments, the patient has one or more of the following pulmonary arterial hypertension types: In some embodiments, the patient is being treated with multiple vasodilators. catalyzer type 5 inhibitor, soluble guanylate cyclase stimulator, prostacyclin receptor activator and endothelin receptor antagonists. is being treated with multiple agents. In some embodiments, one or more of the agents is: Bosentan, sildenafil, beraprost, macitentan, selexipag, epopros Tenor, treprostinil, iloprost, ambrisentan, and tadalafil In some embodiments, the method comprises administering one or more vasodilators to a subject. In some embodiments, the method further comprises administering a phosphodiesterase type 5 inhibitor, Soluble guanylate cyclase stimulators, prostacyclin receptor agonists, and endothelial cells and administering one or more agents selected from the group consisting of serine receptor antagonists. In some embodiments, the one or more agents include bosentan, sildenafil , beraprost, macitentan, selexipag, epoprostenol, treprostinil , iloprost, ambrisentan, and tadalafil. In some embodiments, the patient has a 6-minute walk distance of 150 to 400 meters. In an embodiment, the method includes measuring the patient's 6-minute walk distance by at least 10 meters (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150 , 175, 200, 250, 300, or more than 400 meters). In this embodiment, the patient has a hemoglobin level greater than 8 g / dl and less than 15 g / dl. In some embodiments, the method slows the clinical worsening of pulmonary arterial hypertension. In an embodiment, the method comprises determining the clinical significance of pulmonary hypertension according to the World Health Organization Functional Classification System for Pulmonary Hypertension. In some embodiments, the method comprises slowing the progression of one or more of the following disorders associated with pulmonary arterial hypertension: In some embodiments, the ActRIIA potency reduces the risk of hospitalization due to multiple complications. The peptides include activin A, activin B, GDF11, GDF8, BMP10, and and BMP6.

[0009] In some embodiments, the present disclosure provides a method of treating pulmonary hypertension, comprising administering to a subject in need thereof and administering to a patient suffering from the condition an effective amount of a GDF / BMP antagonist or a GDF / BMP antagonist. In certain aspects, the present disclosure relates to a method for treating pulmonary hypertension comprising administering a combination of The present invention relates to a method for preventing a GDF / BMP disorder, comprising administering to a patient in need thereof an effective amount of a GDF / BMP antagonist. The present invention relates to a method for treating a rheumatoid arthritis comprising administering a GDF / BMP antagonist or a combination of GDF / BMP antagonists to a patient. In certain aspects, the present disclosure provides a method for reducing the rate of progression of pulmonary hypertension, comprising administering to a subject a and administering to a patient in need thereof an effective amount of a GDF / BMP antagonist or a GDF / BMP antagonist. In some embodiments, the present disclosure relates to a method for treating an interstitial A method for treating a chronic pulmonary disease, comprising administering to a patient in need thereof an effective amount of a GDF / BMP antagonist. The method comprises administering a GDF / BMP antagonist to a subject, the GDF / BMP antagonist being a GDF / BMP antagonist selected from the group consisting of activin, GD F8, GDF11, GDF3, BMP6, BMP15, BMP10, ActRIIA, A Inhibits one or more of ctRIIB, ALK4, ALK5, and ALK7 In some embodiments, the present disclosure provides a method for treating one or more complications of interstitial lung disease. In a method for treating, preventing, or reducing the rate of progression and / or severity of a disease administering an effective amount of a GDF / BMP antagonist to a patient in need thereof. and the GDF / BMP antagonist is selected from the group consisting of activin, GDF8, GDF11, and GDF 3, BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK4, The present invention provides a method for inhibiting one or more of ALK5 and ALK7. In embodiments, the interstitial lung disease is idiopathic pulmonary fibrosis. A method for reducing the severity of pulmonary hypertension, comprising administering to a patient in need thereof an effective amount of GDF / The method includes administering a BMP antagonist or a combination of GDF / BMP antagonists. In certain aspects, the present disclosure relates to a method for treating one or more complications of pulmonary hypertension. (e.g., proliferation of smooth muscle and / or endothelial cells in the pulmonary arteries, neovascularization of the pulmonary arteries) Treating lung disease (e.g., pulmonary vascular remodeling, right ventricular hypertrophy, pulmonary fibrosis, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis) The method further comprises administering to a patient in need thereof an effective amount of a GDF / BMP antagonist or G In certain embodiments, the method comprises administering a combination of DF / BMP antagonists. In such a manner, the present disclosure provides a method for treating one or more complications of pulmonary hypertension (e.g., pulmonary artery vasculitis), including the treatment of pulmonary artery vasculitis, ... Muscle and / or endothelial cell proliferation, angiogenesis in the pulmonary arteries, dyspnea, chest pain, pulmonary vascular recurrence A method for preventing pulmonary fibrosis (e.g., pulmonary fibrosis, right ventricular hypertrophy, and pulmonary fibrosis) in a patient in need thereof. and administering to a patient suffering from the condition an effective amount of a GDF / BMP antagonist or a GDF / BMP antagonist. In certain aspects, the present disclosure relates to a method for treating pulmonary hypertension comprising administering a combination of one or more complications of the disease (e.g., proliferation of smooth muscle and / or endothelial cells in the pulmonary arteries) proliferation, pulmonary artery angiogenesis, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis), comprising administering to a patient in need thereof an effective amount of G. administering a GDF / BMP antagonist or a combination of GDF / BMP antagonists In certain aspects, the present disclosure relates to a method for treating one or more of the following conditions: Complications (e.g., proliferation of smooth muscle and / or endothelial cells in the pulmonary arteries, The severity of these diseases (angiogenesis, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis) The present invention relates to a method for reducing the severity of a disease, comprising administering to a patient in need thereof an effective amount of a GDF / BMP antagonist. and the method comprises administering to a subject a combination of a GDF / BMP antagonist or a GDF / BMP antagonist. In certain preferred embodiments, the methods described herein involve treating a patient with pulmonary arterial hypertension. In some embodiments, the methods described herein relate to patients who: mmHg (e.g., at least 25, 30, 35, 40, 45, or 50 mmHg) In some embodiments, the present invention relates to a patient having a resting pulmonary artery pressure (PAP). The method reduces PAP in a patient with pulmonary hypertension. PAP in patients with hypertension should be maintained at least 3 mmHg (e.g., at least 3 , 5, 7, 10, 12, 15, 20, or 25 mmHg). In accordance with the present invention, the methods described herein reduce pulmonary vascular resistance in patients with pulmonary hypertension. In some embodiments, the methods described herein are directed to treating a patient with pulmonary hypertension. In some embodiments, the methods described herein include: Increases left ventricular end-diastolic pressure in patients with pulmonary hypertension. The methods described herein increase exercise capacity (capacity, tolerance) in patients with pulmonary hypertension. For example, the method may be used to improve the 6-minute walking distance in patients with pulmonary hypertension. Increase the distance and optionally increase the walking distance to at least 10 meters (e.g., at least Also 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 meters or more In addition, the method may optionally be evaluated after a 6-minute walk test. In some embodiments, the method may reduce the patient's Borg Dyspnea Index (BDI). Borg Dyspnea Index (BDI) of at least 0.5 index points (e.g., at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 index points). In some embodiments, the methods described herein may be used in combination with other methods, such as those for Class I and Class II diabetes, as recognized by the World Health Organization. In some embodiments, the present invention relates to patients with grade III or IV pulmonary hypertension. The methods described herein may be used to assess the clinical progression (worsening) of pulmonary hypertension (e.g., the World Health Organization's In some embodiments, the method relates to slowing the progression of pulmonary Prevent or slow the progression of hypertension classification (e.g., as recognized by the World Health Organization) Pulmonary hypertension from grade I to grade II, grade II to grade III, or grade III to grade IV In some embodiments, the method includes preventing or slowing the progression of pulmonary hypertension. Promote or increase (e.g., from stage IV to stage III as recognized by the World Health Organization) , promote or increase regression of pulmonary hypertension from grade III to grade II or from grade II to grade I In some embodiments, the patient is administered one or more GDF / BMP antagonists. In addition to the above, one or more supportive therapies or active agents for treating pulmonary hypertension may also be administered. For example, patients may be administered prostacyclin and its derivatives (e.g., epoprin, rostenol, treprostinil, and iloprost); prostacyclin receptor antagonists agonists (e.g., selexipag); endothelin receptor antagonists (e.g., th elin, ambrisentan, macitentan, and bosentan); calcium channels blockers (e.g., amlodipine, diltiazem, and nifedipine); anticoagulants (e.g., , warfarin); diuretics; oxygen therapy; atrial septostomy; pulmonary endarterectomy; phosphodiesterase steroidase type 5 inhibitors (e.g., sildenafil and tadalafil); soluble guanyl Acid cyclase activators (e.g., cinaciguat and riociguat); ASK-1 inhibitors (e.g., CIIA;SCH79797;GS-4997;MSC2032964A;3 H-Naphtho[1,2,3-de]quinyline-2,7-dione, NQDI-1;2-thioxo -thiazolidine, 5-bromo-3-(4-oxo-2-thioxo-thiazolidine-5-yl) lidene)-1,3-dihydro-indol-2-one); NF-κB antagonists (e.g. For example, dh404, CDDO-epoxide; 2,2-difluoropropionamide; C2 8 Imidazole (CDDO-Im); 2-Cyano-3,12-dioxooleane-1,9 -Dien-28-oic acid (CDDO); 3-acetyloleanolic acid; 3-triflic acid Triflouroacetyloleanolic Acid cid);28-methyl-3-acetyloleanane;28-methyl-3-trifluoro Cetyloleanan; 28-methyloxyoleanolic acid; SZC014; SCZ015; SZC017; PEGylated derivative 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]oleanolic acid 28-O-Glucopyranosyl-(1→3)-beta-D-glucopyranosyl]oleanolic acid -beta-D-glucopyranosyl ester; 3-O-[beta-D-glucopyranosyl- (1→2)-beta-D-glucopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl 3-O-[αL-rhamnopyranosyl-(1→3)-β- D-Glucuronopyranosyl]oleanolic acid; 3-O-[alpha-L-rhamnopyranosyl]oleanolic acid; 28-O-beta-[(1→3)-beta-D-glucuronopyranosyl]oleanolic acid -D-Glucopyranosyl ester; 28-O-β-D-Glucopyranosyl-oleanol Acid;3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosiduronic acid (CS1); 3-O-β-D-glucopyranosyl(1→3)-β-D-glucan oleanolic acid Leucopyranosiduronic acid (CS2); Methyl 3,11-dioxoolean-12-ene 28-Olate (DIOXOL); ZCVI4-2; Benzyl 3-dehydro-oxy-1 ,2,5-oxadiazolo[3',4':2,3]oleanolate), lung and / or and administering one or more supportive care or active agents selected from the group consisting of: cardiac transplantation. In some embodiments, the patient may also be administered a BMP9 polypeptide. In embodiments, the BMP9 polypeptide is a mature BMP9 polypeptide. In some embodiments, the BMP9 polypeptide comprises a BMP9 prodomain polypeptide. In embodiments, the BMP9 polypeptide optionally comprises a BMP9 prodomain polypeptide. The BMP9 prodomain polypeptide is administered in a pharmaceutical preparation that may contain the BMP9 prodomain polypeptide. In such pharmaceutical preparations of BMP9, the BMP9 polypeptide comprises a BMP9 prodomain In some embodiments, the BMP9 pharmaceutical preparation may be non-covalently associated with the polypeptide. are substantially free of or free of BMP9 prodomain polypeptides. In embodiments, the patient may also be administered oleanolic acid or a derivative thereof.

[0010] In certain embodiments, the GDF used in accordance with the methods and uses described herein / BMP antagonist or combination of antagonists inhibits at least GDF11 The effect of GDF11 inhibition is , e.g., in an assay described herein (e.g., a Smad signaling reporter assay). The determination can be made using a cell-based assay, including a cell-based assay. In some embodiments, the GDF / BMP antagonist or combination of antagonists of the present disclosure may be Ligand binding activity can be measured using, for example, the uptake assays described herein. In some embodiments, the affinity of the antibody can be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists of the present disclosure comprises at least one x10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, less Tomo1×10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)'s K D As described herein, for example, Ligand traps (e.g., ActRII polypeptides, GDF traps, follistatin polypeptide, FLRG polypeptide, and ALK4:ActRIIB heteromultimer ), including antibodies, small molecules, nucleotide sequences, and combinations thereof, that inhibit GDF11. Various GDF / BMP antagonists that have the potential to inhibit or inhibit the growth of GDF / BMP are described in accordance with the methods and uses described herein. In certain embodiments, GDF / B, which inhibits GDF11, can be used. The MP antagonist or combination of antagonists may be an activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3 , BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK4, A LK5, ALK7, and one or more Smads (e.g., Smad2 and 3) may further inhibit one or more of:

[0011] In certain embodiments, the GDF used in accordance with the methods and uses described herein A BMP antagonist or combination of antagonists inhibits at least GFF8. The effects of GFF8 inhibition are e.g., For example, assays described herein (e.g., Smad signaling reporter assays) Thus, in some embodiments, the ATP concentration can be determined using a cell-based assay including: The GDF / BMP antagonist or combination of antagonists of the present disclosure may comprise at least Ligand binding activity can be measured using assays including, for example, those described herein. In some embodiments, the binding affinity of the present disclosure may be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists should be at least 1×10 - 7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1× 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)'s K D As described herein, for example, ligand-transferases can bind to at least GFF8. TRAP (e.g., ActRII polypeptide, GDF TRAP, follistatin polypeptide) tide, FLRG polypeptide, and ALK4:ActRIIB heteromultimer), antibodies, Various GFF8 inhibitors, including small molecules, nucleotide sequences, and combinations thereof, are disclosed. DF / BMP antagonists are used in accordance with the methods and uses described herein. In certain embodiments, GDF / BMP antagonists that inhibit GFF8 can be used. The combination of inhibitors or antagonists may be activin (e.g., activin A, activin B, B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF11, GDF3, BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK4, ALK5, AL K7 and one of one or more Smads (e.g., Smad2 and 3) Or, more than one may be inhibited.

[0012] In certain embodiments, the GDF used in accordance with the methods and uses described herein A BMP antagonist or combination of antagonists inhibits at least GFF3. The effects of GFF3 inhibition are e.g., For example, assays described herein (e.g., Smad signaling reporter assays) Thus, in some embodiments, the ATP concentration can be determined using a cell-based assay including: The GDF / BMP antagonist or combination of antagonists of the present disclosure may comprise at least Ligand binding activity can be measured using assays including, for example, those described herein. In some embodiments, the binding affinity of the present disclosure may be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists should be at least 1×10 - 7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1× 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)'s K D As described herein, for example, ligand-transferases can bind to GFF3. TRAP (e.g., ActRII polypeptide, GDF TRAP, follistatin polypeptide) tide, FLRG polypeptide, and ALK4:ActRIIB heteromultimer), antibodies, Various GFF3 inhibitors, including small molecules, nucleotide sequences, and combinations thereof, are disclosed. DF / BMP antagonists are used in accordance with the methods and uses described herein. In certain embodiments, GDF / BMP antagonists that inhibit GFF3 can be used. The combination of inhibitors or antagonists may be activin (e.g., activin A, activin B, B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF11, BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK4, ALK5, AL K7 and one of one or more Smads (e.g., Smad2 and 3) Or, more than one may be inhibited.

[0013] In certain embodiments, the GDF used in accordance with the methods and uses described herein The BMP antagonist or combination of antagonists inhibits at least BMP6. The effects of BMP6 inhibition are For example, assays described herein (e.g., Smad signaling reporter assays) Thus, in some embodiments, the ATP concentration can be determined using a cell-based assay including: The GDF / BMP antagonist or combination of antagonists of the present disclosure may comprise at least Ligand binding activity can be measured using assays including, for example, those described herein. In some embodiments, the binding affinity of the present disclosure may be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists should be at least 1×10 - 7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1× 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)'s K D As described herein, for example, ligand-transferases can bind to BMP6. TRAP (e.g., ActRII polypeptide, GDF TRAP, follistatin polypeptide) tide, FLRG polypeptide, and ALK4:ActRIIB heteromultimer), antibodies, Various G inhibitors that inhibit BMP6, including small molecules, nucleotide sequences, and combinations thereof DF / BMP antagonists are used in accordance with the methods and uses described herein. In certain embodiments, GDF / BMP antagonists that inhibit BMP6 can be used. The combination of inhibitors or antagonists may be activin (e.g., activin A, activin B, B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, BMP15, BMP10, ActRIIA, ActRIIB, ALK4, ALK5, AL K7 and one of one or more Smads (e.g., Smad2 and 3) Or, more than one may be inhibited.

[0014] In certain embodiments, the GDF used in accordance with the methods and uses described herein / BMP antagonist or combination of antagonists inhibits at least BMP15 The effect of BMP15 inhibition is , e.g., in an assay described herein (e.g., a Smad signaling reporter assay). The determination can be made using a cell-based assay, including a cell-based assay. In some embodiments, the GDF / BMP antagonist or combination of antagonists of the present disclosure may be Ligand binding activity can be measured using, for example, the uptake assays described herein. In some embodiments, the affinity of the antibody can be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists of the present disclosure comprises at least one x10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, less Tomo1×10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)'s K D As described herein, for example, Ligand traps (e.g., ActRII polypeptides, GDF traps, follistatin polypeptide, FLRG polypeptide, and ALK4:ActRIIB heteromultimer ), including antibodies, small molecules, nucleotide sequences, and combinations thereof, that inhibit BMP15. Various GDF / BMP antagonists that have the potential to inhibit or inhibit the growth of GDF / BMP are described in accordance with the methods and uses described herein. In certain embodiments, GDF / B, which inhibits BMP15, can be used. The MP antagonist or combination of antagonists may be an activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3 , GDF11, BMP6, BMP10, ActRIIA, ActRIIB, ALK4, A LK5, ALK7, and one or more Smads (e.g., Smad2 and 3) may further inhibit one or more of:

[0015] In certain embodiments, the GDF used in accordance with the methods and uses described herein / BMP antagonist or combination of antagonists inhibits at least BMP10 The effect of BMP10 inhibition is , e.g., in an assay described herein (e.g., a Smad signaling reporter assay). The determination can be made using a cell-based assay, including a cell-based assay. In some embodiments, the GDF / BMP antagonist or combination of antagonists of the present disclosure may be Ligand binding activity can be measured using, for example, the antibody described herein. In some embodiments, the affinity of the antibody can be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists of the present disclosure comprises at least one x10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, less Tomo1×10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10-12 M)'s K D As described herein, for example, Ligand traps (e.g., ActRII polypeptides, GDF traps, follistatin polypeptide, and FLRG polypeptide, and ALK4:ActRIIB heterodimer Multimeric FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof Various GDF / BMP antagonists that inhibit BMP10 are described herein, including In certain embodiments, the BM can be used in accordance with the methods and uses described herein. GDF / BMP antagonists or combinations of antagonists that inhibit P10 have been shown to inhibit activity. activin (e.g., activin A, activin B, activin AB, activin C, activin Activin AC, Activin BC, Activin E, Activin AE, and / or Activin BE), GDF8, GDF3, GDF11, BMP6, BMP15, ActRIIA, A ctRIIB, ALK4, ALK5, ALK7, and one or more Smads (e.g. For example, one or more of Smad2 and 3) may be further inhibited.

[0016] In certain embodiments, the GDF used in accordance with the methods and uses described herein The BMP antagonist or combination of antagonists may be at least activin (e.g., For example, activin A, activin B, activin AB, activin C, activin AC, Inhibits activin BC, activin E, activin AE, and / or activin BE The effect of activin inhibition is , e.g., in the assays described herein (e.g., Smad signaling reporter assays). The determination can be made using cell-based assays, including cell-based assays. In some embodiments, the GDF / BMP antagonist or combination of antagonists of the present disclosure may be administered intravenously or intramuscularly. Ligand binding activity can be measured, for example, by the methods described herein. In some embodiments, the binding affinity assay may be determined using a binding affinity assay, including a ELISA assay. In other words, the GDF / BMP antagonist or combination of antagonists of the present disclosure may be at least Also 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, small At least 1 x 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 - 12 M)'s K D As described herein, For example, ligand traps (e.g., ActRII polypeptides, GDF traps, follistatinocytes, etc.) tin polypeptide, FLRG polypeptide, and ALK4:ActRIIB heteromer and activin inhibitors, including activin receptor agonists, antibodies, small molecules, nucleotide sequences, and combinations thereof. Various GDF / BMP antagonists that inhibit the growth of the thyroid gland are suitable for use in the methods and uses described herein. In certain embodiments, GDF / s that inhibit activin can be used. The BMP antagonist or combination of antagonists may be BMP15, GDF8, GDF 3, GDF11, BMP6, BMP10, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, and one or more Smads (e.g., Smad2 and 3) In certain preferred embodiments, the present invention may further inhibit one or more of the following: GDF / BMP antagonists used in accordance with the methods and uses described in the literature or The combination of antagonists is an agent that inhibits at least activin B. In one embodiment, the GDF / BMP antigen used in accordance with the methods and uses described herein The combination of agonists or antagonists does not substantially bind to activin A (e.g., 1×10 -7 K exceeds M D binds to activin A with relatively low binding capacity , for example, about 1 × 10 -8 M or approximately 1 x 10 -9 M), and / or active In certain preferred embodiments, the methods described herein do not inhibit bin A activity. and the GDF / BMP antagonist or combination of antagonists used according to the method of use. The combination inhibits at least activin B but does not substantially bind to activin A (e.g., For example, 1 x 10 -7 K exceeds M D Binds to activin A at or with relatively low binding Sex, for example, about 1 × 10 -8 M or approximately 1 x 10 -9 M), and / or This is a drug that does not inhibit thiamine A activity.

[0017] In certain embodiments, the GDF used in accordance with the methods and uses described herein A BMP antagonist or combination of antagonists inhibits at least ActRII. Agents that inhibit the growth of the thyroid gland (e.g., ActRIIA and / or ActRIIB) (e.g., Act The effects of GDF11 inhibition are described, for example, in Cell-based assays, including those involving Smad signaling reporter assays Thus, in some embodiments, the GDFs of the present disclosure can be determined using the assay The BMP antagonist or combination of antagonists binds to at least ActRII. Ligand binding activity can be measured using binding affinity assays, including, for example, the assays described herein. In some embodiments, the GDF / BM activity of the present disclosure can be determined using a GDF / BM activity assay. The P antagonist or combination of antagonists should be at least 1 × 10 -7 M (e.g. , at least 1 x 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M , at least 1 x 10 -11 M, or at least 1 × 10 -12 M)'s K D At least As described herein, for example, ligand traps ( For example, ActRII polypeptides, GDF traps, follistatin polypeptides, F LRG polypeptide, and ALK4:ActRIIB heteromultimer), antibodies, small molecules, Various GDFs that inhibit ActRII, including nucleotide sequences and combinations thereof / BMP antagonists according to the methods and uses described herein In certain embodiments, GDF / BMP antagonists that inhibit ActRII can be used. The combination of inhibitors or antagonists may be activin (e.g., activin A, activin B, B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, BMP6, BMP15, BMP10, ALK4, ALK5, ALK7, and one or more further inhibits one or more of several Smads (e.g., Smad2 and 3). possible.

[0018] In certain embodiments, the GDF used in accordance with the methods and uses described herein The BMP antagonist or combination of antagonists inhibits at least ALK4. The effects of ALK4 inhibition are For example, assays described herein (e.g., Smad signaling reporter assays) Thus, in some embodiments, the ATP concentration can be determined using a cell-based assay including: The GDF / BMP antagonist or combination of antagonists of the present disclosure may comprise at least Ligand binding activity can be measured using assays including, for example, those described herein. In some embodiments, the binding affinity of the present disclosure may be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists should be at least 1×10 - 7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1× 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)'s K D As described herein, for example, ligand-transferases bind to ALK4. TRAP (e.g., ActRII polypeptide, GDF TRAP, follistatin polypeptide) tide, FLRG polypeptide, and ALK4:ActRIIB heteromultimer), antibodies, Various GAGs that inhibit ALK4, including small molecules, nucleotide sequences, and combinations thereof DF / BMP antagonists are used in accordance with the methods and uses described herein. In certain embodiments, GDF / BMP antagonists that inhibit ALK4 can be used. The combination of inhibitors or antagonists may be activin (e.g., activin A, activin B, B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK5, AL K7 and one of one or more Smads (e.g., Smad2 and 3) Or, more than one may be inhibited.

[0019] In certain embodiments, the GDF used in accordance with the methods and uses described herein The BMP antagonist or combination of antagonists inhibits at least ALK5. The effects of ALK5 inhibition are For example, assays described herein (e.g., Smad signaling reporter assays) Thus, in some embodiments, the ATP concentration can be determined using a cell-based assay including: The GDF / BMP antagonist or combination of antagonists of the present disclosure may comprise at least Ligand binding activity can be measured using assays including, for example, those described herein. In some embodiments, the binding affinity of the present disclosure may be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists should be at least 1×10 - 7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1× 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)'s K D As described herein, for example, ligand-transferases bind to ALK5. TRAP (e.g., ActRII polypeptide, GDF TRAP, follistatin polypeptide) tide, FLRG polypeptide, and ALK4:ActRIIB heteromultimer), antibodies, Various GAGs that inhibit ALK5, including small molecules, nucleotide sequences, and combinations thereof DF / BMP antagonists are used in accordance with the methods and uses described herein. In certain embodiments, GDF / BMP antagonists that inhibit ALK5 can be used. The combination of inhibitors or antagonists may be activin (e.g., activin A, activin B, B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK4, AL K7 and one of one or more Smads (e.g., Smad2 and 3) Or, more than one may be inhibited.

[0020] In certain embodiments, the GDF used in accordance with the methods and uses described herein The BMP antagonist or combination of antagonists inhibits at least ALK7. The effects of ALK7 inhibition are For example, assays described herein (e.g., Smad signaling reporter assays) Thus, in some embodiments, the ATP concentration can be determined using a cell-based assay including: The GDF / BMP antagonist or combination of antagonists of the present disclosure may comprise at least Ligand binding activity can be measured using assays including, for example, those described herein. In some embodiments, the binding affinity of the present disclosure may be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists should be at least 1×10 - 7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1× 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)'s K D As described herein, for example, ligand-transferases can bind to ALK7. TRAP (e.g., ActRII polypeptide, GDF TRAP, follistatin polypeptide) tide, FLRG polypeptide, and ALK4:ActRIIB heteromultimer), antibodies, Various GAGs that inhibit ALK7, including small molecules, nucleotide sequences, and combinations thereof DF / BMP antagonists are used in accordance with the methods and uses described herein. In certain embodiments, GDF / BMP antagonists that inhibit ALK7 can be used. The combination of inhibitors or antagonists may be activin (e.g., activin A, activin B, B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK5, AL K4 and one of one or more Smads (e.g., Smad2 and 3) Or, more than one may be inhibited.

[0021] In certain embodiments, the GDF used in accordance with the methods and uses described herein The BMP antagonist or combination of antagonists comprises at least one or more Smad inhibitors are drugs that inhibit Smad proteins (e.g., Smad2 and 3). The effect on toxicity can be assessed, for example, by assays described herein (e.g., Smad signaling assays). This can be determined using cell-based assays, including transduction reporter assays. Thus, in some embodiments, the GDF / BMP antagonists or antagonists of the present disclosure The combination of compounds includes at least one or more Smad proteins (e.g., Smad2 and Smad3). and 3). Ligand binding activity can be measured using assays including, for example, those described herein. In some embodiments, the binding affinity of the present disclosure may be determined using a binding affinity assay, including The GDF / BMP antagonist or combination of antagonists should be at least 1×10 - 7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1× 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)'s K D at least one or more Smad proteins (e.g., Smad2 and 3) As described herein, for example, ligand traps (e.g., ActR II polypeptide, GDF trap, follistatin polypeptide, FLRG polypeptide and ALK4:ActRIIB heteromultimers), antibodies, small molecules, nucleotide sequences and combinations thereof. Various GDF / BMP antagonists that inhibit d2 and 3) are described herein. In certain embodiments, one or GDF / BMP inhibitors inhibit multiple Smad proteins (e.g., Smad2 and 3). The antagonist or combination of antagonists may be an activin (e.g., activin A, activin B, activin C, activin D, activin E, activin F, activin G, activin H, activin I ... Activin B, Activin AB, Activin C, Activin AC, Activin BC, Activin activin E, activin AE, and / or activin BE), GDF8, GDF3, GD F11, BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK 5, and ALK4.

[0022] In certain embodiments, the GDF used in accordance with the methods and uses described herein The BMP antagonist is an ActRII polypeptide. "Peptides" include naturally occurring ActRIIA and ActRIIB polypeptides and and the like, such as the polypeptides described herein (e.g., GDF trap polypeptides). Preferably, the ActRII polypeptide is a truncated form of the ActRII polypeptide. Contains the ligand-binding domain of the tRII polypeptide or a modified (variant) form thereof , consisting essentially of, or consisting of. For example, in some embodiments, Act The RIIA polypeptide is an ActRIIA polypeptide having an ActRIIA ligand-binding activity. domain, e.g., comprising or consisting essentially of a portion of the ActRIIA extracellular domain Similarly, an ActRIIB polypeptide may be an ActRIIB polypeptide. The peptide ActRIIB ligand binding domain, e.g., the ActRIIB extracellular domain It may comprise, consist essentially of, or consist of a part of the Alternatively, the ActRII polypeptides used in accordance with the methods described herein may be It is a soluble polypeptide.

[0023] In certain aspects, the disclosure provides compositions comprising ActRIIA polypeptides and For example, in some embodiments, the ActRIIA polypeptides of the present disclosure relate to: The sequence of amino acids 30 to 110 of SEQ ID NO: 9 is at least 70%, 75%, 80%, 85%, %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 In some embodiments, the Ac of the present disclosure comprises an amino acid sequence that is 100% identical to or 100% identical to the Ac of the present disclosure. The tRIIA polypeptide corresponds to any one of amino acids 21 to 30 of SEQ ID NO:9. Starting from the corresponding residue (e.g., amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), amino acid 11 of SEQ ID NO:9 Ending at a position corresponding to any one of amino acids 0 to 135 (e.g., amino acid 110, 1 11, 112, 113, 114, 115, 116, 117, 118, 119, 120, 1 21, 122, 123, 124, 125, 126, 127, 128, 129, 130, 1 (Ending in one of the following: 31, 132, 133, 134, or 135) ActR At least 70%, 75%, 80%, 85%, 86%, 87%, 88% of the IIA portion ,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% , 99%, or 100% identical amino acid sequence to ActR. The IIA polypeptide may have at least 70%, 75%, 80%, or 90% similarity to the amino acid sequence of SEQ ID NO:9. %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100% identical amino acid sequence. The ctRIIA polypeptide has at least 70%, 75%, or more amino acid residues that match the amino acid sequence of SEQ ID NO:10. %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 96%, 97%, 98%, 99%, or 100% identical amino acid sequence. %, 98%, 99%, or 100% identical amino acid sequences. In one embodiment, the ActRIIA polypeptide has at least the amino acid sequence of SEQ ID NO:11. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, It may comprise an amino acid sequence that is 96%, 97%, 98%, 99%, or 100% identical. In yet other embodiments, the ActRIIA polypeptide has the amino acid sequence of SEQ ID NO: 32. to at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 9 Amino acid sequences that are 4%, 95%, 96%, 97%, 98%, 99%, or 100% identical In still other embodiments, the ActRIIA polypeptide may comprise the sequence of SEQ ID NO: 36 amino acid sequences have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% may comprise, consist essentially of, or consist of an amino acid sequence that is identical to In still yet other embodiments, the ActRIIA polypeptide is obtained from the actinic acid sequence of SEQ ID NO: 39. The amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical It may comprise, consist essentially of, or consist of an amino acid sequence.

[0024] In other aspects, the disclosure provides compositions comprising ActRIIB polypeptides and uses thereof. For example, in some embodiments, the ActRIIB polypeptide of the present disclosure is The sequence of amino acids 29 to 109 of No. 1 is at least 70%, 75%, 80%, 85%, or 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or or 100% identical amino acid sequence. The peptide has at least 70%, 75%, or 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It may contain an amino acid sequence that is 98%, 99%, or 100% identical to ActRI. The IB polypeptide has an acidic amino acid [naturally occurring (E or or D), or an artificial acidic amino acid. The peptide has at least 70%, 75%, or 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, In some embodiments, the amino acid sequence may be 98%, 99%, or 100% identical. The ActRIIB polypeptide has at least one amino acid sequence corresponding to amino acids 25 to 131 of SEQ ID NO: 1. At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9 Contains amino acid sequences that are 5%, 96%, 97%, 98%, 99%, or 100% identical and the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In some embodiments, the ActRIIB polypeptide comprises amino acid 2 of SEQ ID NO:1. Any one of 0, 21, 22, 23, 24, 25, 26, 27, 28, or 29 starting from residues corresponding to amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, It may comprise an amino acid sequence that is 96%, 97%, 98%, 99%, or 100% identical. In other embodiments, the ActRIIB polypeptide comprises amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 Corresponds to one of 22, 23, 24, 25, 26, 27, 28, or 29 Starting with the residues 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 1 14, 115, 116, 117, 118, 119, 120, 121, 122, 123, 1 24, 125, 126, 127, 128, 129, 130, 131, 132, 133, At least 70%, 70%, or 70% of the sequences ending with a residue corresponding to one of the sequences listed in Table 1 or 134 5%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 7%, 98%, 99%, or 100% identical amino acid sequence, The tRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In one embodiment, the ActRIIB polypeptide has at least 7 amino acid residues in the amino acid sequence of SEQ ID NO:1. 0%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 9 The amino acid sequence may be 6%, 97%, 98%, 99%, or 100% identical. In some embodiments, the ActRIIB polypeptide has at least one amino acid sequence similar to that of SEQ ID NO:1. At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9 Contains amino acid sequences that are 5%, 96%, 97%, 98%, 99%, or 100% identical and the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In yet other embodiments, the ActRIIB polypeptide comprises the amino acid sequence of SEQ ID NO:2. The sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, and 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids In some embodiments, the ActRIIB polypeptide may comprise the sequence of SEQ ID NO:2. The amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, and 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical The ActRIIB polypeptide can comprise an amino acid sequence represented by SEQ ID NO: 1. In yet another embodiment, the ActRIIB polypeptide comprises an acidic amino acid at position 79. , at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 In other instances, an ActRIIB polypeptide may comprise an amino acid sequence that is 0.001% identical to the sequence of the ActRIIB polypeptide. The amino acid sequence in column 3 contains at least 70%, 75%, 80%, 85%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: In another embodiment, the ActRIIB polypeptide comprises an acidic amino acid at position 79 relative to The amino acid sequence of SEQ ID NO: 4 is at least 70%, 75%, 80%, 85%, 90%, %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and In some embodiments, the ActRIIB polypeptide may comprise an amino acid sequence that is 100% identical. The polypeptide has at least 70%, 75%, 80%, 85%, or 90% similarity to the amino acid sequence of SEQ ID NO:4. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 100%, or 100% identical amino acid sequence to the ActRIIB polypeptide. In another embodiment, the ActR comprises an acidic amino acid at position 79 with respect to SEQ ID NO: 4. The IIB polypeptide may have at least 70%, 75%, 80%, or 90% similarity to the amino acid sequence of SEQ ID NO:5. %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 In some embodiments, the amino acid sequence may be 25%, 99%, or 100% identical to the amino acid sequence of the target gene. The ActRIIB polypeptide has at least 70%, 75%, or more amino acid sequence identical to the amino acid sequence of SEQ ID NO:5. %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100% identical amino acid sequence, and The RIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:5. In some embodiments, the ActRIIB polypeptide has at least 70 amino acid residues in the amino acid sequence of SEQ ID NO:6. %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% identical amino acid sequence. In this embodiment, the ActRIIB polypeptide has at least the amino acid sequence of SEQ ID NO:6. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 96%, 97%, 98%, 99%, or 100% identical amino acid sequence. and the ActRIIB polypeptide can comprise an acidic amino acid at position 79 with respect to SEQ ID NO:6. In still other embodiments, the ActRIIB polypeptide comprises the actinic acid sequence of SEQ ID NO: 40. The amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In still other embodiments, the ActRIIB polypeptide may comprise the amino acid sequence , at least 70%, 75%, 80%, 85%, 90% to the amino acid sequence of SEQ ID NO: 42 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In still yet another embodiment, ActRI may comprise an amino acid sequence that is 100% identical. The IB polypeptide has at least 70%, 75%, 80%, or 90% similarity to the amino acid sequence of SEQ ID NO:45. %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100% identical amino acid sequences. In some embodiments, the ActRIIB polypeptide has at least 7 amino acid residues in the amino acid sequence of SEQ ID NO:46. 0%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 9 It may contain amino acid sequences that are 6%, 97%, 98%, 99%, or 100% identical. In yet other embodiments, the ActRIIB polypeptide has the amino acid sequence of SEQ ID NO:46. In the columns, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, Amino acids that are 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical and the ActRIIB polypeptide can comprise the sequence In still yet other embodiments, the ActRIIB polypeptide comprises the amino acid sequence of SEQ ID NO: At least 70%, 75%, 80%, 85%, 90%, and 91% of the amino acid sequence of No. 47 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In some embodiments, the ActRIIB polypeptide may comprise an amino acid sequence that is identical to the ActRIIB polypeptide. is at least 70%, 75%, 80%, 85%, 90%, or more of the amino acid sequence of SEQ ID NO: 47. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and and ActRIIB polypeptides can comprise amino acid sequences that are 100% identical to the sequences of ActRIIB. In still yet another embodiment, the ActR The IIB polypeptide may have at least 70%, 75%, 80%, or 90% similarity to the amino acid sequence of SEQ ID NO:48. 0%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 In some embodiments, the amino acid sequence may be 8%, 99%, or 100% identical. , the ActRIIB polypeptide has at least 70% identity to the amino acid sequence of SEQ ID NO: 48; 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence, The tRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In another embodiment, the ActRIIB polypeptide has the amino acid sequence of SEQ ID NO: 69: At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences In still other embodiments, the ActRIIB polypeptide may comprise SEQ ID NO: 74. The amino acid sequence of %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In some embodiments, the ActRIIB polypeptide may comprise an amino acid sequence. The amino acid sequence of sequence number 74 contains at least 70%, 75%, 80%, 85%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 100% identical to SEQ ID NO: 1, and the ActRIIB polypeptide can comprise an amino acid sequence identical to SEQ ID NO: 1. In still yet another embodiment, ActRIIB comprises an acidic amino acid at position 79 relative to ActRIIB. The polypeptide may have at least 70%, 75%, 80%, or 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences. The ActRIIB polypeptide has at least 70% identity to the amino acid sequence of SEQ ID NO: 77. ,75%,80%,85%,90%,91%,92%,93%,94%,95%,96% , 97%, 98%, 99%, or 100% identical amino acid sequence. In embodiments, the ActRIIB polypeptide has at least the amino acid sequence of SEQ ID NO:77. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 % , 96%, 97%, 98%, 99%, or 100% identical amino acid sequence. and the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In still other embodiments, the ActRIIB polypeptide comprises the amino acid sequence of SEQ ID NO:69. The acid sequence should be at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In still other embodiments, the ActRIIB polypeptide may comprise the sequence The amino acid sequence of sequence number 78 contains at least 70%, 75%, 80%, 85%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 In some embodiments, the ActRIIB polypeptide may comprise an amino acid sequence that is 0.01 to 0.10% identical to the ActRIIB polypeptide. The peptide has at least 70%, 75%, 80%, 85%, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence, and the ActRIIB polypeptide , which contains an acidic amino acid at position 79 with respect to SEQ ID NO: 1. The tRIIB polypeptide has at least 70%, 75%, or , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% identical amino acid sequence. In embodiments, the ActRIIB polypeptide has at least the amino acid sequence of SEQ ID NO:79. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 96%, 97%, 98%, 99%, or 100% identical amino acid sequence. In some embodiments, the ActRIIB polypeptide has the amino acid sequence of SEQ ID NO:79. to at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 9 Amino acid sequences that are 4%, 95%, 96%, 97%, 98%, 99%, or 100% identical and the ActRIIB polypeptide can comprise an acidic amino acid sequence at position 79 relative to SEQ ID NO:1. In certain embodiments, in accordance with the methods and uses described herein, The ActRIIB polypeptide used contains an acidic amino acid at the position corresponding to L79 of SEQ ID NO:1. Does not contain amino acids.

[0025] As described herein, an ActRII polypeptide, an ALK4 polypeptide, and and their variants (GDF traps) are homomultimers, e.g., homodimers, homotrimers, , homotetramers, homopentamers, and higher order homomultimeric complexes. In a preferred embodiment, the ActRII polypeptide and variants thereof are homodimers. In certain embodiments, the ActRII polypeptide dimers described herein comprise a second a first ActR covalently or non-covalently associated with an ActRII polypeptide of The first polypeptide comprises an ActRII domain and an interacting polypeptide. the first member (or second member) of a functional pair (e.g., constant domain of an immunoglobulin) the second polypeptide comprises an amino acid sequence of ActRII polypeptide, and and the amino acid sequence of the second member (or first member) of the interacting pair.

[0026] In certain embodiments, the GDF used in accordance with the methods and uses described herein The / BMP antagonist is an ALK4:ActRIIB heteromultimer. As described, the ALK4:ActRIIB heterodimeric protein complex Differential ligand binding profile compared to the ActRIIB and ALK4 homodimers In particular, ALK4:ActRIIB heterodimers have been found to be selective. exhibited enhanced binding to activin B compared to either homodimer, and Ac Activin A, GDF8, and GDF as observed for tRIIB homodimers 11, as well as BMP9, BMP10, and GDF3. In particular, BMP9 exhibits substantially reduced binding to the ALK4:ActRIIB heterodimer. Although it exhibits low to no observable affinity for the dimer, this ligand The ribosomal ATP binds strongly to the ActRIIB homodimer. The ALK4:ActRIIB heterodimer retains intermediate levels of binding to BMP6. See Figure 19. Therefore, these results support the conclusion that ALK4:ActRIIB The heterodimers are more potent than the ActRIIB homodimers in activin A, activin B, and G. Demonstration that it is a more selective antagonist (inhibitor) of GDF8 and GDF11 Therefore, the ALK4:ActRIIB heterodimer may be able to exert such selective antagonism. In certain applications, it may be advantageous to use ActRIIB homodimers. activin (e.g., activin A, activin B, activin AB, activin AC), GDF8, and GDF11, but retains antagonistic activity Minimizing antagonism of one or more of BMP9, BMP10, and GDF3 Furthermore, there are therapeutic applications in which it is desirable to The mers have been shown to treat PAH in patients. However, activin (e.g., activin A, activin B, activin AB) , activin AC), GDF8, and / or GDF11 ALK4:ActRIIB heteromultimers and their variants bind to multiple ALK4-ActRIIB. It is anticipated that this will be a useful agent for promoting beneficial effects in patients.

[0027] Thus, the present disclosure provides a method for the preparation of a medicament for the treatment of a malaria parasite comprising administering to a patient a therapeutically effective amount of at least one ALK4 polypeptide and at least one ALK4 polypeptide. Heteromultimeric complexes (heteromultimers) containing ActRIIB polypeptides (ALK4 The present invention provides an ALK4 polypeptide, an ActRIIB heteromultimer, and uses thereof. The polypeptide is a polypeptide that binds to the ligand-binding domain of the ALK4 receptor, e.g., the ALK4 extracellular domain. Similarly, ActRIIB polypeptides generally include portions of ActRIIB receptors. The ligand-binding domain of the ActRIIB antibody includes a portion of the extracellular domain of ActRIIB. or such ALK4 and ActRIIB polypeptides, and the resulting polypeptides thereof. The heteromultimer is soluble.

[0028] In certain embodiments, the ALK4:ActRIIB heteromultimer comprises the ALK4:ActRIIB heteromultimer of SEQ ID NO: 100. At least 70%, 75%, 80%, 85%, 86%, 87%, or 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, In other embodiments, the amino acid sequence of A is 98%, 99%, or 100% identical. The LK4:ActRIIB heteromultimer may be at least 70%, 75%, or ,80%,85%,86%,87%,88%,89%,90%,91%,92%,93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ALK In another embodiment, the ALK4:ActRIIB heteromultimer comprises a 4 amino acid sequence: SEQ ID NO: 105, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100% identical to an ALK4 amino acid sequence. ALK4:ActRIIB heteromultimers are at least 70%, 7 5%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 A that is 3%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In another embodiment, the ALK4:ActRIIB heteromultimer comprises the ALK4 amino acid sequence. is at least 70%, 75%, 80%, 85%, 86%, 87%, or 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical ALK4 amino acid sequence. In SEQ ID NO: 116, the ALK4:ActRIIB heteromultimer has at least 70% ,75%,80%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In yet another embodiment, the ALK4:ActRIIB heterotransferase comprises an ALK4 amino acid sequence comprising the ALK4:ActRIIB heterotransferase. The multimer may be at least 70%, 75%, 80%, 85%, 86%, or 90% SEQ ID NO: 117. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical ALK4 amino acid sequence. In embodiments, the ALK4:ActRIIB heteromultimer is at least as sequenced as SEQ ID NO:111. Also 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In yet another embodiment, the ALK4:ActRI comprises an ALK4 amino acid sequence that is identical to the ALK4:ActRI. The IB heteromultimer may be at least 70%, 75%, 80%, 85%, or 90% identical to SEQ ID NO: 113. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Contains ALK4 amino acid sequences that are 96%, 97%, 98%, 99%, or 100% identical nothing.

[0029] In certain embodiments, the ALK4:ActRIIB heteromultimer comprises the amino acid sequence of SEQ ID NO:1. Acid 29~109, 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 ActRIIB amino acid sequence. In this embodiment, the ALK4:ActRIIB heteromultimer has at least 70% identity to SEQ ID NO:2, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In another embodiment, the ALK4:ActRIIB amino acid sequence is The multimer may be at least 70%, 75%, 80%, 85%, 86%, 87%, or 90% of SEQ ID NO: 3. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100% identical ActRIIB amino acid sequence. In one embodiment, the ALK4:ActRIIB heteromultimer comprises at least one ALK4:ActRIIB heteromultimer in SEQ ID NO:5. 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In another embodiment, the ALK4:ActRIIB amino acid sequence is identical to the ALK4:ActRIIB amino acid sequence. The IB heteromultimer may be at least 70%, 75%, 80%, 85%, 86%, or 90% of the total amino acid sequence of SEQ ID NO: 6. %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% identical ActRIIB amino acid sequences In another embodiment, the ALK4:ActRIIB heteromultimer comprises the sequence set forth in SEQ ID NO: 118. , at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and In yet another embodiment, A comprises an ActRIIB amino acid sequence that is 100% identical. The LK4:ActRIIB heteromultimer has at least 70%, 75% ,80%,85%,86%,87%,88%,89%,90%,91%,92%,93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical Act In another embodiment, the ALK4:ActRIIB heteromer comprises the RIIB amino acid sequence. The body is at least 70%, 75%, 80%, 85%, 86%, 87% ,88%,89%,90%,91%,92%,93%,94%,95%,96%,97% , 98%, 99%, or 100% identical ActRIIB amino acid sequence. In embodiments, the ALK4:ActRIIB heteromultimer is selected from the group consisting of SEQ ID NO: 115 and at least one ALK4:ActRIIB heteromultimer. Also 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In another embodiment, the ALK4:ActRIIB amino acid sequence may be identical to that of the ALK4:ActRIIB. The tRIIB heteromultimer may be at least 70%, 75%, 80%, 85%, 90%, 95 ... 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 ActRIIB ami that is 5%, 96%, 97%, 98%, 99%, or 100% identical In another embodiment, the ALK4:ActRIIB heteromultimer comprises the acid sequence SEQ ID NO: No. 110, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9%, or 100% identical to the ActRIIB amino acid sequence. In another embodiment, the ALK4:ActRIIB heteromultimer corresponds to L79 of SEQ ID NO:1. and ActRIIB polypeptides containing an acidic amino acid (e.g., E or D) at positions corresponding to the Not at all.

[0030] As described herein, the ALK4:ActRIIB heteromultimeric structure can be, for example, , heterodimers, heterotrimers, heterotetramers, heteropentamers, and higher-order heteromultimers See, for example, Figures 21-23. In certain preferred embodiments, In certain embodiments, the ALK4:ActRIIB heteromultimer is a heterodimer. The ALK4 and / or ActRIIB polypeptide may be a fusion protein. .

[0031] In certain embodiments, ActRII polynucleotides, including variants thereof (e.g., GDF traps), The peptide, ALK4 polypeptide, can be a fusion protein. For example, in some embodiments In this embodiment, the ActRII (or ALK4) polypeptide is 4) a polypeptide domain and one or more heterologous (non-ActRII) polypeptides In some embodiments, the ActRII (or ActRII-binding domain) may be a fusion protein comprising the ActRII (or ActRII-binding domain). The ActRII (or ALK4) polypeptide is composed of the ActRII (or ALK4) polypeptide as one domain. The amino acid sequence derived from the peptide (e.g., the linker of the ActRII (or ALK4) receptor) and improved pharmacokinetics, easy purification, particularly It has one or more heterologous domains that provide desired properties, such as targeting to a specific tissue. For example, the domain of the fusion protein may be a fusion protein that is stable in vivo. Qualitative, in vivo half-life, uptake / administration, tissue localization or distribution, protein complexation The present invention provides a method for enhancing one or more of the following: formation of a target protein, multimerization of the fusion protein, and / or purification. Optionally, the ActRII (or ALK4) polypeptide of the fusion protein The domain may be directly connected (fused) to one or more heterologous polypeptide domains, or Alternatively, an intervening sequence such as a linker may be inserted between the amino acid sequence of the ActRII (or ALK4) polypeptide. and the amino acid sequence of one or more heterologous domains. In certain embodiments, the ActRII (or ALK4) fusion protein is a heterologous A relatively unstructured linker located between the ALK4 domain and the ActRII (or ALK4) domain This unstructured linker binds to the extracellular domain of ActRII (or ALK4). This may correspond to an unstructured region of approximately 15 amino acids at the C-terminus of the protein, or may be a region that does not exhibit secondary structure. an artificial sequence of 3 to 15, 20, 30, 50 or more amino acids that does not contain any amino acid pair The linker may be rich in glycine and / or proline residues, For example, it may contain repeat sequences of threonine / serine and glycine. The sequences TGGG (SEQ ID NO: 23), SGGG (SEQ ID NO: 24), TGGGG (SEQ ID NO: 25), 21), SGGGG (SEQ ID NO: 22), GGGGS (SEQ ID NO: 25), GGGG (SEQ ID NO: No. 20), and GGG (SEQ ID NO: 19). In embodiments, the ActRII (or ALK4) fusion protein is a fusion protein that is capable of binding to, for example, an immunoglobulin. For example, the amino acid sequence are IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgA1 or are derived from the Fc domain of IgA2), IgE, or IgM immunoglobulins. For example, The Fc portion of the immunoglobulin domain is selected from the group consisting of SEQ ID NOs: 14 to 18. , at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences Such an immune system may comprise, consist essentially of, or consist of The globulin domain confers altered Fc activity, e.g., one or more Fc domains. One or more amino acid modifications (e.g., deletions, additions, and deletions) that decrease effector function. In some embodiments, the ActRII (or ALK4) fusion protein may comprise a nucleotide sequence (nucleotide sequence and / or substitution). The conjugated protein comprises the amino acid sequence shown in the formula ABC. For example, the B portion is For example, the N- and C-terminal truncated ActRII (or ALK4) polypeptides described herein The A and C moieties are independently zero, one, or more than one amino acid. and both A and C moieties are heterologous to B. Moieties A and / or C may be In certain embodiments, the Act B moiety can be attached to the B moiety via an anchor sequence. The RII (or ALK4) fusion protein contains a leader sequence. The leader sequence may be the native ActRII (or ALK4) leader sequence or a heterologous leader sequence. In certain embodiments, the leader sequence is a tissue plasminogen activator (TPA) A leader sequence (e.g., SEQ ID NO: 34).

[0032] The ActRII polypeptide or ALK4 polypeptide containing the variant has an epitope Purified subsequences such as nucleotide tags, FLAG tags, polyhistidine sequences, and GST fusions Optionally, the ActRII polypeptide or the ALK4 polypeptide may comprise a glycoprotein. Cosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, and / or amino acids conjugated to a lipid moiety. and ActRII polypeptides, each of which contains one or more modified amino acid residues. The ALK4 polypeptide can include at least one N-linked sugar, and can include two, three, or Such polypeptides may also contain O-linked sugars. Generally, ActRII and ALK4 polypeptides are preferred in patients. Proper native glycosylation of polypeptides to reduce the possibility of an undesired immune response Preferably, ActRII and ALK are expressed in mammalian cell lines that mediate expression of the gene. 4 Polypeptides can be expressed in engineered insect or yeast cells, as well as in COS cells, CHO cells, Suitable cell lines for use in patients include mammalian cells such as HEK cells and NSO cells. In some embodiments, the glycosylated protein may be produced in a variety of cell lines that glycosylate proteins. In this study, ActRII or ALK4 polypeptides were glycosylated and The glycosylation pattern is comparable to that obtained from a human hamster ovary cell line. In embodiments, the ActRII or ALK4 polypeptides of the disclosure are administered to mammals (e.g., , mouse, or human) Optionally, the ActRII or ALK4 polypeptide exhibits a serum half-life in hours. At least 6, 8, 10, 12, 14, 2 in mammals (e.g., mice or humans) It may exhibit a serum half-life of 0, 25, or 30 days.

[0033] In certain aspects, the present disclosure provides a method for treating a cancer, comprising administering to a patient a therapeutically effective amount of one or more GDF / BMP antagonists of the present disclosure. The present invention provides a pharmaceutical preparation comprising a pulmonary steroid and a pharmaceutically acceptable carrier. To treat hypertension, particularly one or more complications of pulmonary hypertension (e.g., pulmonary artery smooth muscle and / or endothelial cell proliferation in the lungs, angiogenesis in the pulmonary arteries, dyspnea, chest pain to treat or prevent pulmonary fibrosis (pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis) The compounds used, such as prostacyclin, epoprostenol, and sildenafil vasodilators such as benzodiazepine; endothelin receptor antagonists such as bosentan; calcium channel blockers such as benzodiazepine, diltiazem, and nifedipine; warfarin anticoagulants such as benzodiazepines; diuretics; BMP9 polypeptides; BMP10 polypeptides; solon methyl; and one or more additional active compounds, such as compounds containing oleanolic acid. Generally, pharmaceutical preparations are preferably pyrogen-free (for therapeutic use). pyrogen-free to the extent required by regulations governing the quality of products for human consumption. (This means that

[0034] In certain instances, the GDF / BMP antagonist or combination of antagonists of the present disclosure When the combination is administered to a disorder or condition described herein, the GDF / BMP antagonist During administration of the drug, the effect on red blood cells should be monitored, or the desired effect on red blood cells should be monitored. Determine or adjust the dosage of GDF / BMP antagonists to reduce adverse effects For example, it may be desirable to measure red blood cell levels, hemoglobin levels, or hematopoietic Increased tocrit levels can cause an undesirable increase in blood pressure.

[0035] In certain embodiments, the GDF / BMP antigens used in accordance with the methods and uses of the present disclosure In some embodiments, the antibody is an antibody or a combination of antibodies. also binds to ActRII (ActRIIA and / or ActRIIB). In this embodiment, the antibody that binds ActRII optionally binds to an antibody described herein. ActRII signaling as measured in cell-based assays such as the In certain embodiments, the antibody that binds to ActRII inhibits one or more The binding of GDF / BMP ligands, type I receptors, or co-receptors to ActRII In certain embodiments, the antibody that binds to ActRII inhibits one or more The GDF / BMP ligands are activins (e.g., activin A, activin B, activin Activin C, Activin AB, Activin AC, Activin BC, Activin E, Activin activin AE, and activin BE), GDF8, GDF11, BMP6, BMP15, BM inhibits binding to ActRII selected from the group consisting of P10, and GDF3 In some embodiments, the antibody binds to at least ALK4. The antibodies that bind to ALK4 can optionally be assayed in cells, such as in assays described herein. inhibits ALK4 signaling as measured in a specific antibody-based assay. In one embodiment, the antibody that binds ALK4 binds one or more GDF / BMP ligands. In certain embodiments, the ALK4 receptor inhibits the type II receptor or co-receptor from binding to ALK4. In one embodiment, the antibody that binds to ALK4 is a GDF / BMP ligand that binds to ALK4. activin (e.g., activin A, activin B, activin C, activin AB, activin activin AC, activin BC, activin E, activin AE, and activin BE) , consisting of GDF8, GDF11, BMP6, BMP15, BMP10, and GDF3 In some embodiments, the antibody inhibits binding to ALK4 selected from the group consisting of at least In certain embodiments, the antibody that binds to ALK5 binds to any of Alternatively, when measured in a cell-based assay such as the assays described herein, In certain embodiments, the ALK5 signaling inhibitor binds to ALK5. The antibody may be selected from the group consisting of one or more GDF / BMP ligands, type II receptors, or co-receptors. In certain embodiments, an antibody that binds to ALK5 inhibits binding to ALK5. In some embodiments, one or more GDF / BMP ligands may be activin (e.g., activin A, activin B). Activin B, Activin C, Activin AB, Activin AC, Activin BC, Activin activin E, activin AE, and activin BE), GDF8, GDF11, BMP6, Binds to ALK5 selected from the group consisting of BMP15, BMP10, and GDF3 In some embodiments, the antibody binds to at least ALK7. In this embodiment, the antibody that binds to ALK7 is optionally administered in a manner similar to that described herein. inhibits ALK7 signaling as measured in cell-based assays such as In certain embodiments, antibodies that bind to ALK7 bind to one or more GDF / B It inhibits the binding of MP ligands, type II receptors, or co-receptors to ALK7. In certain embodiments, the antibody that binds to ALK7 is The enzyme is activin (e.g., activin A, activin B, activin C, activin B). Activin AB, Activin AC, Activin BC, Activin E, Activin AE, and Activin Chibin BE), GDF8, GDF11, BMP6, BMP15, BMP10, and GD In some embodiments, the compound inhibits binding to ALK7 selected from the group consisting of: F3. The antibody binds to at least GDF11. In certain embodiments, the antibody binds to GDF11. The antibodies can optionally be used in cell-based assays such as those described herein. In certain embodiments, the inhibitor inhibits ActRII signaling as measured by The antibody that binds to GDF11 may be GDF11-ActRII binding and / or GDF11 ALK binding (e.g., GDF11-ALK4, GDF11-ALK5, and / or In some embodiments, the antibody inhibits at least GDF11-ALK7 binding. In certain embodiments, the antibody that binds to GDF8 optionally comprises the Act when measured in a cell-based assay, such as the assays described herein. In certain embodiments, the antibody that binds to GDF8 inhibits RII signaling. GDF8-ActRII binding and / or GDF8-ALK binding (e.g., GDF8- ALK4, GDF8-ALK5, and / or GDF8-ALK7 binding). In some embodiments, the antibody binds to at least BMP6. The antibodies that bind to BMP6 can optionally be assayed in cell-based assays such as those described herein. inhibits ActRII signaling as measured in a specific assay. In this embodiment, the antibody that binds to BMP6 is BMP6-ActRII binding and / or BMP6-ALK binding (e.g., BMP6-ALK4, BMP6-ALK5, and / or or BMP6-ALK7 binding). In certain embodiments, the antibody that binds to BMP15 optionally and optionally, when measured in a cell-based assay such as the assays described herein. In certain embodiments, the inhibitor binds to BMP15 and inhibits ActRII signaling. The antibodies may be BMP15-ActRII binding and / or BMP15-ALK binding (e.g., For example, BMP15-ALK4, BMP15-ALK5, and / or BMP15-ALK 7) binding. In some embodiments, the antibody binds to at least GDF3. In certain embodiments, the antibody that binds to GDF3 is optionally an antibody described herein. ActRII signaling as measured in cell-based assays such as the In certain embodiments, the antibody that binds to GDF3 inhibits GDF3-ActRI. I binding and / or GDF3-ALK binding (e.g., GDF3-ALK4, GDF3- In some embodiments, the inhibitor inhibits ALK5, ALK6, and / or GDF3-ALK7 binding. The antibody binds to at least BMP10. In certain embodiments, the antibody binds to BMP10. The antibodies can optionally be used in cell-based assays such as those described herein. In certain embodiments, the inhibitor inhibits ActRII signaling as measured by The antibody that binds to BMP10 is a BMP10-ActRII binding antibody and / or a BMP10 ALK binding (e.g., BMP10-ALK4, BMP10-ALK5, and / or In some embodiments, the antibody inhibits activin (e.g., BMP10-ALK7 binding). For example, activin A, activin B, activin C, activin AB, activin AC, Activin BC, activin E, activin AE, and activin BE). In certain embodiments, activins (e.g., activin A, activin B, activin B) are used. Activin C, Activin AB, Activin AC, Activin BC, Activin E, Activin A Antibodies that bind to activin (e.g., activin B, E, and activin B) can optionally be used in combination with the antibodies described herein. inhibits ActRII signaling as measured in cell-based assays such as In certain embodiments, activins (e.g., activin A, activin B, Activin C, Activin AB, Activin AC, Activin BC, Activin E, Activin Antibodies that bind to activin-ActRII (activin AE, and activin BE) are and / or activin-ALK binding (e.g., activin-ALK4, activin- In some embodiments, the ALK5 and activin-ALK7 binding are inhibited. In certain embodiments, the antibody binds to activin B. The antibodies are optionally used in cell-based assays such as those described herein. In certain embodiments, the inhibitor inhibits ActRII signaling as measured by the ATP-dependent agonist (ATP). Antibodies that bind to activin B are known to bind to activin B-ActRII and / or activin B. B-ALK binding (e.g., activin B-ALK4, activin B-ALK5, and / or or activin B-ALK7 binding). RIIB, ActRIIA, ALK4, ALK5, ALK7, GDF11, GDF8, one or more of: cutin, BMP6, GDF3, BMP10, and BMP15 In certain embodiments, the antibody or combination of antibodies binds to Multispecific antibodies or combinations of multispecific antibodies can be used to detect Ac in cell-based assays. tRIIB, GDF11, GDF8, activin, BMP6, GDF3, BMP10, and and BMP15. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. , single-chain antibodies, F(ab')2 fragments, single-chain diabodies, tandem single-chain Fv fragments, tandem single-chain diabodies, or single-chain diabodies and immunoglobulins It is a fusion protein containing at least a portion of the purine heavy chain constant region.

[0036] In certain embodiments, the GDF / BMP antagonist is a small molecule inhibitor or In some embodiments, the small molecule inhibitor is a combination of inhibitors that inhibit at least ActRII ( For example, an inhibitor of ActRIIA and / or ActRIIB. In some embodiments, the small molecule inhibitor is an inhibitor of at least ALK4. The molecular inhibitor is an inhibitor of at least ALK5. In some embodiments, the small molecule inhibitor In some embodiments, the small molecule inhibitor is an inhibitor of at least ALK7. In some embodiments, the small molecule inhibitor is an inhibitor of at least GDF11. In some embodiments, the small molecule inhibitor is an inhibitor of at least BMP6. In some embodiments, the small molecule inhibitor is an inhibitor of at least BMP15. In some embodiments, the small molecule inhibitor is an inhibitor of at least BMP10. In embodiments, the small molecule inhibitor is an inhibitor of at least GDF3. In this study, small molecule inhibitors were identified that inhibited at least one of the activins (e.g., activin A, activin B, activin B). Activin C, Activin AB, Activin AC, Activin BC, Activin E, Activin In some embodiments, the small molecule inhibitor is an inhibitor of activin (activin AE and activin BE). In some embodiments, the small molecule inhibitor is an inhibitor of at least activin B. At the very least, they are inhibitors of Smad proteins (eg, Smad2 and 3).

[0037] In certain embodiments, the GDF / BMP antagonist is a nucleic acid inhibitor or a nucleic acid inhibitor. In some embodiments, the nucleic acid inhibitor is a combination of at least ActRII (e.g., , ActRIIA and / or ActRIIB). In some embodiments, the nucleic acid inhibitor is an inhibitor of at least ALK4. is an inhibitor of at least ALK5. In some embodiments, the nucleic acid inhibitor is at least In some embodiments, the nucleic acid inhibitor is an inhibitor of at least GDF11. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least GDF8. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least BMP6. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least BMP15. The nucleic acid inhibitor is an inhibitor of at least BMP10. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least GDF3. Also activins (e.g., activin A, activin B, activin C, activin AB, Activin AC, Activin BC, Activin E, Activin AE, and Activin B E) In some embodiments, the nucleic acid inhibitor is an inhibitor of at least activin B. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least one or more Smads. (e.g., Smad2 and 3) inhibitors.

[0038] In certain embodiments, the GDF / BMP antagonist is a follistatin polypeptide. In some embodiments, the follistatin polypeptide is the amino acid sequence of SEQ ID NO: 26. The sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, and 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids In some embodiments, the follistatin polypeptide comprises the amino acid sequence of SEQ ID NO: 27. The amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In some embodiments, the follistatin polypeptide comprises the amino acid sequence SEQ ID NO:2. 8 amino acid sequences with at least 70%, 75%, 80%, 85%, 90%, 91%, 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In some embodiments, the follistatin polypeptide comprises an amino acid sequence having the sequence The amino acid sequence of No. 29 has at least 70%, 75%, 80%, 85%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 In some embodiments, the follistatin polypeptide comprises an amino acid sequence that is % identical to , at least 70%, 75%, 80%, 85%, 90% to the amino acid sequence of SEQ ID NO: 30 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or Contains amino acid sequences that are 100% identical.

[0039] In certain embodiments, the GDF / BMP antagonist is a FLRG polypeptide. In some embodiments, the FLRG polypeptide has at least one amino acid sequence similar to the amino acid sequence of SEQ ID NO:31. At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9 Contains amino acid sequences that are 5%, 96%, 97%, 98%, 99%, or 100% identical . [Brief explanation of the drawings]

[0040] [Figure 1] Figure 1 shows an alignment of the extracellular domains of human ActRIIB (sequence number 2) and human ActRIIA (sequence number 10), with residues predicted herein to directly contact the ligand based on synthetic analysis of multiple ActRIIB and ActRIIA crystal structures indicated by boxes.

[0041] [Figure 2] Figure 2 shows a multiple sequence alignment of ActRIIB proteins (sequence numbers 53-58) from various vertebrates and human ActRIIA (sequence number 59), as well as the consensus ActRII sequence derived from the alignment (sequence number 60).

[0042] [Figure 3]Figure 3 shows a multiple sequence alignment of ActRIIA proteins from various vertebrates and human ActRIIA (sequence numbers 61-68).

[0043] [Figure 4] Figure 4 shows a multiple sequence alignment of Fc domains from human IgG isotypes using Clustal 2.1. The hinge region is indicated by a dotted underline. Double underlines indicate examples of positions that can be engineered in IgG1 Fc to promote asymmetric chain pairing, and corresponding positions for other isotypes, IgG2, IgG3, and IgG4.

[0044] [Figure 5] Figure 5 shows the purification of ActRIIA-hFc expressed in CHO cells. The protein purifies as a single, distinct peak when visualized by granulation column (upper panel) and Coomassie-stained SDS-PAGE (lower panel) (left lane: molecular weight standards; right lane: ActRIIA-hFc).

[0045] [Figure 6] FIG. 6 shows the binding of ActRIIA-hFc to activin (upper panel) and GDF-11 (lower panel) as measured by Biacore™ assay.

[0046] [Figure 7] Figure 7 shows the complete, unprocessed amino acid sequence of ActRIIB(25-131)-hFc (SEQ ID NO: 69). The TPA leader (residues 1-22) and the doubly truncated ActRIIB extracellular domain (residues 24-131, using numbering based on the native sequence in SEQ ID NO: 1) are each underlined. The glutamic acid at position 25 compared to SEQ ID NO: 1, which sequencing revealed to be the N-terminal amino acid of the mature fusion protein, is highlighted.

[0047] [Figure 8A]Figures 8A and 8B show the nucleotide sequence encoding ActRIIB(25-131)-hFc (coding strand shown above, SEQ ID NO:70; complement shown below, 3'-5', SEQ ID NO:71). The sequences encoding the TPA leader (nucleotides 1-66) and the ActRIIB extracellular domain (nucleotides 73-396) are underlined. The corresponding amino acid sequence of ActRIIB(25-131) is also shown. [Figure 8B] Figures 8A and 8B show the nucleotide sequence encoding ActRIIB(25-131)-hFc (coding strand shown above, SEQ ID NO:70; complement shown below, 3'-5', SEQ ID NO:71). The sequences encoding the TPA leader (nucleotides 1-66) and the ActRIIB extracellular domain (nucleotides 73-396) are underlined. The corresponding amino acid sequence of ActRIIB(25-131) is also shown.

[0048] [Figure 9A] Figures 9A and 9B show an alternative nucleotide sequence encoding ActRIIB(25-131)-hFc (coding strand shown above, SEQ ID NO:72; complement shown 3'-5' below, SEQ ID NO:73). This sequence confers greater levels of protein expression in initial transformants and makes cell line development a more rapid process. The sequences encoding the TPA leader (nucleotides 1-66) and ActRIIB extracellular domain (nucleotides 73-396) are underlined, and substitutions in the wild-type nucleotide sequence of the ECD (see Figure 8) are highlighted. The corresponding amino acid sequence of ActRIIB(25-131) is also shown. [Figure 9B]Figures 9A and 9B show an alternative nucleotide sequence encoding ActRIIB(25-131)-hFc (coding strand shown above, SEQ ID NO:72; complement shown 3'-5' below, SEQ ID NO:73). This sequence confers greater levels of protein expression in initial transformants and makes cell line development a more rapid process. The sequences encoding the TPA leader (nucleotides 1-66) and ActRIIB extracellular domain (nucleotides 73-396) are underlined, and substitutions in the wild-type nucleotide sequence of the ECD (see Figure 8) are highlighted. The corresponding amino acid sequence of ActRIIB(25-131) is also shown.

[0049] [Figure 10] 10 shows the complete amino acid sequence of GDF trap ActRIIB(L79D 20-134)-hFc (SEQ ID NO: 74), including the TPA leader sequence (double underlined), the ActRIIB extracellular domain (residues 20-134 of SEQ ID NO: 1; single underlined), and the hFc domain. The aspartic acid substituted at position 79 of the native sequence is double underlined and highlighted, as is the glycine, which sequencing revealed to be the N-terminal residue of the mature fusion protein.

[0050] [Figure 11A] Figures 11A and 11B show the nucleotide sequence encoding ActRIIB(L79D 20-134)-hFc. SEQ ID NO:75 corresponds to the sense strand, and SEQ ID NO:76 corresponds to the antisense strand. The TPA leader (nucleotides 1-66) is double underlined, and the ActRIIB extracellular domain (nucleotides 76-420) is single underlined. [Figure 11B] Figures 11A and 11B show the nucleotide sequence encoding ActRIIB(L79D 20-134)-hFc. SEQ ID NO:75 corresponds to the sense strand, and SEQ ID NO:76 corresponds to the antisense strand. The TPA leader (nucleotides 1-66) is double underlined, and the ActRIIB extracellular domain (nucleotides 76-420) is single underlined.

[0051] [Figure 12] 12 shows the complete amino acid sequence of truncated GDF trap ActRIIB(L79D 25-131)-hFc (SEQ ID NO:77), which includes the TPA leader sequence (double underlined), the truncated ActRIIB extracellular domain (residues 25-131 of SEQ ID NO:1; single underlined), and the hFc domain. The aspartic acid substituted at position 79 of the native sequence is double underlined and highlighted, as is the glutamic acid, which sequencing revealed to be the N-terminal residue of the mature fusion protein.

[0052] [Figure 13] Figure 13 shows the amino acid sequence of truncated GDF trap ActRIIB(L79D 25-131)-hFc without the leader (SEQ ID NO: 78). The truncated ActRIIB extracellular domain (residues 25-131 of SEQ ID NO: 1) is underlined. The aspartic acid substituted at position 79 of the native sequence is double underlined and highlighted, as is the glutamic acid, which sequencing revealed to be the N-terminal residue of the mature fusion protein.

[0053] [Figure 14] 14 shows the amino acid sequence of truncated GDF trap ActRIIB (L79D 25-131) without the leader, hFc domain, and linker (SEQ ID NO: 79). The aspartic acid substituted at position 79 of the native sequence is underlined and highlighted, as is the glutamic acid, which sequencing revealed to be the N-terminal residue of the mature fusion protein.

[0054] [Figure 15A]Figures 15A and 15B show the nucleotide sequence encoding ActRIIB(L79D 25-131)-hFc. SEQ ID NO:80 corresponds to the sense strand, and SEQ ID NO:81 corresponds to the antisense strand. The TPA leader (nucleotides 1-66) is double underlined, and the truncated ActRIIB extracellular domain (nucleotides 76-396) is single underlined. The amino acid sequence of the ActRIIB extracellular domain (residues 25-131 of SEQ ID NO:1) is also shown. [Figure 15B] Figures 15A and 15B show the nucleotide sequence encoding ActRIIB(L79D 25-131)-hFc. SEQ ID NO:80 corresponds to the sense strand, and SEQ ID NO:81 corresponds to the antisense strand. The TPA leader (nucleotides 1-66) is double underlined, and the truncated ActRIIB extracellular domain (nucleotides 76-396) is single underlined. The amino acid sequence of the ActRIIB extracellular domain (residues 25-131 of SEQ ID NO:1) is also shown.

[0055] [Figure 16A] Figures 16A and 16B show alternative nucleotide sequences encoding ActRIIB(L79D 25-131)-hFc. SEQ ID NO:82 corresponds to the sense strand, and SEQ ID NO:83 corresponds to the antisense strand. The TPA leader (nucleotides 1-66) is double underlined, the truncated ActRIIB extracellular domain (nucleotides 76-396) is single underlined, and substitutions in the wild-type nucleotide sequence of the extracellular domain are double underlined and highlighted (SEQ ID NO:81, compare with Figure 15). The amino acid sequence of the ActRIIB extracellular domain (residues 25-131 of SEQ ID NO:1) is also shown. [Figure 16B]Figures 16A and 16B show alternative nucleotide sequences encoding ActRIIB(L79D 25-131)-hFc. SEQ ID NO:82 corresponds to the sense strand, and SEQ ID NO:83 corresponds to the antisense strand. The TPA leader (nucleotides 1-66) is double underlined, the truncated ActRIIB extracellular domain (nucleotides 76-396) is single underlined, and substitutions in the wild-type nucleotide sequence of the extracellular domain are double underlined and highlighted (SEQ ID NO:81, compare with Figure 15). The amino acid sequence of the ActRIIB extracellular domain (residues 25-131 of SEQ ID NO:1) is also shown.

[0056] [Figure 17] Figure 17 shows nucleotides 76-396 (SEQ ID NO:84) of the alternative nucleotide sequence shown in Figure 16 (SEQ ID NO:82). The same nucleotide substitutions shown in Figure 16 are again underlined and highlighted. SEQ ID NO:84 encodes only the truncated ActRIIB extracellular domain (corresponding to residues 25-131 of SEQ ID NO:1) with the L79D substitution, e.g., ActRIIB(L79D 25-131).

[0057] [Figure 18] FIG. 18 shows a multiple sequence alignment of ALK4 proteins from various vertebrates and human ALK4 (SEQ ID NOs: 126-132).

[0058] [Figure 19]Figure 19 shows ligand binding data for the ALK4-Fc:ActRIIB-Fc heterodimer protein complex compared to the ActRIIB-Fc homodimer and ALK4-Fc homodimer. For each protein complex, the ligands are ranked by koff, the rate constant that correlates well with ligand signaling inhibition, and listed in descending order of binding affinity (the tightest-binding ligands are listed at the top). On the left, yellow, red, green, and blue lines indicate the magnitude of the off-rate constants. Solid black lines indicate ligands whose binding to the heterodimer is enhanced or unchanged compared to the homodimer, while dashed red lines indicate substantially reduced binding compared to the homodimer. As shown, the ALK4-Fc:ActRIIB-Fc heterodimer displays enhanced binding to activin B compared to either homodimer, retains the strong binding to activin A, GDF8, and GDF11 observed with the ActRIIB-Fc homodimer, and exhibits substantially reduced binding to BMP9, BMP10, and GDF3. Like the ActRIIB-Fc homodimer, the heterodimer retains a moderate level of binding to BMP6.

[0059] [Figure 20]Figure 20 shows comparative data of IC50 of ALK4-Fc:ActRIIB-Fc heterodimer / ActRIIB-Fc:ActRIIB-Fc homodimer as determined by the A-204 reporter gene assay described herein. Similar to ActRIIB-Fc:ActRIIB-Fc homodimer, ALK4-Fc:ActRIIB-Fc heterodimer inhibits activin A, activin B, GDF8, and GDF11 signaling pathways. However, the inhibition of BMP9 and BMP10 signaling pathways by ALK4-Fc:ActRIIB-Fc heterodimer is significantly reduced compared to ActRIIB-Fc:ActRIIB-Fc homodimer. These data demonstrate that the ALK4:ActRIIB heterodimer is a more selective antagonist of activin A, activin B, GDF8, and GDF11 compared to the corresponding ActRIIB:ActRIIB homodimer.

[0060] [Figure 21A] Figures 21A and 21B show two schematic examples of heteromeric protein complexes containing type I and type II receptor polypeptides. Figure 21A depicts a heterodimeric protein complex containing one type I receptor fusion polypeptide and one type II receptor fusion polypeptide, which can be assembled covalently or noncovalently via multimerization domains contained within each polypeptide chain. The two assembled multimerization domains form an interaction pair that can be guided or unguided. Figure 21B depicts a heterotetrameric protein complex similar to Figure 21A, containing two heterodimeric complexes. Higher order complexes can be envisioned. [Figure 21B]Figures 21A and 21B show two schematic examples of heteromeric protein complexes containing type I and type II receptor polypeptides. Figure 21A depicts a heterodimeric protein complex containing one type I receptor fusion polypeptide and one type II receptor fusion polypeptide, which can be assembled covalently or noncovalently via multimerization domains contained within each polypeptide chain. The two assembled multimerization domains form an interaction pair that can be guided or unguided. Figure 21B depicts a heterotetrameric protein complex similar to Figure 21A, containing two heterodimeric complexes. Higher order complexes can be envisioned.

[0061] [Figure 22]Figure 22 shows a schematic example of a heteromeric protein complex comprising a type I receptor polypeptide (indicated as "I") (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ALK4 protein from a human or other species, such as those described herein) and a type II receptor polypeptide (indicated as "II") (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species, such as those described herein). In the illustrated embodiment, the type I receptor polypeptide is part of a fusion polypeptide comprising the first member ("C1") of the interaction pair, and the type II receptor polypeptide is part of a fusion polypeptide comprising the second member ("C2") of the interaction pair. In each fusion polypeptide, a linker can be disposed between the type I or type II receptor polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair can be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other rather than self-associate; alternatively, the interaction pair can be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference and can have the same or different amino acid sequences. While traditional Fc fusion proteins and antibodies are examples of unguided interaction pairs, various engineered Fc domains have been designed as guided (asymmetric) interaction pairs (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106).

[0062] [Figure 23A]Figures 23A-23D show schematic examples of heteromeric protein complexes comprising an ALK4 polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ALK4 protein from a human or other species, such as those described herein), and an ActRIIB polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species, such as those described herein). In the illustrated embodiment, the ALK4 polypeptide is part of a fusion polypeptide comprising a first member ("C1") of the interaction pair, and the ActRIIB polypeptide is part of a fusion polypeptide comprising a second member ("C2") of the interaction pair. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncated forms and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker can be disposed between the ALK4 or ActRIIB polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair can be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and both can have the same or different amino acid sequences. See Figure 23A. Alternatively, the interaction pair can be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other rather than self-associate. See Figure 23B. Higher order complexes can be envisioned, see Figures 23C and 23D. [Figure 23B]Figures 23A-23D show schematic examples of heteromeric protein complexes comprising an ALK4 polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ALK4 protein from a human or other species, such as those described herein), and an ActRIIB polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species, such as those described herein). In the illustrated embodiment, the ALK4 polypeptide is part of a fusion polypeptide comprising a first member ("C1") of the interaction pair, and the ActRIIB polypeptide is part of a fusion polypeptide comprising a second member ("C2") of the interaction pair. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncated forms and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker can be disposed between the ALK4 or ActRIIB polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair can be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and both can have the same or different amino acid sequences. See Figure 23A. Alternatively, the interaction pair can be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other rather than self-associate. See Figure 23B. Higher order complexes can be envisioned, see Figures 23C and 23D. [Figure 23C]Figures 23A-23D show schematic examples of heteromeric protein complexes comprising an ALK4 polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ALK4 protein from a human or other species, such as those described herein), and an ActRIIB polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species, such as those described herein). In the illustrated embodiment, the ALK4 polypeptide is part of a fusion polypeptide comprising a first member ("C1") of the interaction pair, and the ActRIIB polypeptide is part of a fusion polypeptide comprising a second member ("C2") of the interaction pair. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncated forms and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker can be disposed between the ALK4 or ActRIIB polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair can be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and both can have the same or different amino acid sequences. See Figure 23A. Alternatively, the interaction pair can be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other rather than self-associate. See Figure 23B. Higher order complexes can be envisioned, see Figures 23C and 23D. [Figure 23D]Figures 23A-23D show schematic examples of heteromeric protein complexes comprising an ALK4 polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ALK4 protein from a human or other species, such as those described herein), and an ActRIIB polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species, such as those described herein). In the illustrated embodiment, the ALK4 polypeptide is part of a fusion polypeptide comprising a first member ("C1") of the interaction pair, and the ActRIIB polypeptide is part of a fusion polypeptide comprising a second member ("C2") of the interaction pair. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncated forms and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker can be disposed between the ALK4 or ActRIIB polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair can be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and both can have the same or different amino acid sequences. See Figure 23A. Alternatively, the interaction pair can be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other rather than self-associate. See Figure 23B. Higher order complexes can be envisioned, see Figures 23C and 23D. DETAILED DESCRIPTION OF THE INVENTION

[0063] Detailed Description of the Invention 1. Overview The TGF-β superfamily includes TGF-beta, activin, nodal, and osteogenic Proteins (BMPs), growth differentiation factors (GDFs), and anti-Müllerian hormone (AMH) ) [Weiss et al. (2013) Devel Opmental Biology, Vol. 2 (No. 1): pp. 47-63. Members of the superfamily are found in both vertebrates and mammals and are ubiquitous in diverse tissues. TGF-β is expressed in the early stages of development and functions throughout the life of the animal. The -β superfamily proteins mediate stem cell self-renewal, gastrulation, differentiation, and organ morphogenesis. , and is a key mediator of adult tissue homeostasis. Consistent with this ubiquitous activity, Aberrant TGF-beta superfamily signaling is a risk factor for, for example, autoimmune diseases, cardiovascular It is associated with a wide range of human pathologies, including vascular disease, fibrotic diseases, and cancer.

[0064] Ligands of the TGF-beta superfamily share the same dimeric structure, and their structure In this case, the central 3-1 / 2 turn helix of one monomer is connected to the beta-helix of the other monomer. -It is packed against the concave surface formed by the strands. Most of the members are further stabilized by intermolecular disulfide bonds. The sulfide bond crosses the ring formed by two other disulfide bonds, forming a "cis- This generates what is called a "taint knot" motif [Lin et al. (2006) Rep induction, 132:179-190; and Hinck et al. (2012) FEBS Letters, vol. 586: pp. 1860-1870].

[0065] TGF-beta superfamily signaling is mediated by type I and type II serine / threonine receptors. This is mediated by a heteromeric complex of kinase receptors, which is downregulated by ligand stimulation. SMAD proteins (e.g., SMAD proteins 1, 2, 3, 5, and 8) Massague (2000) Nat. Rev. Mol Cell Biol., vol. 1:169-178]. These type I and type II receptors The body comprises a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase specificity. Generally, type I receptors mediate intracellular signal transduction while The type II receptor is required for binding of TGF-beta superfamily ligands. Type I and type II receptors form a stabilized complex after ligand binding, while type II receptors This results in phosphorylation of the type I receptor by ATP.

[0066] The TGF-beta family is composed of type I receptors to which it binds and Sm receptors to which it activates. Based on the ad protein, two phylogenetic branches can be distinguished. A recently evolved branch, e.g., type I, which activates Smad2 and 3 Signaling through receptors, TGF-beta, activin, GDF8, GDF9, G DF11, BMP3, and nodal [Hinck (2012) FEBS L etters, vol. 586: 1860-1870]. The other branch is the superfamily These proteins include more distantly related proteins, e.g., Smad1, 5, and 8, which signal via BMP, BMP2, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b , BMP9, BMP10, GDF1, GDF5, GDF6, and GDF7.

[0067] Activin is a member of the TGF-beta superfamily and a follicle-stimulating hormone. It was first discovered as a regulator of steroid hormone secretion, but has since been shown to regulate a variety of reproductive and non-reproductive hormones. The role of the β-subunits has been characterized. β A β A , β B β B , and β A β B ), which are the three major activin forms (A, B and The human genome contains activin C and activin B, which are expressed primarily in the liver. E also codes, β C or β E Heterodimeric forms containing TGF-beta are also known. In the superfamily, activins mediate hormone production in ovarian and placental cells. stimulates the ATP-dependent ATP synthesis, supporting neuronal cell survival and positively or negatively affecting cell cycle progression depending on the cell type. It can affect embryos and induce mesoderm differentiation at least in amphibian embryos. It is a unique and multifunctional factor. [DePaolo et al. (1991) Proc Soc Ep Biol Med., 198:500-512; Dyson et al. (1997) Curr Biol., 7:81-84; and Woodruff (1998) B Biochem Pharmacol., 55:953-963]. In this study, activin signaling is antagonized by its related heterodimer, inhibin. For example, it acts as an active ingredient in regulating the secretion of follicle-stimulating hormone (FSH) from the pituitary gland. Inhibin stimulates FSH synthesis and secretion, while inhibin inhibits FSH synthesis and secretion. It is possible to modulate activin bioactivity and / or bind to activin. Other proteins that can be identified include follistatin (FS), follistatin-related protein (FS RP, also known as FLRG or FSTL3), and α2-macroglobulin .

[0068] As described herein, an agent that binds to "activin A" is an isolated β A Sub in the context of units or dimeric complexes (e.g., β A β A Homodimer or β A β B heterodimer), or A Specific binding to the subunit Heterodimeric complexes (e.g., β A β B In the case of a heterodimer, Drugs that bind to β- A Specific for epitopes present within the subunit But the non-β A subunits (e.g., β of the complex) B subunit) Similarly, the compounds described herein that antagonize (inhibit) "activin A" do not bind to the agonist. The disclosed agents include isolated β A in the context of subunits or dimeric complexes (For example, β A β A Homodimer or β A β B whether as heterodimers β AThese are agents that inhibit one or more activities mediated by the β subunit. A β B In the case of heterodimers, drugs that inhibit activin A inhibit β A Subunit 1 Specifically inhibits one or more activities of the complex but not the non-β A Subunits (e.g., complex Body beta B This drug does not inhibit the activity of activin B. , "activin C," and "activin E." The present invention also applies to drugs that antagonize "activin AB." A One or more activities mediated by the subunit and β B Catalyzed by subunits These drugs inhibit one or more activities mediated by the ATP.

[0069] Both BMPs and GDFs have a characteristic fold of the TGF-beta superfamily. They form a family of cytokines that share the same cysteine ​​knot [Rider et al. (20 10) Biochem J., Vol. 429 (No. 1): pp. 1-12]. This family is For example, BMP2, BMP4, BMP6, BMP7, BMP2a, BMP3, BMP3b (also known as GDF10), BMP4, BMP5, BMP6, BMP7, BMP8, B MP8a, BMP8b, BMP9 (also known as GDF2), BMP10, BMP11 ( GDF11), BMP12 (also known as GDF7), BMP13 (GDF 6), BMP14 (also known as GDF5), BMP15, GDF1, GD F3 (also known as VGR2), GDF8 (also known as myostatin), GDF9, G DF15, and Decapentaplegic, which gave the BMP its name. In addition to their ability to induce bone formation, BMPs / GDFs play a key role in the development of a wide range of tissues. BMP / GDF homo- and heterodimers display type I and type II receptor activity. The dimers interact with each other to generate multiple possible signaling complexes, allowing the two competing BMP / GDFs are highly They have specific and localized functions, including the developmental restriction of BMP / GDF expression and and several specific BMP antagonists that bind cytokines with high affinity. Curiously, it is regulated in several ways, including by the secretion of ATP proteins. Some of these antagonists resemble TGF-beta superfamily ligands. are.

[0070] Growth differentiation factor-8 (GDF8) is also known as myostatin. GDF8 is involved in bone It is a negative regulator of skeletal muscle mass and is highly expressed in developing and adult skeletal muscle. GDF8 null mutation in transgenic mice results in marked skeletal muscle hypertrophy and hyperplasia McPherron et al., Nature (1997) 387 : pp. 83-90]. Similar increases in skeletal muscle mass have been observed in cattle and, notably, in humans with GD. This is evident in the naturally occurring mutations of F8 [Ashmore et al. (1974) Gr owth, vol. 38: pp. 501-507;Swatland and Kieffer, J. Anim. Sci. (1994) 38:752-757; McPherron et al. Yo and Lee, Proc. Natl. Acad. Sci. USA (1997) 9 4:12457-12461; Kambadur et al., Genome Res. (19 97) 7:910-915; and Schuelke et al. (2004) N En gl J Med, 350:2682-8]. Research has shown that HIV infection in humans The associated muscle wasting has also been shown to be accompanied by increased GDF8 protein expression [Gonza Lez-Cadavid et al., PNAS (1998) 95:14938-43. Additionally, GDF8 modulates the production of muscle-specific enzymes (e.g., creatine kinase). and can modulate cell proliferation of myoblasts [International Patent Application Publication No. WO 00 / 43781]. The GDF8 propeptide is non-covalently linked to the mature GDF8 domain dimer. By binding to the nucleotides, the biological activity of the nucleotides can be inactivated [Miyazono et al. ( 1988) J. Biol. Chem., 263:6407-6415; Wak Efield et al. (1988) J. Biol. Chem., 263:7646-7 654; and Brown et al. (1990) Growth Factors, Vol. 3:3 5-43]. Binds to GDF8 or structurally related proteins and inhibits their biological activity. Other proteins that inhibit NF-κB include follistatin and potentially follistatin-related proteins. Protein [Gamer et al. (1999) Dev. Biol. 208:222-232 ] is included.

[0071] GDF11, also known as BMP11, is expressed in the tail bud, limb bud, maxillary arch, and It is a secreted protein expressed in the mandibular arch and dorsal root ganglia [McPherr on et al. (1999) Nat. Genet., 22:260-264; and Na Kashima et al. (1999) Mech. Dev., Vol. 80: 185-189]. GDF11 plays unique roles in the patterning of both mesodermal and neural tissues [Gamer et al. (1999) Dev Biol. 208:222-32]. GD F11 is a negative regulator of chondrogenesis and myogenesis in the developing chick limb It was shown [Gamer et al. (2001) Dev Biol., 229:407-20 GDF11 expression in muscle is also involved in regulating muscle growth in a manner similar to GDF8. In addition, the expression of GDF11 in the brain suggests that GDF11 may play a role in neuronal regulation. Interestingly, GDF11 also has activities related to the function of the olfactory system. It has been found to inhibit neurogenesis in epithelia [Wu et al. (2003) Neuron. 37:197-207]. Therefore, GDF11 may be involved in muscle and neurodegenerative diseases. in vitro and in vivo in the treatment of diseases such as encephalopathy (e.g., amyotrophic lateral sclerosis) It may have in vivo applications.

[0072] As demonstrated herein, soluble ActRIIA polypeptides and AL The K4:ActRIIB heterodimer is a heterodimer that is composed of various ActRIIA and ActRI Binds to IB-interacting ligands and reduces blood pressure and cardiac hypertrophy in PAH models While not wishing to be limited to any particular mechanism, the effects of these agents are This is expected to be mainly due to its antagonistic effect on ctRIIA / B signaling. Regardless, ActRIIA / B signaling antagonists (GDF / BMP antagonists) nisto) reduces blood pressure, reduces cardiac hypertrophy, and has been shown to be effective in treating pulmonary hypertension. The positive effects of steroids on blood pressure and Blood pressure and hypertrophy are dynamic and there are factors that increase blood pressure and hypertrophy and factors that decrease blood pressure and hypertrophy. It should be noted that blood pressure and cardiac hypertrophy change depending on the balance of factors involved. Promotes elevated blood pressure and cardiac hypertrophy by increasing factors that reduce blood pressure and cardiac hypertrophy This can be reduced by reducing the factors that cause it, or both. The terms "reduction in blood pressure" and "reduction in cardiac hypertrophy" refer to observable physical changes in blood pressure and cardiac tissue. refers to a change in the environment and is intended to be neutral with respect to the mechanism by which the change occurs.

[0073] The rat model of PAH used in the studies described herein is a promising model for efficacy in humans. It is believed to be predictive, and therefore the present disclosure provides an ActRIIA polypeptide, ALK4:A ctRIIB heterodimer and other GDF / BMP antagonists were used to investigate the effects of pulmonary hypertension. and treating one or more complications of hypertension (e.g., PAH), particularly pulmonary hypertension in humans. The present invention provides methods for treating, preventing, or reducing the severity or duration of rheumatoid arthritis. As disclosed in, for example, one or more GDF / BMP antagonists. ActRIIA / B ligands [e.g., activins (activin A, activin B, activin Activin AB, Activin C, Activin AC, Activin BC, Activin E, Activin activin AE, and / or activin BE), GDF8, GDF11, GDF3, BMP6 antagonists that inhibit one or more type I and / or type II BMPs; or type II receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK7, and ALK5); and one or more downstream signals Antagonists that inhibit signaling components (e.g., Smad proteins such as Smad2 and 3) Various compounds that can be used to antagonize ActRIIA / B signaling, including agonists The GDF / BMP antagonists used in accordance with the methods and uses of the present disclosure The antibody can be in various forms, such as a ligand trap (e.g., a soluble ActRIIA polypeptide). ActRIIB polypeptide, ALK4:ActRIIB heterodimer, follistatin tyrosine polypeptides, and FLRG polypeptides), antibody antagonists (e.g., Chibin, GDF8, GDF11, GDF3, BMP6, BMP15, BMP10, Act One or more of RIIA, ActRIIB, ALK4, ALK7, and ALK5 antibodies that inhibit a number of GDF1 receptors), small molecule antagonists [e.g., activin, GDF8, GDF1 1, GDF3, BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK4, ALK7, ALK5, and one or more Smad proteins (e.g., small molecules that inhibit one or more of the Smads (Smad2 and Smad3), and nucleotides antagonists [e.g., activin, GDF8, GDF11, GDF3, BMP6, BMP15, BMP10, ActRIIA, ActRIIB, ALK4, ALK7, AL K5, and one or more Smad proteins (e.g., Smad2 and 3). The present invention also includes a nucleotide sequence that inhibits one or more of the following:

[0074] The terms used herein generally refer to the general meaning of the term within the context of this disclosure and the meaning of each term. In the particular context in which it is used, it has its ordinary meaning in the art. In describing the compositions and methods, and how to make and use them, To provide further guidance, certain terms are used as they are discussed below or elsewhere in this specification. The scope or meaning of any use of a term will be clear from the specific context in which it is used. It is.

[0075] Homologous means "common" in all its grammatical forms and spelling variations. It refers to the relationship between two proteins that have a common evolutionary origin and are superfamily proteins of the same species. This includes proteins from the same family as well as homologous proteins from different species. The proteins (and nucleic acids encoding them) are identical in structure, whether in terms of percent identity or not. Whether due to the presence of conserved residues or motifs and positions, the sequence similarity However, as reflected by common usage and this application In the literature, the term "homologous" when modified by an adverb such as "highly" indicates sequence similarity. They may refer to different species and may or may not be related to a common evolutionary origin.

[0076] The term "sequence similarity," in all its grammatical forms, refers to sequences that share a common evolutionary origin. refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not be shared .

[0077] "Percent (%) sequence identity with respect to a reference polypeptide (or nucleotide) sequence" " aligns sequences, introduces gaps if necessary, and determines what is part of the sequence identity. After achieving the maximum percent sequence identity, the reference polypeptide is The candidate sequence is identical to the amino acid residue (or nucleic acid) in the nucleotide sequence. It is defined as the percentage of amino acid residues (or nucleic acids) in a molecule. Alignment for purposes of determining amino acid sequence identity can be accomplished by a variety of techniques within the skill of the art. On the other hand, for example, BLAST, BLAST-2, ALIGN or Megalign (DN Achieved using publicly available computer software such as ASTAR software. Those skilled in the art will appreciate that achieving maximal alignment over the full length of the sequences being compared is feasible. Determine appropriate parameters for sequence alignment, including any algorithms required for its construction. However, for purposes herein, % amino acid (nucleic acid) sequence identity can be used. Identity values ​​are generated using the sequence comparison computer program ALIGN-2. The LIGN-2 sequence comparison computer program is a registered trademark of Genentech, Inc. The source code is copyrighted by the U.S. Copyright Office. ce), Washington, DC 20559, filed by user documentation. where the U.S. Copyright Registration Number is The ALIGN-2 program is registered under the information number TXU510087. Genentech, Inc., South San Francisco, Ca. It is available from lif. or can be compiled from source code. The LIGN-2 program is compatible with UNIX operating systems, including Digital UNIX V4.0D. It should be compiled for use on the ® operating system. All sequence comparison parameters are set by the ALIGN-2 program. Do not fluctuate.

[0078] "Agonize," in all its grammatical forms, is a protein and / or activate a gene (e.g., activate or inhibit gene expression of the protein) by amplifying or inducing an inactive protein to enter an active state. refers to the process of increasing the activity of a protein and / or gene.

[0079] "Antagonize" in all its grammatical forms refers to proteins and / or genes inhibiting a gene (e.g., by inhibiting or reducing gene expression of the protein) or by inducing active proteins to enter an inactive state) or It refers to the process of decreasing the activity of proteins and / or genes.

[0080] Throughout this specification and claims, the terms "about" and "approximately" are used in connection with numerical values. "Approximately" denotes an interval of accuracy familiar and accepted by those skilled in the art. Such an interval of accuracy is ±10%. Alternatively, and particularly in biological systems, the term " "About" and "approximately" mean within an order of magnitude of a given value, preferably ≦5-fold, more preferably ≦ It can mean twice the value.

[0081] The numerical ranges disclosed herein are inclusive of the numbers defining the range.

[0082] The terms "a" and "an" are used to refer to Unless clearly indicated otherwise, the term "a" (or "singular") includes plural referents. or "an" and the terms "one or more" and "at least one" and can be used interchangeably herein. Furthermore, "and / or" means When used in the specification, two or more features or components, with or without other features or components, are included. should be construed as a specific disclosure of each specified feature or component beyond that. Therefore, when used herein in phrases such as "A and / or B," The term "and / or" means "A and B," "A or B," "A" (alone and Similarly, terms such as "A, B and / or C" are intended to include "B" (alone). The term "and / or" as used in the phrase is intended to include each of the following aspects: Contemplated: 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 (alone); B (alone); and C (alone).

[0083] Throughout this specification, the words "comprise" or "comprises" Variations such as "rises" or "comprising" are used to refer to implies the inclusion of any integer or group of integers listed, but the exclusion of any other integer or group of integers. It will be understood that this does not imply

[0084] 2. ActRII polypeptide, ALK4 polypeptide, ALK4:ActRIIB polypeptide Multimers and their modifications In certain aspects, the present disclosure provides ActRII polypeptides and uses thereof (e.g., Pulmonary hypertension or one or more complications of pulmonary hypertension and / or interstitial lung disease (e.g., idiopathic pulmonary fibrosis) The term "ActR" as used herein refers to the use of ActR to reduce the severity of atopic dermatitis. "II" refers to the family of type II activin receptors. This family is comprised of type IIA activin receptors. Activin receptor (ActRIIA) and type IIB activin receptor (ActRIIB) Includes:

[0085] As used herein, the term "ActRIIB" refers to activin receptor I from any species. Type IB (ActRIIB) proteins and their use in the identification of such Ac by mutagenesis or other modifications ActR as used herein refers to a family of variants derived from the tRIIB protein. Reference to IIB is understood to refer to any one of the presently identified forms. ActRIIB family members generally bind to ligands containing cysteine-rich regions. The peptide-binding extracellular domain, transmembrane domain, and predicted serine / threonine kinase domain It is a transmembrane protein composed of a cytoplasmic domain with enzyme activity.

[0086] The term "ActRIIB polypeptide" refers to any of the ActRIIB family members. In addition to naturally occurring polypeptides, any variants (mutants, The term "antibody" includes polypeptides containing the polypeptides described herein, including fragments, fusions, and peptidomimetic forms. Examples of such variant ActRIIB polypeptides are provided throughout this disclosure and by reference. International Patent Application Publication No. WO2006 / 01262, the entire contents of which are incorporated herein by reference. 7, WO2008 / 097541, WO2010 / 151426 and WO2011 / 0 20045. Any ActRIIB-related polypeptide described herein The amino acid numbering of the peptides is based on that of human ActRI, as shown below, unless otherwise specified. Based on the numbering of the IB precursor protein sequence (SEQ ID NO: 1). The human ActRIIB precursor protein sequence is shown below: [ka]

[0087] The signal peptide is indicated by a single underline; the extracellular domain is indicated by a bold font. Potential endogenous N-linked glycosylation sites are indicated by double underlining. The processed (mature) extracellular ActRIIB polypeptide sequence is shown below:

[0088] [ka]

[0089] In some embodiments, the protein is produced with an "SGR..." sequence at the N-terminus. The C-terminal "tail" of the extracellular domain is indicated by a single underline. The sequence with the "tail" deleted (Δ15 sequence) is shown below:

[0090] [ka]

[0091] A form of ActRIIB having an alanine at position 64 of SEQ ID NO: 1 (A64) has also been reported in the literature. For example, Hilden et al. (1994) Blood, 83(8):2 See pages 163-2170. Applicants have identified ActRIIB having an A64 substitution. The ActRIIB-Fc fusion protein, which contains the extracellular domain of activin and GD It was confirmed that the 64th position of the ATPase had a relatively low affinity for F11. The same ActRIIB-Fc fusion protein with arginine (R64) showed low nanomolar Affinity for activin and GDF11 in the high picomolar to high picomolar range Therefore, the sequence having R64 is a "wild type" of human ActRIIB in the present disclosure. The "raw type" is used as the reference sequence. A form of ActRIIB with an alanine at position 64 is shown below: [ka]

[0092] The signal peptide is indicated by a single underline and the extracellular domain is indicated by a bold font. It is indicated by.

[0093] Alternative A64 form of processed (mature) extracellular ActRIIB polypeptide sequence The columns are: [ka]

[0094] In some embodiments, the protein is produced with an "SGR..." sequence at the N-terminus. The C-terminal "tail" of the extracellular domain is indicated by a single underline. The sequence with the "tail" deleted (Δ15 sequence) is shown below: [ka]

[0095] The nucleic acid sequence encoding the human ActRIIB precursor protein is shown below (SEQ ID NO:7): ), which encodes amino acids 1 to 513 of the ActRIIB precursor, Genbank It represents nucleotides 25 to 1560 of the reference sequence NM_001106.3. The sequence provides an arginine at position 64, and is modified to provide an alanine instead. The signal sequence is underlined. [ka] [ka]

[0096] The nucleic acid sequence encoding the processed extracellular human ActRIIB polypeptide is: (SEQ ID NO: 8). The sequence shown provides an arginine at position 64 and instead It can be modified to provide an alanine instead. [ka]

[0097] Amino acids of human ActRIIB extracellular domain and human ActRIIA extracellular domain The alignment of the amino acid sequences is illustrated in Figure 1. This alignment is The amino acid residues in both receptors that are thought to directly contact the I ligand are indicated. For example, the composite ActRII structure shows that the ActRIIB ligand-binding pocket is bounded by residue Y 31, N33, N35, L38 to T41, E47, E50, Q53 to K55, L57 , H58, Y60, S62, K74, W78 to N83, Y85, R87, A92 and It is indicated that the positions are partially defined by E94 to F101. It is expected that conservative mutations will be tolerated.

[0098] In addition, ActRIIB is well conserved among vertebrates, and its extracellular domain Large stretches are completely preserved. For example, Figure 2 shows various Acts Multiple sequence alignment of the human ActRIIB extracellular domain compared with RIIB orthologues Many of the ligands that bind to ActRIIB are also highly conserved. Therefore, these alignments reveal the importance of normal ActRIIB ligand binding activity. It predicts the important amino acid positions in the ligand-binding domain, and also predicts the normal Act May be tolerant of substitutions that do not significantly alter RIIB ligand binding activity It is therefore possible to predict the amino acid positions according to the presently disclosed method. Active human ActRIIB variant polypeptides useful in this context may be derived from other vertebrate ActRIIB polypeptides. It may contain one or more amino acids at the corresponding positions from the sequence, or may be human or other It may contain residues similar to those in the vertebrate sequence. The following example illustrates this approach to defining active ActRIIB variants. L46 in the main sequence (SEQ ID NO: 2) is Xenopus ActRIIB (SEQ ID NO: 58) is a valine, this position can be varied and is optional. Alternatively, change to another hydrophobic residue such as V, I, or F, or to a non-polar residue such as A. E52 in the human extracellular domain is K in Xenopus, and this site , E, D, K, R, H, S, T, P, G, Y and possibly A, including polar residues. This indicates that the T93 in the human extracellular domain is a nucleotide sequence that is highly tolerant to various changes. In Mega laevis, it is K, and a wide range of structural variations are tolerated at this position, including S, K, R, E, D, This indicates a preference for polar residues such as H, G, P, G, and Y. Human extracellular domain F108 in is Y in Xenopus, and therefore Y or I, V or L Other hydrophobic groups such as α- and β-terminal amino acids should be acceptable. In the case of the F. megaterus, the residue is K, and charged residues including D, R, K, and H, as well as Q and N, are present at this position. This indicates that R112 in the human extracellular domain is likely to be tolerated by the claw. In frogs, it is K, indicating that basic residues, including R and H, are tolerated at this position. The A at position 119 in the human extracellular domain is relatively less conserved, Since it appears as P in rodents and V in Xenopus, it is basically impossible to find a suitable site at this position. Other amino acids should also be tolerated.

[0099] Furthermore, the ActRII protein has many structural and functional characteristics, particularly with respect to the ligation of the ActRII protein. It has been characterized in the art for bond binding [Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) Na ture Structural Biology 6(1):18-22;Al Lendorph et al. (2006) PNAS 103 (20:7643-7648) Thompson et al. (2003) The EMBO Journal, Vol. 22, No. 7 ): pp. 1555-1566; and U.S. Pat. Nos. 7,709,605 and 7,612 ,041 and 7,842,663]. In addition to the teachings herein, these references The literature describes ActRs that retain one or more normal activities (e.g., ligand binding activity). Guidance on how to generate IIB variants is provided.

[0100] For example, a distinct structural motif known as the three-finger toxin fold is Type I The extracellular domain of each monomeric receptor is important for ligand binding by the type II and type III receptors. It is formed by conserved cysteine ​​residues located at variable positions within the Nwald et al. (1999) Nat Struct Biol 6:18-22; Yobi Hinck (2012) FEBS Lett 586:1860-1870 Therefore, the human A region bounded by the outermost of these conserved cysteines The core ligand binding domain of ActRIIB is SEQ ID NO: 1 (ActRIIB precursor) Corresponding to positions 29 to 109. These cysteines are adjacent to the core sequence bounded by The structurally less ordered amino acids that interact with the ligand may alter the binding of the ligand, without necessarily altering the ligand binding. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 at the N-terminus 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27If and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 at the C-terminus. 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 residues, which can be truncated. Exemplary Ac residues for N- and / or C-terminal truncations The tRIIB extracellular domain comprises SEQ ID NOs: 2, 3, 5 and 6.

[0101] Attisano et al. found that the proline amino acid sequence at the C-terminus of the extracellular domain of ActRIIB This study showed that deletion of the activin receptor reduced the affinity of the receptor for activin. An ActRIIB-Fc fusion protein containing amino acids 20 to 119 of SEQ ID NO: 1. ActRIIB(20-119)-Fc contains the proline knot region and the complete juxtamembrane domain. Compared with ActRIIB(20-134)-Fc containing the domain, GDF11 and ActRIIB have reduced binding to thiamine (see, e.g., U.S. Pat. No. 7,842,663 However, the ActRIIB(20-129)-Fc protein has a proline knot. Although the region is disrupted, it retains similar, but somewhat reduced, activity compared to the wild type.

[0102] Therefore, amino acids 134, 133, 132, 131, 130 and 129 (SEQ ID NO: All ActRIIB extracellular domains ending at (with respect to No. 1) are predicted to be active. However, constructs stopping at 134 or 133 may be most active. Mutations in any of positions 9 to 134 (with respect to SEQ ID NO: 1) may enhance ligand binding affinity. In support of this, P129 and P130 (distribution It is well known in the art that mutations in the above (with respect to column number 1) do not substantially reduce ligand binding. Therefore, the ActRIIB polypeptide of the present disclosure has a structure similar to that of amino acid 109 (the most It can end as short as 109 and 119 (terminal cysteine), or between 109 and 119 (terminal cysteine). For example, 109, 110, 111, 112, 113, 114, 115, 116, 117, Forms ending in 118 or 119 are predicted to have reduced ligand binding. Amino acid 119 (with respect to this SEQ ID NO: 1) is not well conserved and is therefore easily altered. or truncated. ActRI ending at or after 128 (relative to SEQ ID NO: 1) The IB polypeptide should retain ligand binding activity. and 127 or between (e.g., 119, 120, 121, 122, 123, 12 ActRIIB polypeptides ending in 124, 125, 126, or 127 are associated with moderate binding. Depending on the clinical or experimental setting, one of these forms may be , would be desirable for use.

[0103] At the N-terminus of ActRIIB, amino acid 29 or earlier (with respect to SEQ ID NO: 1) ) is expected to retain ligand binding activity. Amino acid 29 represents the first cysteine. Mutation of α-asparagine to asparagine significantly inhibits N-linked ligand binding without substantially affecting ligand binding. A glycosylation sequence is introduced [U.S. Patent No. 7,842,663]. in the region between the signal cleavage peptide and the cysteine ​​bridge region, corresponding to ~29 Ensure that mutations are well tolerated, especially at positions 20, 21, 22, 23, and 24. The ActRIIB polypeptide beginning with (with respect to SEQ ID NO: 1) is a general ligand binding polypeptide. The binding activity should be maintained at positions 25, 26, 27, 28 and 29 (relative to SEQ ID NO: 1). ) is also expected to retain ligand-binding activity. For example, in U.S. Patent No. 7,842,663, it is surprisingly found that It was demonstrated that ActRIIB constructs starting with 4 or 25 would be most active. was done.

[0104] In summary, the general formula for the active portion (e.g., ligand-binding portion) of ActRIIB is: The ActRIIB polypeptide comprises amino acids 29 to 109 of SEQ ID NO: 1. For example, a sequence starting from a residue corresponding to any one of amino acids 20 to 29 of SEQ ID NO: 1 (e.g., amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29), amino acids 109 to 134 of SEQ ID NO: 1 Ending at a position corresponding to one of the amino acids (e.g., amino acids 109, 110, 111, 11 2, 113, 114, 115, 116, 117, 118, 119, 120, 121, 12 2, 123, 124, 125, 126, 127, 128, 129, 130, 131, 13 132, 133, or 134) At least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 % identical to, consisting essentially of, or consisting of an amino acid sequence As another example, 20 to 29 of SEQ ID NO: 1 (for example, 20, 21, 22, 23, Any one of 24th, 25th, 26th, 27th, 28th or 29th) or 21st to 29th ( For example, any of positions 21, 22, 23, 24, 25, 26, 27, 28, or 29 Starting from position 119 to 134 (e.g., 119, 120, 121) of SEQ ID NO: 1 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133 or 134), 119 to 133 (e.g., 1 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 1 Any one of positions 29, 130, 131, 132 or 133), 129-134 (e.g., any one of positions 129, 130, 131, 132, 133, or 134) 1) or 129-133 (e.g., 129, 130, 131, 132, or 133) Other examples include polypeptides ending at any one of positions 1 to 4 of SEQ ID NO: 1. 20-24 (e.g., any one of positions 20, 21, 22, 23, or 24), 21-24 (e.g., any one of positions 21, 22, 23, or 24) or 22 Starting at position ~25 (e.g., any one of positions 22, 22, 23, or 25), 109 to 134 of SEQ ID NO: 1 (e.g., 109, 110, 111, 112, 113, 11 4, 115, 116, 117, 118, 119, 120, 121, 122, 123, 12 4, 125, 126, 127, 128, 129, 130, 131, 132, 133 or Any one of the 134 positions), 119-134 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, any one of positions 132, 133, or 134) or 129-134 (e.g., Finish in one of the following places: 129th, 130th, 131st, 132nd, 133rd or 134th Variants within these ranges, particularly constructs that are similar to the corresponding portion of SEQ ID NO: 1, are also included. , at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or comprising, consisting essentially of, or consisting of an amino acid sequence with 100% identity Further variants are also contemplated.

[0105] The variants described herein can be combined in various ways. In this state, the ActRIIB variants bind to the first, second, fifth, sixth, and seventh ligand-binding pockets. , 8, 9, 10, or 15 or fewer conservative amino acid changes, optionally, a ligand-binding polypeptide One zero in the 40th, 53rd, 55th, 74th, 79th and / or 82nd positions in a ket or multiple non-conservative changes. The side sites are the amino and carboxy termini of the extracellular domain (as described above), and positions 42 to 46 and 65 to 73 (with respect to SEQ ID NO: 1); asparagus at position 65 A sucralose to alanine change (N65A) improves ligand binding in the R64 background. This change is likely due to the fact that Glycosylation of N65 in the A64 background was eliminated, thereby minimizing the This demonstrates that significant changes in R64A can be tolerated. However, R64K is well tolerated, allowing another basic residue, such as H, to be placed at position 64. [U.S. Patent No. 7,842,663]. The results of the mutagenesis program conducted in ActRIIB have been beneficially preserved. This indicates that there are amino acid positions with a high number of . As such, positions 80 (acidic or hydrophobic amino acids), 78 (hydrophobic, especially tryptophan) , 37th position (acidic, especially aspartic or glutamic acid), 56th position (basic amino acids) , 60 (hydrophobic amino acids, especially phenylalanine or tyrosine). Thus, the present disclosure provides a method for identifying amino acid sequences that may be conserved in ActRIIB polypeptides. Other positions that may be desirable to preserve are: position 52 (acid (polar or charged amino acid), 55 (basic amino acid), 81 (acidic), 98 (polar or charged, particularly or E, D, R or K), all relative to SEQ ID NO:1.

[0106] Additional N-linked glycosylation sites (NXS / T) were introduced into the ActRIIB extracellular domain. It has been demonstrated that addition to amines is well tolerated (e.g., U.S. Pat. No. 5,629,139). (See Patent No. 7,842,663.) Therefore, the NXS / T sequence is generally Specifically, the ligand binding pocket defined in FIG. 1 is present in the ActRIIB polypeptide of the present disclosure. It may also be introduced into a location outside the nucleotide sequence. The regions affected are amino acids 20-29, 20-24, 22-25, 109-134, and 120-1 34, or 129 to 134 (with respect to SEQ ID NO: 1). The NXS / T sequence also , in the linker between the ActRIIB sequence and the Fc domain or other fusion component, Such sites may be pre-existing or may be introduced into the fusion moiety itself. by introducing N in the correct position relative to the S or T that This can be introduced with minimal effort by introducing S or T at the position corresponding to N. Therefore, desirable changes to create N-linked glycosylation sites are: A24N, R64 N, S67N (possibly in combination with N65A modification), E105N, R112N, G1 20N, E123N, P129N, A132N, R112S, and R112T (Sequence No. (Regarding No. 1). Due to the protection afforded by glycosylation, glycosylated Any S predicted to be involved can be changed to a T without creating an immunogenic site. Similarly, any T that is predicted to be glycosylated can be changed to S. Thus, the alterations S67T and S44T (with respect to SEQ ID NO: 1) are contemplated. In the A24N variant, the S26T alteration may be used. The IIB polypeptide may contain one or more additional non-endogenous N-linked glycoproteins, as described above. It may be a variant having a cosylation consensus sequence.

[0107] In certain embodiments, the present disclosure provides fragments, functional variants, and modified forms thereof. GDF / BMP antagonists (inhibitors) including ActRIIB polypeptides containing and uses thereof (e.g., to treat or prevent PH or one or more PH-related complications). Preferably, the ActRIIB polypeptide is soluble (e.g., In some embodiments, the extracellular domain of ActRIIB is included. The polypeptide may be one or more GDF / BMP ligands [e.g., GDF11, GD F8, activin (activin A, activin B, activin AB, activin C, activin activity (e.g., S mad signaling). Thus, in some embodiments, ActRIIB polypeptides The polypeptide may bind to one or more GDF / BMP ligands [e.g., GDF11, GDF 8. Activin (activin A, activin B, activin AB, activin C, activin Bin E), BMP6, GDF3, BMP15, and BMP10. In embodiments, the ActRIIB polypeptide of the disclosure comprises amino acids 20-29 of SEQ ID NO:1. Starting from the residue corresponding to (e.g., amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 6, 27, 28, or 29), and Ending at positions corresponding to 109-134 (e.g., amino acids 109, 110, 111, 1 12, 113, 114, 115, 116, 117, 118, 119, 120, 121, 1 22, 123, 124, 125, 126, 127, 128, 129, 130, 131, 1 (ending in one of the following: 32, 133, or 134) At least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or Containing, consisting essentially of, or not consisting of an amino acid sequence that is 100% identical In some embodiments, the ActRIIB polypeptide comprises amino acids 29 to 30 of SEQ ID NO:1. 109, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, or 100% identical to an amino acid sequence In some embodiments, the ActRIIB polypeptide of the disclosure consists essentially of SEQ ID NO: 1 amino acids 29-109, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 Containing or consisting of an amino acid sequence that is 7%, 98%, 99%, or 100% identical to or consisting essentially of, wherein the position corresponding to L79 in SEQ ID NO: 1 is an acidic amino acid (naturally occurring acidic amino acids D and E, or artificial acidic amino acids). In certain embodiments, the ActRIIB polypeptide of the disclosure comprises amino acid 25 of SEQ ID NO:1. ~131, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9%, or 100% identical to the amino acid sequence of the In certain embodiments, the ActRIIB polypeptides of the disclosure consist essentially of the sequence At least 70%, 75%, 80%, 85%, 86% of amino acids 25-131 of sequence number 1 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 97%, 98%, 99%, or 100% identical amino acid sequence to or and wherein the position corresponding to L79 of SEQ ID NO: 1 is an acidic amino acid. In some embodiments, the ActRIIB polypeptide of the disclosure is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 40, 42, 45, 46, 47, 48, 69, 74, 77, 7 Any one of 8, 79, 108, 110, 114, 115, 118, and 120 The amino acid sequence of the sequence has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence to the In some embodiments, the ActRIIB polypeptide of the disclosure consists essentially of the sequence Numbers 1, 2, 3, 4, 5, 6, 40, 42, 45, 46, 47, 48, 69, 74, 77 , 78, 79, 108, 110, 114, 115, 118, and 120 or one amino acid sequence has 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 the Essentially, the position corresponding to L79 in SEQ ID NO: 1 is an acidic amino acid. In some embodiments, the ActRIIB polypeptides of the present disclosure comprise at least one ActR IIB polypeptide, comprising, consisting of, or consisting essentially of SEQ ID NO: The position corresponding to L79 in 1 is not an acidic amino acid (i.e., a naturally occurring amino acid acids D or E or artificial acidic amino acid residues).

[0108] In certain embodiments, the present disclosure relates to ActRIIA polypeptides. The term "ActRIIA" as used herein refers to activin receptor type IIA (A) from any species. ActRIIA) proteins, and such ActRIs by mutagenesis or other modifications. ActRIIA as used herein refers to a family of variants derived from the IA protein. Reference to is understood to refer to any one of the presently identified forms. Members of the tRIIA family generally contain a cysteine-rich domain that binds ligands. Active extracellular domain, transmembrane domain, and predicted serine / threonine kinase activity It is a transmembrane protein composed of a cytoplasmic domain having

[0109] The term "ActRIIA polypeptide" refers to any of the ActRIIA family members. In addition to naturally occurring polypeptides, any variants (mutants, The term "antibody" includes polypeptides containing the polypeptides described herein, including fragments, fusions, and peptidomimetic forms. Examples of such variant ActRIIA polypeptides are provided throughout this disclosure and by reference. International Patent Application Publication No. WO2006 / 012627 and and WO2007 / 062188. The amino acid numbering of RIIA-related polypeptides is as follows, unless otherwise specified: Based on the numbering of the human ActRIIA precursor protein sequence (SEQ ID NO: 9) shown.

[0110] The canonical human ActRIIA precursor protein sequence is shown below: [ka]

[0111] The signal peptide is indicated by a single underline; the extracellular domain is in bold font. potential endogenous N-linked glycosylation sites are indicated by double underlines .

[0112] The processed (mature) extracellular human ActRIIA polypeptide sequence is shown below: [ka]

[0113] The C-terminal "tail" of the extracellular domain is indicated by a single underline. The missing sequence (Δ15 sequence) is shown below: [ka]

[0114] The nucleic acid sequence encoding the human ActRIIA precursor protein is shown below (SEQ ID NO:1): 2), which corresponds to nucleotides 159 to 169 of the Genbank reference sequence NM_001616.4. 1700 is shown. The signal sequence is underlined. [ka] [ka]

[0115] Nuclear coding for the processed soluble (extracellular) human ActRIIA polypeptide The acid sequence is as follows: [ka]

[0116] ActRIIA is well conserved among vertebrates and has a large extracellular domain. For example, Figure 3 shows the structure of various ActRIIA Multiple sequence alignment of the human ActRIIA extracellular domain compared with its orthologues. Many of the ligands that bind to ActRIIA are also highly conserved. These alignments reveal that the ligand binding site is important for normal ActRIIA ligand binding activity. Predicting critical amino acid positions within the ATP-binding domain and normal ActRIIA Amino acids that may be tolerant to substitutions without significantly altering ligand binding activity It is therefore possible to predict the location of a useful Active human ActRIIA variant polypeptides may be derived from the sequence of another vertebrate ActRIIA. or a human or other vertebrate. It may contain residues similar to those in the animal sequence.

[0117] While not meant to be limiting, the following examples illustrate this concept of defining active ActRIIA variants. As illustrated in Figure 3, F13 in the human extracellular domain is Ovis aries (SEQ ID NO: 62), Gallus gallus (SEQ ID NO: 65) , Bos Taurus (SEQ ID NO: 66), Tyto alba (SEQ ID NO: 67), and and Myotis davidii (SEQ ID NO: 68) ActRIIA is Y, and F This indicates that aromatic residues, including C, W, and Y, are tolerated at this position. Q24 in the domain is R in Bos Taurus ActRIIA, D, This indicates that charged residues, including R, K, H, and E, are tolerated at this position. S95 in the ectodomain is a marker for Gallus gallus and Tyto alba In ActRIIA, it is F, and this site is E, D, K, R, H, S, T, P, G, Y, etc. A wide variety of changes involving polar residues and possibly hydrophobic residues such as L, I, or F This indicates that E52 in the human extracellular domain may be tolerant to Ovis a In ActRIIA, it is D, and acidic residues including D and E are tolerated at this position. This indicates that the human extracellular domain of P29 is relatively poorly conserved. , appears as S in Ovis aries ActRIIA, and Myotis davi dii appears as L in ActRIIA, so essentially any amino acid can be It should be acceptable in this position.

[0118] Moreover, as mentioned above, ActRII proteins have the following structural / functional characteristics: It has been characterized in the art, particularly with respect to ligand binding [Attisano et al. (1999) 2) Cell, vol. 68(1): pp. 97-108; Greenwald et al. (1999) ) Nature Structural Biology, Vol. 6 (No. 1): pp. 18-22 Allendorph et al. (2006) PNAS, 103(20):7643-7 648; Thompson et al. (2003) The EMBO Journal, 22 Vol. (7): pp. 1555-1566; and U.S. Pat. No. 7,709,605, No. 7, 612,041, and 7,842,663]. In addition to the teachings herein, these The references describe A's that retain one or more desired activities (e.g., ligand binding activity). It provides ample guidance on how to generate ctRII variants.

[0119] For example, a distinct structural motif known as the three-finger toxin fold is Type I The extracellular domain of each monomeric receptor is important for ligand binding by the type II and type III receptors. It is formed by conserved cysteine ​​residues located at variable positions within the nwald et al. (1999) Nat Struct Biol 6:18-22; Yobi Hinck (2012) FEBS Lett, 586:1860-1870] Therefore, the human A region bounded by the outermost of these conserved cysteines The core ligand binding domain of ActRIIA is SEQ ID NO: 9 (ActRIIA precursor) These correspond to positions 30 to 110. Structurally less ordered amino acids adjacent to the amino acid sequence do not necessarily alter ligand binding. Approximately 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, and about 1, 2, 3, 4, 5, 6, or 7 residues at the C-terminus. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 It can be truncated by 1, 22, 23, 24, or 25 residues. A extracellular domain truncations include SEQ ID NOs: 10 and 11.

[0120] Thus, the general formula for the active portion (e.g., ligand binding) of ActRIIA is SEQ ID NO: a polypeptide comprising, consisting essentially of, or consisting of amino acids 30 to 110 of SEQ ID NO: 9; Thus, an ActRIIA polypeptide is, for example, SEQ ID NO: 9 Starting from a residue corresponding to any one of amino acids 21 to 30 (e.g., amino acid Any of the following: 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 1), corresponding to any one of amino acids 110 to 135 of SEQ ID NO: 9. Ending at a position (e.g., 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 1 35) in the ActRIIA section, at least 70%, 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical It may comprise, consist essentially of, or consist of a sequence of amino acids. is 21 to 30 of SEQ ID NO: 9 (e.g., amino acids 21, 22, 23, 24, 25, 26, Starting with any one of 27, 28, 29, or 30), 22-30 (e.g. , any of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30 starting with one of the amino acids), 23-30 (e.g., amino acids 23, 24, 25, 26, 27, Starting from any one of 28, 29, or 30), 24-30 (e.g., Amii Starting with one of the following acids: 24, 25, 26, 27, 28, 29, or 30 9), starting at a position selected from 111 to 135 (e.g., amino acid 11 1, 112, 113, 114, 115, 116, 117, 118, 119, 120, 12 1, 122, 123, 124, 125, 126, 127, 128, 129, 130, 13 (Ending in one of the following: 1, 132, 133, 134 or 135), 112-1 35 (e.g., amino acids 112, 113, 114, 115, 116, 117, 118, 1 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 1 Ending in one of 29, 130, 131, 132, 133, 134 or 135 ), 113-135 (e.g., amino acids 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, Any of 128, 129, 130, 131, 132, 133, 134 or 135 or ending in one), 120-135 (e.g., amino acids 120, 121, 122, 123 , 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , 134 or 135), 130-135 (e.g., Ami Ending in one of the following acids: 130, 131, 132, 133, 134 or 135 ), 111 to 134 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 13 15, 116, 117, 118, 119, 120, 121, 122, 123, 124, 1 25, 126, 127, 128, 129, 130, 131, 132, 133, or 13 4), 111 to 133 (e.g., 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 to 132 (e.g., Amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 1 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 1 29, 130, 131, or 132), or 111~ 131 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, (ending in one of the following: 127, 128, 129, 130, or 131) Variants within these ranges, particularly those ending at the corresponding positions of SEQ ID NO: 9, are also included. At least 70%, 75%, 80%, 85%, 86%, 87%, 88% of the ,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% , 99%, or 100% identity to an amino acid sequence of Variants that are or consist of are also contemplated. Thus, in some embodiments, Act The RIIA polypeptide has at least 70%, 75%, or 80% repeat sequence(s) between amino acids 30 and 110 of SEQ ID NO:9. 5%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical It may comprise, consist essentially of, or consist of a polypeptide. In certain embodiments, the ActRIIA polypeptide comprises at least one amino acid sequence selected from the group consisting of amino acids 30 to 110 of SEQ ID NO:9. Both are 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, and 91%. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical and have no more than 1, 2, 5, 10, or 15 conserved amino acids in the ligand-binding pocket The term "polypeptide" includes polypeptides containing complementary amino acid changes.

[0121] In certain embodiments, the present disclosure provides fragments, functional variants, and modified forms thereof. a GDF / BMP antagonist (inhibitor) comprising an ActRIIA polypeptide containing the agonist; and uses thereof (e.g., increasing an immune response in a patient in need thereof, and treating cancer). Preferably, the ActRIIA polypeptide is In some embodiments, the ActIa protein is soluble (e.g., the extracellular domain of ActRIIA). The RIIA polypeptide may be a polypeptide that binds to one or more GDF / BMP ligands [e.g., GDF1 1, GDF8, activin (activin A, activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP15, and / or BMP10] In some embodiments, the ActRIIA polypeptide inhibits (e.g., inhibits Smad signaling). The peptide may bind to one or more GDF / BMP ligands [e.g., GDF11, GDF8 , activin (activin A, activin B, activin AB, activin C, activin It binds to BMP6, GDF3, BMP15, and / or BMP10. In some embodiments, the ActRIIA polypeptide of the disclosure comprises amino acid 21 of SEQ ID NO:9. Starting with the residue corresponding to ~30 (e.g., amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 4 6, 27, 28, 29, or 30), and the amino acid sequence of SEQ ID NO: 9 Ending at a position corresponding to any one of amino acids 110 to 135 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, Ending in one of the following: 130, 131, 132, 133, 134, or 135 ) ActRIIA portion at least 70%, 75%, 80%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 98%, 99%, or 100% identical to or essentially identical to In some embodiments, the ActRIIA polypeptide is or consists of: At least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 11 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 contain or have an amino acid sequence that is 6%, 97%, 98%, 99%, or 100% identical to In certain embodiments, the ActRIIA polypeptide comprises an ActRIIA polypeptide. The polypeptide is a polypeptide that is at least 70%, 75%, 80%, or 90% of the amino acids 21 to 135 of SEQ ID NO: 9. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences In some embodiments, the method comprises: The RIIA polypeptide is any one of SEQ ID NOs: 9, 10, 11, 32, 36, and 39. The amino acid sequence of any one of the sequences has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, Containing, consisting of, or containing an amino acid sequence that is 98%, 99%, or 100% identical to Or from now on it will become essential.

[0122] In certain aspects, the present disclosure provides GDF trap polypeptides (referred to as "GDF traps"). In some embodiments, the GDF traps of the present disclosure relate to variant Act RII polypeptide has one or more mutations in comparison to the corresponding wild-type ActRII polypeptide. ActRII polypeptides (e.g., the extracellular domain (ligand binding domain) of the "wild-type" or unmodified ActRII polypeptide one or more mutations (e.g., amino acid additions, deletions) in the Variant ActRII polypeptides (e.g., ActRII polypeptides) containing ActRII deletions, substitutions, and combinations thereof In a preferred embodiment, the present disclosure The GDF trap polypeptides have at least one amino acid sequence similar to the corresponding wild-type ActRII polypeptide. For example, preferred GDF traps may be GDF11 and GDF2. and / or GDF8 and inhibit (e.g., antagonize) their function. In some embodiments, the GDF traps of the present disclosure may further comprise one or more of the ligands of GDF / BMP. Thus, the present disclosure provides a method for the preparation of one or more ActRII ligands. The present invention provides GDF trap polypeptides with altered binding specificity for GDF.

[0123] To illustrate, activin (activin A, activin B, activin AB, activin one or more activins, such as activin C, and / or activin E), in particular activin A Altered ligands of GDF11 and / or GDF8 for tRII-binding ligands One or more mutations may be selected that increase the selectivity of the dopamine-binding domain. Alternatively, the altered ligand-binding domain may have a relative affinity for the wild-type ligand-binding domain. At least 2, 5, 10, 20, 50, 100, or even 1000 times greater than the K for activin binding d of GDF11 and / or GDF8 binding d against Optionally, the altered ligand-binding domain has a ratio of wild-type ligand-binding At least 2, 5, 10, 20, 50, 100, or even 100 compared to the gender domain 0-fold greater IC for activin inhibition 50 of GDF11 and / or GDF8 inhibition C 50 Optionally, the altered ligand-binding domain has a ratio of IC of thiamine inhibition 50 At least 2, 5, 10, 20, 50, 100, or more 1000 times smaller IC 50 inhibits GDF11 and / or GDF8.

[0124] Amino acid residues of the ActRIIB protein (e.g., E39, K5 with respect to SEQ ID NO: 1) 5, Y60, K74, W78, L79, D80, and F101) are listed in the ActRIIB It resides in the Gd-binding pocket and binds, for example, activin A, GDF11, and GDF8. Thus, the present disclosure provides a method for the preparation of such a compound, which serves to mediate binding to its ligand, including the use of a compound of formula (I). Altered ligands of the ActRIIB receptor containing one or more mutations at amino acid residues GDF traps containing a GDF-binding domain (e.g., a GDF8 / GDF11 binding domain) The present invention provides a polypeptide.

[0125] As a specific example, the positively charged amino acid residue As in the ligand-binding domain of ActRIIB p(D80) was mutated to a different amino acid residue to preferentially bind GDF8 but not It is possible to produce GDF trap polypeptides that do not bind to activin. The D80 residue of SEQ ID NO: 1 is replaced by an uncharged amino acid residue, a negative amino acid residue, and a hydrophobic amino acid residue. In a further specific example, the amino acid residue is changed to an amino acid residue selected from the group consisting of , by altering the hydrophobic residue L79 of SEQ ID NO: 1 to form a modified activin-GDF11 / GD For example, the L79P substitution can confer F8 binding properties over activin binding. In contrast, replacing L79 with an acidic amino acid [asparagine] reduces GDF11 binding to a small extent. L79D or L79E substitution], Greatly reduces binding affinity but retains GDF11 binding affinity. In an exemplary embodiment, the methods described herein optionally include one or more additional at a position corresponding to position 79 of SEQ ID NO: 1 in combination with an amino acid substitution, addition, or deletion G, a variant ActRIIB polypeptide containing an acidic amino acid (e.g., D or E). DF trap polypeptides are utilized.

[0126] In certain aspects, the present disclosure relates to ALK4 polypeptides and uses thereof. The term "ALK4" as used herein refers to an activin receptor-like kinase (ALK4) from any species. ALK4 protein and such proteins by mutagenesis or other modifications References herein to ALK4 refer to a family of variants derived from the currently identified ALK4 gene. It is understood that the term "ALK4 family member" refers to any one of the above forms. They generally consist of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and It consists of a cytoplasmic domain with predicted serine / threonine kinase activity. It is a transmembrane protein.

[0127] The term "ALK4 polypeptide" refers to any naturally occurring member of the ALK4 family. In addition to the polypeptides described above, any variants (mutants, fragments) thereof that retain useful activity are also included. The present invention also includes polypeptides containing the polypeptides described herein, including ... The amino acid numbering of any ALK4-related polypeptides listed herein is the same as that of any ALK4-related polypeptide listed herein unless otherwise specified. If not, use the numbering based on the human ALK4 precursor protein sequence (SEQ ID NO: 100) below. Ku.

[0128] Human ALK4 precursor protein sequence (NCBI Ref Seq NP_004293 ) is as follows: [ka]

[0129] The signal peptide is indicated by a single underline and the extracellular domain is indicated by a bold font. Point out.

[0130] The processed extracellular human ALK4 polypeptide sequence is shown below: [ka]

[0131] The nucleic acid sequence encoding the ALK4 precursor protein is shown below (SEQ ID NO: 102): This is located at nucleotides 78 to 1592 of the Genbank reference sequence NM_004302.4. The signal sequence is underlined and the extracellular domain is indicated in bold font. [ka] [ka]

[0132] The nucleic acid sequence encoding the extracellular ALK4 polypeptide is shown below: [ka]

[0133] Isoform C ( NCBI Ref Seq NP_064732.3) is shown below: [ka] [ka]

[0134] The extracellular domain is indicated in bold font.

[0135] The processed extracellular ALK4 polypeptide sequence is as follows: [ka]

[0136] Genbank Reference Sequence NM_020327.3 ALK4 precursor protein (isoform) corresponding to nucleotides 186 to 1547 The nucleic acid sequence encoding the extracellular domain of IL-14 is shown below (SEQ ID NO: 106). Nucleotides that correspond to the nucleotide sequence are indicated in bold font. [ka]

[0137] The nucleic acid sequence encoding the extracellular ALK4 polypeptide (isoform B) is: is: [ka]

[0138] ALK4 is well conserved among vertebrates and contains a large section of the extracellular domain (s For example, Figure 18 shows the various ALK4 orthologs. 1 depicts a multiple sequence alignment of the human ALK4 extracellular domain compared to ALK. Many of the ligands that bind to 4 are also highly conserved. From the data, we identified a key site within the ligand-binding domain that is important for normal ALK4 ligand-binding activity. We predicted the amino acid positions that significantly alter normal ALK4 ligand binding activity. It is possible to predict amino acid positions that are likely to tolerate substitutions without Thus, active human ALK4 variant polypeptides useful in accordance with the presently disclosed methods contains one or more amino acids at corresponding positions from the sequence of another vertebrate ALK4. It may contain residues similar to those in human or other vertebrate sequences.

[0139] While not meant to be limiting, the following examples illustrate this approach to defining active ALK4 variants. As illustrated in Figure 18, the human ALK4 extracellular domain (SEQ ID NO: 12) 6) V6 is isoform in Mus musculus ALK4 (SEQ ID NO: 130). leucine, so the position may be varied, optionally with another amino acid such as L, I or F. or the hydrophobic residues of Gallus gallus ALK4 (SEQ ID NO: As observed in No. 129, it may be changed to a non-polar residue such as A. E40 in ALK4 is K in Gallus gallus ALK4, and this site , polar residues such as E, D, K, R, H, S, T, P, G, Y, and possibly non- This indicates that the protein can tolerate a wide variety of changes, including polar residues. S15 in Gallus gallus ALK4 is D, and the width at this position is A wide range of structural variations is tolerated, including polar species such as S, T, R, E, K, H, G, P, G, and Y. This indicates that E40 in the human extracellular domain is a Gallus In C. gallus ALK4, it is K, and in C. gallus ALK4, it is D, R, K, H, and charged amino acids containing Q and N. This indicates that a residue is allowed at this position. R80 in the human extracellular domain is C and K in Ondylura cristata ALK4 (SEQ ID NO: 127), and R, This indicates that basic residues, including K and H, are tolerated at this position. Y77 in the ALK4 gene is F in Sus scrofa ALK4 (SEQ ID NO: 131). This indicates that aromatic residues, including F, W, and Y, are tolerated at this position. The extracellular domain of P93 is relatively poorly conserved, and Erinaceu s europaeus ALK4 (SEQ ID NO: 128) and S and Gallus ga In llus ALK4, it appears as N, meaning that essentially any amino acid can be used at this position. Acids should also be acceptable.

[0140] Moreover, the ALK4 protein has many structural and functional characteristics, particularly with regard to ligand binding. The cases have been characterized in the art [e.g., Harrison et al. (2003) J Biol Chem, vol. 278 (issue 23): 21129-21135; Roman o et al. (2012) J Mol Model, vol. 18(no. 8): pp. 3617-3625; and Calvanese et al. (2009) Vol. 15(3): pp. 175-183]. In addition to the teachings in the specification, these references also include one or more normal activities (e.g., Guidance on how to generate ALK4 variants that retain ligand binding activity is provided. Provide enough.

[0141] For example, a distinct structural motif known as the three-finger toxin fold is Type I The extracellular domain of each monomeric receptor is important for ligand binding by the type II and type III receptors. It is formed by conserved cysteine ​​residues located at variable positions within the nwald et al. (1999) Nat Struct Biol 6:18-22; Yobi Hinck (2012) FEBS Lett, 586:1860-1870] Therefore, the human A region bounded by the outermost of these conserved cysteines The core ligand binding domain of ALK4 is 34-1 of SEQ ID NO: 100 (ALK4 precursor). The structure adjacent to the core sequence bounded by these cysteines corresponds to position 01. Less ordered amino acids can be added to the N-terminus without necessarily altering ligand binding. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33 residues, and / or 1, 2, 3, 4, 5, 6 at the C-terminus ,7,8,9,10,11,12,13,14,15,16,17,18,19,20, The N-terminus and / or Exemplary ALK4 extracellular domains for C-terminal truncations are SEQ ID NOs: 101 and 102. Including 05.

[0142] Thus, the general formula for an active portion (e.g., ligand-binding portion) of ALK4 is: With respect to SEQ ID NO: 100, it contains amino acids 34 to 101. Thus, the ALK4 polypeptide The code may be, for example, a residue corresponding to any one of amino acids 24 to 34 of SEQ ID NO: 100. Starting from base (e.g., amino acids 24, 25, 26, 27, 28, 29, 30, 31, 3 2, 33, or 34), and amino acid 1 of SEQ ID NO: 100 Ending at a position corresponding to any one of amino acids 01 to 126 (e.g., amino acid 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, (Ending in one of 122, 123, 124, 125, or 126) ALK 4 parts, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9%, or 100% identical amino acid sequence. In another example, the amino acid sequence may be 24 to 34 of SEQ ID NO: 100 (e.g., 2 4th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, or 34th place Any one), 25-34 (e.g., 25, 26, 27, 28, 29, 30, 31, 32 , 33, or 34), or 26-34 (e.g., 26, 27 , 28th, 29th, 30th, 31st, 32nd, 33rd, or 34th place) That is, 101 to 126 of SEQ ID NO: 100 (for example, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 05, 106, 107, 108, 109, 110, 111, 112, 113, 114, 1 15, 116, 117, 118, 119, 120, 121, 122, 123, 124, 1 25, or 126), 102 to 126 (e.g., 102, 10 3, 104, 105, 106, 107, 108, 109, 110, 111, 112, 11 3, 114, 115, 116, 117, 118, 119, 120, 121, 122, 12 3, 124, 125, or 126), 101 to 125 (e.g. , 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 , 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 , 121, 122, 123, 124, or 125th place), 101~ 124 (e.g., 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119th, 120th, 121st, 122nd, 123rd, or 124th place), 1 01 to 121 (e.g., 101, 102, 103, 104, 105, 106, 107, 1 08, 109, 110, 111, 112, 113, 114, 115, 116, 117, 1 18, 119, 120, or 121), 111-126 (e.g. 111, 112, 113, 114, 115, 116, 117, 118, 119, 12 (One of the following: 0, 121, 122, 123, 124, 125, or 126th place) , 111-125 (e.g., 111, 112, 113, 114, 115, 116, 117 , 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, or 125th place Any one), 111 to 124 (e.g., 111, 112, 113, 114, 115, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, or 124th place Any one of these), 121-126 (for example, 121, 122, 123, 124, 1 25, or 126), 121-125 (e.g., 121, 12 2, 123, 124, or 125), 121-124 (e.g. , 121, 122, 123, or 124), or 124 to 1 Constructs ending at position 26 (e.g., any one of positions 124, 125, or 126) Variants within these ranges, particularly to the corresponding portions of SEQ ID NO: 100, include: At least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or Variants with 100% identity are also contemplated.

[0143] The variants described herein can be combined in various ways. In this embodiment, the ALK4 variants are 1, 2, 5, 6, 7, 8, and 9 in the ligand-binding pocket. Contains no more than 9, 10, or 15 conservative amino acid changes. Variability is particularly well tolerated. The sites outside the binding pocket are the amino and carboxyl groups of the extracellular domain (as described above). Contains carboxyl terminus.

[0144] In certain embodiments, the present disclosure provides fragments, functional variants, and modified forms thereof. BMP / G is a heteromultimer containing at least one ALK4 polypeptide, including DF antagonists and uses thereof (e.g., for the treatment of PAH or one or more of (treating, preventing, or reducing the severity of complications). In some cases, the ALK4 polypeptide is soluble (e.g., the extracellular domain of ALK4). In some embodiments, the heteromultimer comprising an ALK4 polypeptide is a heteromultimer of one or more TGF- β superfamily ligands [e.g., GDF11, GDF8, activin (activin Activin A, Activin B, Activin AB, Activin C, Activin E), BMP6, GD F3, BMP10, and / or BMP9 (e.g., Smad signaling) In some embodiments, heteromultimers comprising one or more ALK4 polypeptides may be used. Several TGFβ superfamily ligands [e.g., GDF11, GDF8, activin (Activin A, Activin B, Activin AB, Activin C, Activin E), BM In some embodiments, the IL-16 receptor binds to a IL-16 receptor, ... The heteromultimer has at least 70%, 75%, or 80% amino acids 34 to 101 of SEQ ID NO: 100. 5%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 At least one that is 3%, 94%, 95%, 97%, 98%, 99%, or 100% identical In some embodiments, the heteromultimer comprises an ALK4 polypeptide. 101, 104, 105, 111, 113, 116, 117, 122, and 124 The amino acid sequence has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or at least one ALK4 polypeptide that is 100% identical. In this embodiment, the heteromultimer comprises SEQ ID NOs: 100, 101, 104, 105, 111, 113, 116, 117, 122, and 124 amino acid sequences, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, At least one that is 94%, 95%, 97%, 98%, 99%, or 100% identical at least one ALK consisting of or consisting essentially of an ALK4 polypeptide Contains 4 polypeptides.

[0145] In certain aspects, the present disclosure provides methods for the use thereof (e.g., for the prevention of immune deficiency in patients in need thereof). and treating cancer. 4 receptor polypeptides (e.g., SEQ ID NOs: 100, 101, 104, 105, 111, 1 13, 116, 117, 122, and 124 and variants thereof), and one or more or multiple ActRIIB receptor polypeptides (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 58, 59, 60, 63, 64, 65, 66, 68, 69, 70, 71, 73, 77 , 78, 108, 110, 114, 115, 118, and 120 and modifications thereof. The present invention relates to heteromultimeric complexes containing ALK4 :ActRIIB heteromultimer complex" or "ALK4:ActRIIB heteromultimer complex" Preferably, the ALK4:ActRIIB heteromultimer is soluble. For example, heteromultimeric complexes may be formed by combining the soluble portion (domain) of the ALK4 receptor and Ac soluble portion (domain) of the tRIIB receptor. Generally, ALK4 and Act The extracellular domain of RIIB corresponds to the soluble portion of these receptors. In some embodiments, the ALK4:ActRIIB heteromultimer is an extracellular In some embodiments, the A domain and the extracellular domain of the ActRIIB receptor. The LK4:ActRIIB heteromultimer binds one or more of the TGFβ superfamily members. Ligands [e.g., GDF11, GDF8, activin (activin A, activin B, Activin AB, Activin C, Activin E), BMP6, GDF3, BMP10, and and / or BMP9] (e.g., Smad signaling). In this study, ALK4:ActRIIB heteromultimers were identified as a fusion protein of one or more TGFβ superfamily members. Family ligands [e.g., GDF11, GDF8, activin (activin A, activin activin B, activin AB, activin C, activin E), BMP6, GDF3, BMP 10, and / or BMP9. IIB heteromultimers are identified by SEQ ID NOs: 100, 101, 104, 105, 111, 113, 1 The amino acid sequences of 16, 117, 122, and 124 were at least 70%, 75%, and 8 0%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 Contains or contains sequences that are 4%, 95%, 97%, 98%, 99%, or 100% identical to comprising at least one ALK4 polypeptide consisting essentially of, or consisting of, In some embodiments, the ALK4:ActRIIB heteromultimeric complex of the present disclosure has the sequence Any one of amino acids 24-34, 25-34, or 26-34 of number 100 Starting from residues corresponding to 101 to 126, 102 to 126, 101 of SEQ ID NO: 100 ~125, 101~124, 101~121, 111~126, 111~125, 111 ~124th, 121st to 126th, 121st to 125th, 121st to 124th, or 124th to 126th The portion of ALK4 ending in ALK4 is at least 70%, 75%, 80%, 85%, 86%, 87% %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98 %, 99%, or 100% identical to, or consisting essentially of, a sequence In some embodiments, the ALK4 polypeptide comprises at least one ALK4 polypeptide consisting of: The ActRIIB heteromultimer comprises at least one amino acid sequence selected from amino acids 34 to 101 of SEQ ID NO: 100. 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical At least one ALK4 polypeptide comprising, consisting essentially of, or consisting of the sequence In some embodiments, the ALK4-ActRIIB heteromultimer comprises a peptide of SEQ ID NO: No. 1, 2, 3, 4, 5, 6, 58, 59, 60, 63, 64, 65, 66, 68, 69, 70, 71, 73, 77, 78, 108, 110, 114, 115, 118, and 12 0, or %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 97%, 98%, 99%, or 100% identical to or substantially identical to and at least one ActRIIB polypeptide consisting of or consisting of: In embodiments, the ALK4:ActRIIB heteromultimer complex of the present disclosure has the structure of SEQ ID NO:1. Of amino acids 20-29, 20-24, 21-24, 22-25, or 21-29 Starting from a residue corresponding to any one of 109 to 134, 119 to 134 of SEQ ID NO: 1 , 119-133, 129-134, or 129-133 of ActRIIB Parts include at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, and 89% , 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or at least one sequence that contains, consists essentially of, or consists of a sequence that is 100% identical to In some embodiments, the ALK4:ActRII polypeptide comprises two ActRIIB polypeptides. The B heteromultimer has at least 70%, 75%, or 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or contain sequences that are 94%, 95%, 97%, 98%, 99%, or 100% identical to the sequence and at least one ActRIIB polypeptide consisting essentially of or consisting of: In some embodiments, the ALK4:ActRIIB heteromultimer comprises the amino acid sequence of SEQ ID NO:1. At least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 11 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 9 9%, or 100% identical to a sequence In certain embodiments, the present disclosure provides a method for the treatment of a cancer, comprising administering to a patient a therapeutically effective amount of at least one ActRIIB polypeptide. The ALK4:ActRIIB heteromultimer complex of this invention is located at the position corresponding to L79 in SEQ ID NO:1. The position is not an acidic amino acid (i.e., a naturally occurring D or E amino acid residue, or at least one ActRIIB polypeptide (which is not an artificial acidic amino acid residue) The ALK4:ActRIIB heteromultimers of the present disclosure include, for example, heterodimers, heterodimers, This includes trimers, heterotetramers, and higher oligomeric structures. See, e.g., Figures 21-23 In certain preferred embodiments, the heteromultimeric complex of the present disclosure comprises: It is an ALK4:ActRIIB heterodimer.

[0146] In some embodiments, the present disclosure provides therapeutic efficacy or stability (e.g., shelf life and efficacy). ActRI is used for purposes such as enhancing resistance to proteolysis in vivo. Creating functional variants by modifying the structure of I and / or ALK4 polypeptides Variants may be produced by amino acid substitution, deletion, addition, or a combination thereof. For example, the substitution of leucine with isoleucine or valine for glutamic acid can be achieved. Isolated replacement of aspartic acid by phosphate, of threonine by serine, or Similar substitutions of amino acids with structurally related amino acids (e.g., conservative mutations) It is reasonable to expect that the resulting Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether or not a change in the amino acid sequence of a polypeptide of the present disclosure results in a functional homologue is not known. The variant polypeptides produce a response in cells in a manner similar to the wild-type polypeptide. Ability, or for example, BMP2, BMP2 / 7, BMP3, BMP4, BMP4 / 7, B MP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF 3, GDF5, GDF6 / BMP13, GDF7, GDF8, GDF9b / BMP15, GDF11 / BMP11, GDF15 / MIC1, TGF-β1, TGF-β2, TGF -β3, activin A, activin B, activin C, activin E, activin AB, Activin AC, nodal, glial cell line-derived neurotrophic factor (GDNF), Neurturin one or more TGF-β receptors, including α-glucan, ... - This can be easily determined by assessing the ability of the antibody to bind to a beta-ligand.

[0147] In certain embodiments, the present disclosure provides a method for modifying the glycosylation of a polypeptide. Consider specific mutations in ActRII and / or ALK4 polypeptides that may be involved in the treatment of cancer. Such mutations may involve one or more mutations, such as O-linked or N-linked glycosylation sites. The glycosylation sites of asparagi can be introduced or eliminated. Amino-linked glycosylation recognition sites are generally specifically linked to glycosylation sites by appropriate cellular glycosylation enzymes. The recognized tripeptide sequence is asparagine-X-threonine or asparagine-X- The alteration includes a serine (where "X" is any amino acid). by the addition of or substitution by one or more serine or threonine residues in the sequence. It can also be made by adding a glycosylation recognition site (for O-linked glycosylation sites). Various amino acid substitutions or deletions at either or both of the first or third amino acid positions (and / or an amino acid deletion at the second position) to form a modified tripeptide sequence. Another method for increasing the number of carbohydrate moieties in a polypeptide is to by chemical or enzymatic coupling of glycosides to the polypeptide. Depending on the coupling mode used, the sugar(s) may be: (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as in cysteine; (d ) a free hydroxyl group, such as in serine, threonine, or hydroxyproline; (e) an aromatic residue, such as in phenylalanine, tyrosine, or tryptophan; or (f) It can be attached to the amide group of glutamine. Removal of some carbohydrate moieties can be accomplished chemically and / or enzymatically. Chemical deglycosylation can be carried out using, for example, the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment may involve exposing the polypeptide to a compound that leaves the amino acid sequence intact. The linking sugar (N-acetylglucosamine or N-acetylgalactosamine) is removed while Enzymes that cleave most or all of the sugars from carbohydrate moieties on polypeptides. The cleavage by α-tocopherol was reported by Thotakura et al. [Meth. Enzymol. (1987) 1 38:350] of various endo- and exoglycosidases. Mammalian, yeast, insect and plant cells can all be used to It is possible to introduce different glycosylation patterns that can be influenced by the amino acid sequence of the peptide. Therefore, the sequence of the polypeptide may be adjusted, if necessary, depending on the type of expression system used. Generally, polypeptides of the present disclosure for use in humans can be expressed in HEK In mammalian cell lines that provide proper glycosylation, such as 293 or CHO cell lines Although it is possible to express the ribosomal protein in mammalian cells, other mammalian expression cell lines are expected to be useful as well. .

[0148] The present disclosure provides variants, particularly combinatorial variants of ActRII and / or ALK4 polypeptides. Further considerations are given to methods for generating sets of combinatorial mutants as well as truncation mutants. The pool of natorial mutants is functionally active (e.g., GDF / BMP ligand binding) Such combinatorial libraries are particularly useful for identifying ActRII sequences. The purpose of screening for ligands is to identify, for example, altered pharmacokinetics or altered ligand binding. The objective of this invention may be to generate polypeptide variants with altered properties, such as modified bond binding. Various screening assays are provided below, and variants can be evaluated using such assays. For example, ActRII and / or ALK4 variants, and Heteromultimers containing one or more GDF / BMP ligands (e.g., BMP2, BM P2 / 7, BMP3, BMP4, BMP4 / 7, BMP5, BMP6, BMP7, BMP 8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6 / BMP13 , GDF7, GDF8, GDF9b / BMP15, GDF11 / BMP11, GDF15 / MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, activin B , activin AB, activin AC, nodal, glial cell line-derived neurotrophic factor (GDN F), neurturin, artemin, persephin, MIS, and lefty) ActRII and / or ALK4 polypeptides and heteropolymers thereof Ability to prevent binding of GDF / BMP ligands to the body and / or GDF / BMP can be screened for their ability to interfere with ligand-induced signaling. do.

[0149] ActRII polypeptide, ALK4 polypeptide, and ALK4:ActRIIB The activity of the heterodimers can be examined in cell-based or in vivo assays. For example, the role of the ActRII polypeptide in the expression of genes involved in the pathogenesis of PH can be investigated. The effects of ALK4 ActRIIB, ALK4 polypeptide, or ALK4:ActRIIB heterodimer were evaluated. This can optionally be achieved by one or more recombinant ligand proteins (e.g., B MP2, BMP2 / 7, BMP3, BMP4, BMP4 / 7, BMP5, BMP6, BM P7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6 / BMP13, GDF7, GDF8, GDF9b / BMP15, GDF11 / BMP11 , GDF15 / MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, Activin B, Activin C, Activin E, Activin AB, Activin AC, Nod al, glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin, and parsley. The assay may be performed in the presence of ActRII polypeptides (Fin, MIS, and Lefty). an ALK4 polypeptide, an ALK4:ActRIIB heterodimer, and any Optionally, cells may be transfected to produce GDF / BMP ligands. Similarly, an ActRII polypeptide, an ALK4 polypeptide, or an ALK4:ActR IIB heterodimers may be administered to mice or other animals to assess their effect on PH pathogenesis. The activity of the ActRII polypeptide may be assessed using art-recognized methods. an ALK4 polypeptide, an ALK4:ActRIIB heterodimer, or The activity of the variants in blood cell progenitor cells was assessed for any effect on the growth of these cells. For example, by the assays described herein and assays well known in the art. SMAD-responsive reporter genes may be used in such cell lines. to monitor the effect on downstream signal transduction.

[0150] Reference ActRII polypeptide, ALK4 polypeptide, or ALK4:ActRI Combinations with increased selectivity or overall increased potency compared to IB heterodimers. Nutritionally derived variants can be generated. Such variants can be used in recombinant DNA constructs. When expressed from a gene, it can be used in gene therapy protocols. Mutagenesis may be performed on the corresponding unmodified ActRII polypeptide, ALK4 polypeptide, or is a variant with a dramatically different intracellular half-life than the ALK4:ActRIIB heterodimer For example, the modified protein may be a protein of the unmodified polypeptide. by proteolysis or other cellular processes that result in destruction or otherwise inactivation. Such variants and their analogs can be made either more stable or less stable. The half-life of a polypeptide can be modulated by utilizing the gene encoding it. The level of the peptide complex can be altered. For example, a shorter half-life may result in a more transient It is capable of producing a biological effect and, when part of an inducible expression system, of expressing the recombinant polypeptide in cells. This allows for tighter control of the peptide complex levels. Mutations may be made in the linker (if any) and / or the Fc portion to produce A ActRII polypeptide, ALK4 polypeptide, or ALK4:ActRIIB polypeptide The half-life of the dimer can be altered.

[0151] The combinatorial library contains potential ActRII polypeptides, ALK 4 polypeptide, or at least a portion of the ALK4:ActRIIB heterodimer sequence. a degenerate library of genes encoding a library of polypeptides comprising For example, a potential ActRII- and / or ALK4-encoding nucleotide sequence can be A degenerate set of nucleotide sequences can be synthesized as individual polypeptides or alternatively as larger fusion polypeptides. so that they can be expressed as a set of proteins (e.g., for phage display), A mixture of synthetic oligonucleotides is enzymatically ligated into the gene sequence. can be done.

[0152] A library of potential homologs can be generated from degenerate oligonucleotide sequences. There are many ways to synthesize degenerate gene sequences. Chemical synthesis can be performed in an automated DNA synthesizer. The synthetic gene can then be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is well known in the art. , SA (1983) Tetrahedron 39:3; Itakura et al. (19 81) Recombinant DNA, Proc. 3rd Clevel and S ympos.Macromolecules, edited by A. G. Walton, Amsterdam m: Elsevier, pp. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Sc ience 198:1056 and Ike et al. (1983) Nucleic Acids cid Res. 11:477]. Such techniques have been used in directed evolution of other proteins ( It has been used in directed evolution [Scott et al. (1 990) 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. Pat. No. 5,223,409, Ibid. Nos. 5,198,346 and 5,096,815].

[0153] Alternatively, other forms of mutagenesis are used to generate combinatorial libraries. For example, alanine scanning mutagenesis [Ruf et al. (1994) Biol. Chemistry 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. (199 3) J. Biol. Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085 p.], 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) Sci ence 232:316], saturation mutagenesis [Meyers et al. (1986) Sci ence 232:613]; PCR mutagenesis [Leung et al. (1989) Met hod Cell Mol Biol 1:11-19]; or chemical mutagenesis Random mutagenesis including [Miller et al. (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Sp Ring Harbor, NY; and Greener et al. (1994) Strategy ies in Mol Biol 7:32-34] Thus, for example, the ActRII polypeptide, ALK4 polypeptide, and ALK4:ActRIIB heterodimeric complexes can be generated and isolated from the library. Linker scanning mutagenesis, especially in a combinatorial setting, has been shown to increase the number of cleavage sites (biological activity). ActRII polypeptide, ALK4 polypeptide or ALK4:ActRI This is an attractive method for identifying IB heterodimers.

[0154] The gene products of the combinatorial library generated by point mutation and truncation were screened. In addition, to screen for gene products with certain specific properties. A wide range of techniques for screening cDNA libraries are known in the art. Such techniques are generally used for combinatorial mutagenesis of ActRII polypeptides. It will be adaptable for rapid screening of gene libraries generated by the method. The most widely used technique for screening gene libraries is the typical Next, clone the gene library into a replicable expression vector. and transducing the appropriate cells with the resulting vector library. The steps of transforming and detecting the desired activity are performed by using a vector encoding the gene whose product is detected. The combinatorial genes are expressed under conditions that facilitate the relatively easy isolation of the vectors. A preferred assay involves measuring the activity of a ligand (e.g., BMP2, BMP2 / 7) , BMP3, BMP4, BMP4 / 7, BMP5, BMP6, BMP7, BMP8a, B MP8b, BMP9, BMP10, GDF3, GDF5, GDF6 / BMP13, GDF 7, GDF8, GDF9b / BMP15, GDF11 / BMP11, GDF15 / MIC 1, TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin activin C, activin E, activin AB, activin AC, nodal, glial cell-derived Neurotrophic factors (GDNF), neurturin, artemin, persephin, MIS and Lefty (lefty) binding assays and / or ligand-mediated cell signaling assays nothing.

[0155] As will be appreciated by those skilled in the art, the mutations, variants, Most of the modifications are at the nucleic acid level, or in some cases post-translational or chemical modifications. Such techniques are well known in the art, some of which are In part, this disclosure is directed to other methods within the scope of the inventions described herein. Use as guidance for producing and using variant ActRII polypeptides ActRII polypeptide, ALK4 polypeptide, or ALK4:Ac Functionally active portions (fragments) and variants of the tRIIB heterodimer are identified.

[0156] In certain embodiments, the ActRII polypeptide, ALK4 polypeptide of the present disclosure , and functionally active fragments of the ALK4:ActRIIB heterodimer are ActR II and / or a corresponding fragment of a nucleic acid encoding an ALK4 polypeptide The recombinantly produced polypeptide can be obtained by screening. In addition, the fragments can be synthesized using conventional Merrifield solid phase f-Moc or t-Boc cleavage. The fragments can be synthesized chemically using techniques known in the art, such as chemistry. and producing (recombinantly or chemically synthesized) and testing ActRII and / or or ALK4 receptor and / or one or more ligands (e.g., BMP2, BMP2 / 7, BMP3, BMP4, BMP4 / 7, BMP5, BMP6, BMP7, B MP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6 / BMP 13, GDF7, GDF8, GDF9b / BMP15, GDF11 / BMP11, GDF 15 / MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, activin Activin B, activin C, activin E, activin AB, activin AC, nodal, Glomerular cell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, It can function as an antagonist (inhibitor) of MIS and lefty These peptidyl fragments can be identified.

[0157] In certain embodiments, the ActRII polypeptide, ALK4 polypeptide of the present disclosure and / or the ALK4:ActRIIB heterodimer is an ActRII polypeptide , ALK4 polypeptide, or ALK4:ActRIIB heterodimer In addition to any post-translational modifications present, further post-translational modifications may be included. These include acetylation, carboxylation, glycosylation, phosphorylation, and lipidation. ion), and acylation. II polypeptide, ALK4 polypeptide, or ALK4:ActRIIB heterodimer The body contains a variety of compounds, such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The ligand trap polypeptide may contain non-amino acid elements. The effect of ALK4 on the functionality of ActRII, AKL4, and ALK4:ActR IIB variants can be tested as described herein. When a peptide is produced intracellularly by cleaving a nascent form of a polypeptide, translation occurs. Post-translational processing may also be important for the correct folding and / or function of proteins. Various cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH -3T3 or HEK293) specific cellular machinery for such post-translational activity (c ellular machinery and characteristic mechanisms ic mechanism) that allows for precise modification and proteolysis of the ActRII polypeptide. The frequency may be selected to ensure uniformity.

[0158] In certain aspects, the ActRII and ALK4 polypeptides of the present disclosure are ActR II or ALK4 polypeptide and one or more This includes fusion proteins with heterologous moieties (domains). Examples include 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. Fusion domains can be selected to confer desired properties, including, but not limited to: For example, some fusion domains allow for the isolation of fusion proteins by affinity chromatography. For the purposes of affinity purification, glutathione is particularly useful for the isolation of proteins. , amylase, and nickel or cobalt conjugated resins. A relevant matrix for affinity chromatography is used. Many of the TRICKs were purified using the Pharmacia GST purification system and (HIS6) fusion proteins. systems such as the QIAexpress™ system (Qiagen), which is useful with toners In another example, the fusion domain may be ActRII. Alternatively, the detection domain may be selected to facilitate detection of the ALK4 polypeptide. Main examples include various fluorescent proteins (e.g., GFP), as well as specific antibodies. Examples of suitable epitope tags include those that are available, which are usually short peptide sequences. Well-known epitope tags that are readily available for use with monoclonal antibodies include FLAG, Some of these include the influenza virus hemagglutinin (HA) and c-myc tags. In this case, the fusion domain allows the associated protease to partially digest the fusion protein and Factor Xa or It has a protease cleavage site, such as that for thrombin. is then isolated from the fusion domain by subsequent chromatographic separation. Other types of fusion domains that can be selected include multimerizing (e.g., dimerizing) domains. , tetramerization) domains, and constant domains, e.g., from immunoglobulins (e.g., Fc domains) that confer additional biological functions.

[0159] In certain embodiments, the ActRII and ALK4 polypeptides of the present disclosure are polypeptides The compound contains one or more modifications that can "stabilize" the peptide. This may be due to reduced drug destruction, reduced renal clearance, or other pharmacokinetic effects. Increases in vitro and serum half-lives, whether or not this is due to For example, such modifications may enhance the shelf life of the polypeptide. enhance the circulating half-life of the polypeptide and / or enhance the protein content of the polypeptide. Such stabilizing modifications include fusion proteins (e.g., ActRII polypeptides) that are polypeptide (or ALK4 polypeptide) domain and a stabilizer domain. fusion proteins containing the polypeptides of the present disclosure), modifications of glycosylation sites (e.g., glycosylation sites), and modifications of carbohydrate moieties (e.g., the polynucleotides of the present disclosure) These include, but are not limited to, removal of carbohydrate moieties from peptides. The term "stabilizer domain" as used herein refers to a fusion protein as in the case of a fusion protein. It refers not only to the binding domain (e.g., immunoglobulin Fc domain) but also to the carbohydrate moiety or non-proteinaceous moieties such as polyethylene glycol. In certain preferred embodiments, an ActRII polypeptide (or an ALK4 polypeptide) peptide) with a heterologous domain that stabilizes the polypeptide (a "stabilizer" domain) ), preferably fused with a heterologous domain that increases the stability of the polypeptide in vivo. Fusions with immunoglobulin constant domains (e.g., Fc domains) can be used to bind to a wide range of It is known that these compounds confer desirable pharmacokinetic properties to proteins. Fusion with human serum albumin can also confer desirable properties.

[0160] Examples of naturally occurring amino acid sequences that can be used for the Fc portion of human IgG1 (G1Fc) The sequence is shown below (SEQ ID NO: 14). The dotted underline indicates the hinge region, and the solid underline indicates the In part, the present disclosure refers to positions with naturally occurring variants relative to SEQ ID NO: 14. ,70%,75%,80%,85%,86%,87%,88%,89%,90%,91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% A polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence having the same identity. Naturally occurring variants of G1Fc are provided as peptides with the numbers used in SEQ ID NO: 14. According to the Uniprot coding system, it would contain E134D and M136L ( See P01857). [ka]

[0161] Optionally, the IgG1 Fc domain comprises Asp-265, lysine 322, and As In certain cases, these mutations Mutant IgG1 Fc domains with one or more of the following (e.g., Asp-265 mutations) The Fc domain has reduced binding ability to Fcγ receptors compared to the wild-type Fc domain. In some cases, patients with one or more of these mutations (e.g., Asn-434 mutation) The mutant Fc domains exhibited enhanced MHC class I association compared to the wild-type IgG1 Fc domain. It has increased binding ability to Fc receptors (FcRN).

[0162] Examples of naturally occurring amino acid sequences that can be used for the Fc portion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 15). The dotted underline indicates the hinge region and the double underline indicates the Indicates where in the sequence there is a database conflict (Uniprot P018 59). In part, the present disclosure provides 70%, 75%, 80% ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% , 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity The present invention provides polypeptides comprising, consisting essentially of, or consisting of a sequence. [ka] [ka]

[0163] Two examples of amino acid sequences that can be used for the Fc portion of human IgG3 (G3Fc) The hinge region in G3Fc is the largest of the hinge regions in other Fc chains. It can be four times as long and consists of three identical 15-residue segments preceded by a similar 17-residue segment. The first G3Fc sequence (SEQ ID NO: 16) shown below contains a single base segment. The second G3Fc sequence (sequence Row 17) contains the full length hinge region. In each case, the dotted underline indicates the hinge region. The solid underline indicates the naturally occurring modification according to UniProt P01859. In part, the present disclosure provides SEQ ID NOS: 16 and 17 with respect to 7 0%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical A polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence having the Offer petites. [ka]

[0164] Naturally occurring variants in G3Fc (see, e.g., Uniprot P01860) (reference) when converted to the numbering system used in SEQ ID NO: 16, becomes E68Q, P76 L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169d el, F221Y, and the present disclosure provides G3 containing one or more of these variants. The present invention provides a fusion protein containing an Fc domain. The gene (IGHG3) exhibits structural polymorphism characterized by different hinge lengths [Uniprot In particular, variant WIS contains most of the V region and the entire CH1 region. This is because it has an extra interchain dinucleotide at position 7 in addition to position 11, which is normally present in the hinge region. The variant ZUC has most of the V region, all of the CH1 region, and It lacks a portion of the hinge. The variant OMM may represent an allelic form or a different gamma chain subclass. The present disclosure provides G3Fc domains containing one or more of these variants. Additional fusion proteins containing the domain are provided.

[0165] Examples of naturally occurring amino acid sequences that can be used for the Fc portion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 18). The dotted underline indicates the hinge region. 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Containing or consisting essentially of an amino acid sequence with 99% or 100% identity or a polypeptide consisting thereof. [ka]

[0166] Various engineered mutations in the Fc domain were compared with the G1Fc sequence (SEQ ID NO: 14). and similar mutations in G2Fc, G3Fc, and G4Fc are presented herein. can be derived from their alignment with G1Fc in Figure 4. Because of the hinge length, similar Fc positions based on isotype alignment (Figure 4) are found in the sequence Numbers 14, 15, 16, 17, and 18 have different numbers of amino acids. Injury, the entire IgG1 heavy chain constant domain (C) as well as the Uniprot database H 1. Hi Nji, C H 2 and C H 3 regions), hinge, C H 2 and C H 3 Immunoglobulin sequences consisting of regions (e.g., SEQ ID NOs: 14, 15, 16, 17, and 1 8), a given amino acid position in the For example, the human G1Fc sequence (SEQ ID NO: 14), the human IgG1 complex Chain constant domain (Uniprot P01857) and selection in human IgG1 heavy chains C H The correspondence between the three positions is as follows: [Table 1]

[0167] In certain aspects, the polypeptides disclosed herein comprise at least one Act at least one AL covalently or noncovalently associated with the RIIB polypeptide; A protein complex containing the K4 polypeptide can be formed. The polypeptides disclosed in the document form heterodimeric complexes, but not heterotrimers, heterotrimers, or heterodimers. tetramers, and further oligomeric structures (see, e.g., Figures 21-23), etc. Higher order heteromultimeric complexes (heteromultimers) include, but are not limited to, In some embodiments, ALK4 and / or ActRIIB polypeptides are also included. As disclosed herein, the term "multimer" refers to a multimer that is a polypeptide chain consisting of a polypeptide chain, ... The "merization domain" comprises at least a first polypeptide and at least a second polypeptide. This refers to an amino acid or sequence of amino acids that promotes covalent or non-covalent interactions between amino acids. The polypeptides disclosed herein may be covalently or non-covalently linked to a multimerization domain. Preferably, the multimerization domain is connected to the first polypeptide (e.g., a second polypeptide (e.g., an ActRIIB polypeptide) and a third polypeptide (e.g., an ALK4 polypeptide) promotes interactions between the peptides to form heteromultimers (e.g., heterodimers) promotes and optionally prevents homomultimerization (e.g., homodimer formation) or other processes. This disfavors the desired heteromultimer (see, e.g., Figure 22). Increase the amount.

[0168] Many methods known in the art can be used to produce ALK4:ActRIIB heteromultimers. For example, disulfide bonds can be formed between the first and second polypeptides. As described above, in the first polypeptide (e.g., the ALK4 polypeptide), The ol is a separate release agonist in a second polypeptide (e.g., an ActRIIB polypeptide). with a free thiol-containing residue, such as cysteine, to interact with thiol-containing residues. Non-naturally occurring disulfide bonds by replacing naturally occurring amino acids As an additional example of an interaction that promotes heteromultimer formation, For example, ions such as those described in Kjaergaard et al., WO2007147901 sexual interactions; static interactions such as those described in Kannan et al., US Pat. No. 8,592,562 Electrostatic steering effect; Christ coiled coils such as those described in Ensen et al., US20120302737 Interactions; Pack and Plueckthun (1992) Biochemist 31:1579-1584; and P ack et al. (1993) Bio / Technology 11:1271-1277 These include helix-turn-helix motifs such as those described in For example, chemical crosslinking, peptide linkers, disulfide crosslinking, etc. By covalent attachment or by avidin-biotin or leucine zipper technology, etc. Affinity interactions can result in the linking of various segments.

[0169] In certain aspects, the multimerization domain may comprise one member of an interaction pair. In some embodiments, the polypeptides disclosed herein are administered in combination with a second polypeptide. forming a protein complex containing the first polypeptide covalently or noncovalently associated therewith; the first polypeptide can be made up of an amino acid sequence of an ALK4 polypeptide and the second polypeptide comprises the amino acid sequence of an ActR member of an interacting pair; The amino acid sequence of the IIB polypeptide and the amino acid sequence of the second member of the interacting pair The interaction pair interacts to form a complex, particularly a heterodimeric complex. Although it may be any two polypeptide sequences, a working embodiment is a homodimer. It is also possible to use an interaction pair that can form a complex. A member of the present invention can be selected from, for example, any of SEQ ID NOs: 2, 3, 5, 6, 101, and 103. or one sequence, at least 70%, 75%, 80%, 85%, 86%, 87%, 88% ,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% , 99%, or 100% identical to, or consisting essentially of, an amino acid sequence or a polypeptide sequence consisting of the ALK4 or Ac The interacting pair may be fused to a tRIIB polypeptide to provide a therapeutic effect such as increased serum half-life. to impart improved properties / activity of, or to provide improved properties / activity of, another The portion may be selected to act as an adapter to which the portion is attached. Improved cleavage can be achieved by attaching a phenyl glycol moiety to one or both members of the interaction pair. These compounds may result in improved properties / activity such as increased serum half-life.

[0170] The first and second members of the interacting pair can be an asymmetric pair, which means that This means that members of a pair preferentially associate with each other rather than self-associate. Thus, the first and second members of the asymmetric interaction pair associate to form a heterodimer (See, e.g., Figure 22). Alternatively, the interaction A pair may be unguided, meaning that members of the pair may choose to They may associate with each other or with themselves, and thus have the same or different amino acid sequences. Therefore, the first and second unguided interaction pairs The two members associate to form homodimeric or heterodimeric complexes Optionally, an interaction pair (e.g., an asymmetric pair or an unguided pair) can be The first member of an interaction pair is covalently associated with the second member of the interaction pair. Optionally, interacting pairs (e.g., asymmetric or unguided interacting pairs) The first member of a) associates with the second member of the interaction pair by non-covalent bonding. Optionally, of the interacting pairs (e.g., asymmetric or unguided interacting pairs) The first member is non-covalently associated with the second member of the interaction pair.

[0171] As a specific example, the present disclosure provides human Ig that have been modified to promote heteromultimer formation. CH1, CH2, or CH3 derived from IgG1, IgG2, IgG3, and / or IgG4 A fused to a polypeptide containing an immunoglobulin constant domain, such as a CH3 domain The present invention provides a fusion protein containing LK4 or ActRIIB. A problem that arises in the large-scale production of immunoglobulin-based proteins is the “chain association problem.” The chain association problem, as prominently encountered in the production of bispecific antibodies, arise uniquely when different heavy and / or light chains are produced in a single cell line. This paper addresses the challenge of efficiently producing desired multi-chain proteins from multiple combinations of proteins. [Klein et al. (2012) mAbs, vol. 4: 653-663]. This is most urgent when two different heavy chains and two different light chains are produced in the same cell. In this case, there are a total of 16 possible strand combinations (which Nevertheless, the same principle is applied to two different This causes a reduced yield of the desired multi-chain fusion protein incorporating only the (asymmetric) heavy chain.

[0172] Increased tolerance to desired pairing of Fc-containing fusion polypeptide chains in a single cell line Various methods for producing the preferred asymmetric fusion proteins in acceptable yields are known in the art. [Klein et al. (2012) mAbs 4:653-663; and Spie ss et al. (2015) Molecular Immunology, Vol. 67(Issue 2A): 95-106]. Charge-based methods for obtaining desired pairing of Fc-containing chains are Pair formation (electrostatic steering), "knobs-into-holes" ) Stereo-pairing, SEEDbody pairing, and leucine zipper base pairing These include, but are not limited to, Ridgway et al. (1996) Protein synthesis. in Eng, 9:617-621; Merchant et al. (1998) Nat B iotech, 16:677-681; Davis et al. (2010) Protein Eng Des Sel, vol. 23: pp. 195-202; Gunasekaran et al. (2 285:19637-19646; Wranik et al. (2012) J Biol Chem, vol. 287: pp. 43331-43339; US5932448; WO 1993 / 011162; WO2009 / 089004 and WO2011 / 03460 5]. As described herein, these methods were used to identify ALK4-Fc:ActR IIB-Fc heteromultimeric complexes can be generated. See, e.g., Figure 23. sea ​​bream.

[0173] ALK4:ActRIIB Heteromultimers and Methods for Producing Such Heteromultimers have been previously disclosed, for example, in WO 97 / 04909, the entire teachings of which are incorporated herein by reference. Please refer to 2016 / 164497.

[0174] The fusion protein (e.g., immunoglobulin) may be synthesized in any manner consistent with the desired functionality. It is understood that different elements of the Fc fusion protein can be arranged in different ways. For example, the ActRII polypeptide (or ALK4 polypeptide) domain may be a heterologous domain. Alternatively, the heterologous domain can be located C-terminal to the ActR II polypeptide (or ALK4 polypeptide) domain. ActRII polypeptide (or ALK4 polypeptide) domain and heterologous The domains do not have to be contiguous in the fusion protein, and additional domains or The amino acid sequence is C- or N-terminal to either domain, or between domains. It may be included.

[0175] For example, the ActRII (or ALK4) receptor fusion protein is shown in formula ABC. The B portion may comprise the amino acid sequence of the ActRII (or ALK4) polypeptide. The A and C portions may independently be zero, one, or two or more peptide domains. amino acids, and both the A and C moieties, if present, are heterologous to B. The A and / or C moieties can be attached to the B moiety via a linker sequence. The linker may be a glycine (e.g., 2 to 10, 2 to 5, 2 to 4, or 2 to 3 glycine residues) It can be rich in glycine and proline residues, e.g., threonine / serine and a single sequence of glycine, or repeats of threonine / serine and / or glycine Sequences such as GGG (SEQ ID NO: 19), GGGG (SEQ ID NO: 20), TGGGG (SEQ ID NO: SEQ ID NO: 21), SGGGG (SEQ ID NO: 22), TGGG (SEQ ID NO: 23), SGGG (SEQ ID NO: 24), SEQ ID NO: 24), or GGGGS (SEQ ID NO: 25) singlet or repeats In certain embodiments, an ActRII (or ALK4) fusion tag may be used. The protein comprises the amino acid sequence shown in the formula ABC, where A is the leader ( signal sequence, and B is a sequence derived from the ActRII (or ALK4) polypeptide domain. C is a parameter that describes in vivo stability, in vivo half-life, uptake / administration, and tissue localization. one or more of: aggregation or distribution, protein complex formation, and / or purification In certain embodiments, ActRII (or ActRII) is a polypeptide moiety that enhances the The LK4) fusion protein comprises the amino acid sequence shown in the formula ABC, wherein A is the TPA leader sequence, and B is the ActRII (or ALK4) receptor polypeptide. C is an immunoglobulin Fc domain. The proteins are SEQ ID NOs: 32, 36, 39, 40, 42, 45, 46, 48, 69, 74, 77 , 78, 108, 110, 111, 113, 114, 115, 116, 117, 118, 120, 122, and 124. .

[0176] In a preferred embodiment, the ActRII polypeptide used in accordance with the methods described herein The ALK4 polypeptide, the ALK4:ActRIIB heteromultimer, As used herein, an isolated protein or polypeptide is an isolated polypeptide. The polypeptide has been separated from a component of its natural environment. The polypeptides of the present disclosure may be purified by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing, Ion electrophoresis (IEF, capillary electrophoresis) or chromatography (e.g., ion 95%, 96%, 97%, 98% or higher as determined by exchange or reversed-phase HPLC Purified to greater than 99% purity. Methods for assessing purity are well known in the art [e.g., , Flatman et al. (2007) J. Chromatogr. B, 848:79 [See pages 10-87]. In some embodiments, the method may be used in accordance with the methods described herein. ActRII polypeptide, ALK4 polypeptide, and ALK4:Ac The tRIIB heteromultimer is a recombinant polypeptide.

[0177] TActRII polypeptides, ALK4 polypeptides and ALK4:Act of the present disclosure RIIB heteromultimers can be produced by a variety of techniques known in the art. For example, the polypeptides of the present disclosure may be prepared using methods such as those described in Bodansky, M., Principles of Peptide Synthesis, Springer Verlag, Be rlin (1993) and Grant GA (eds.), Synthetic P eptides: A User's Guide, W. H. Freeman an d Company, New York (1992), and other techniques. It can be synthesized using protein chemistry techniques. In addition, automated peptide synthesizers are commercially available. commercially available (e.g., Advanced ChemTech Model 396; (See Milligen / Biosearch 9600). Alternatively, they Polypeptides of the present disclosure, including fragments or variants of , various expression systems [e.g., E. coli, Chinese hamster ovary (CHO) cells, It can be produced by recombinant means using COS cells, baculovirus, etc. In some embodiments, the modified or unmodified polypeptides of the present disclosure are, for example, proteases, e.g., For example, trypsin, thermolysin, chymotrypsin, pepsin or paired basic Recombinantly produced full-length A by using amino acid converting enzyme (PACE) It can be produced by digestion of the ctRII polypeptide. software, e.g., MacVector, Omega, PCGene, Molec (Using ular Simulation, Inc.) to analyze the proteolytic cleavage sites Alternatively, such polypeptides can be purified by chemical cleavage (e.g., , cyanogen bromide, hydroxylamine, etc.) to generate recombinantly produced full-length Ac It may be produced from a tRII or ALK4 polypeptide.

[0178] 3. Nucleic acids encoding ActRII and ALK4 polypeptides and variants thereof acid In certain embodiments, the present disclosure provides an ActRII and / or ALK4 polypeptide. and isolated polypeptides encoding the polypeptides (including fragments, functional variants, and fusion proteins thereof). Nucleic acids and / or recombinant nucleic acids are provided. For example, SEQ ID NO: 7 is a naturally occurring human While encoding the ActRIIB precursor polypeptide (the R64 variant described above), SEQ ID NO: 8 encodes the processed extracellular domain of ActRIIB (the R64 variant described above). The nucleic acids of interest may be single-stranded or double-stranded. These nucleic acids are referred to as DNA molecules. These nucleic acids may be, for example, nucleic acids described herein, or may be RNA molecules. Use in methods for producing ActRII-based ligand trap polypeptides. This can be done.

[0179] As used herein, an isolated nucleic acid(s) is / are removed from a component of its natural environment. An isolated nucleic acid molecule is one that is not contained in cells that normally contain the nucleic acid molecule. The nucleic acid molecule may be extrachromosomal or a chromosome different from its natural chromosomal location. It is present in the chromophore position.

[0180] In certain embodiments, a nucleic acid encoding an ActRII or ALK4 polypeptide of the disclosure is provided. The nucleic acids are SEQ ID NOs: 7, 8, 12, 13, 37, 43, 49, 70, 71, 72, 73 , 75, 76, 80, 81, 82, 83, 84, 102, 103, 106, 107, 10 9, 112, 119, 121, 123 and 135. Variant nucleotide sequences are understood to include nucleic acids that contain allelic variants. It contains sequences that differ by one or more nucleotide substitutions, additions, or deletions, but SEQ ID NOs: 7, 8, 12, 13, 37, 43, 49, 70, 71, 72, 73, 75 ,76,80,81,82,83,84,102,103,106,107,109,1 12, 119, 121, 123, and 135 It may contain a coding sequence that is different from the nucleotide sequence.

[0181] In certain embodiments, the ActRII or ALK4 polypeptide of the disclosure has the sequence Numbers 7, 8, 12, 13, 37, 43, 49, 70, 71, 72, 73, 75, 76, 8 0, 81, 82, 83, 84, 102, 103, 106, 107, 109, 112, 11 9, 121, 123, and 135, at least 70%, 75%, , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% identical isolated and / or recombinant nucleic acid sequences. Those skilled in the art will recognize that SEQ ID NOs: 7, 8, 12, 13, 37, 43, 49, 70, 71, 72, 73, 75, 76, 80, 81, 82, 83, 84, 102, 103, 10 6, 107, 109, 112, 119, 121, 123, and 135, At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or 100% identical nucleic acid sequences and It will be appreciated that such variations are within the scope of the present disclosure. The nucleic acid sequences of the present disclosure can be isolated, recombinant, and / or heterologous. The nucleic acid sequence may be fused to a DNA sequence or may be present in a DNA library.

[0182] In other embodiments, the nucleic acids of the present disclosure are SEQ ID NOs: 7, 8, 12, 13, 37, 43, 49 , 70, 71, 72, 73, 75, 76, 80, 81, 82, 83, 84, 102, 10 3, 106, 107, 109, 112, 119, 121, 123 and 135 The nucleotide sequences identified are SEQ ID NOs: 7, 8, 12, 13, 37, 43, 49, 70, 7 1, 72, 73, 75, 76, 80, 81, 82, 83, 84, 102, 103, 106 , 107, 109, 112, 119, 121, 123, and 135, or Nucleotides that hybridize to those fragments under highly stringent conditions are identified. As discussed above, those skilled in the art will recognize that DNA hybridization techniques can be used to identify and characterize the sequences of the target gene. Those skilled in the art will readily appreciate that appropriate stringency conditions which promote cleavage may be varied. The author has determined that the appropriate stringency conditions that promote DNA hybridization are For example, 6.0× sodium chloride / chloride at about 45° C. After hybridization with sodium citrate (SSC), 2.0 at 50°C For example, the salt concentration in the wash step may be about 2.5×SSC at 50°C. Low stringency of 0x SSC to high stringency of approximately 0.2x SSC at 50°C Furthermore, the temperature in the washing step can be selected from as low as room temperature (about 22°C). Stringent conditions can be increased to high stringency conditions at about 65°C. When both temperature and salt are changed, the temperature or salt concentration is held constant and the other variable is changed. In one embodiment, the present disclosure provides a method for the preparation of a soluble collagenase (e.g., a collagenase inhibitor) comprising 6×SSC at room temperature followed by 2×SSC at room temperature. Nucleic acids that hybridize under low stringency conditions of washing with SC are provided.

[0183] Due to degeneracy in the genetic code, SEQ ID NOS: 7, 8, 12, 13, 37, 43, 49, 70, 71, 72, 73, 75, 76, 80, 81, 82, 83, 84, 102, 103 , 106, 107, 109, 112, 119, 121, 123, and 135 Isolated nucleic acids that differ from the amino acids are also within the scope of this disclosure. Codons that specify the same amino acid or synonyms ( For example, CAU and CAC are synonyms for histidine) However, in mammalian cells, "silent" mutations can occur that do not affect the amino acid sequence. Within the cells, there are polymorphisms in the DNA sequence that result in changes in the amino acid sequence of the subject protein. Those skilled in the art will appreciate that due to natural allelic variations, individuals of a given species will have different genetic makeups. Between them, one or more nucleotides (approximately 3-5% or It is understood that variations in these nucleotides (at and any such nucleotide variations and resulting amino acids Polymorphisms of are within the scope of this disclosure.

[0184] In certain embodiments, the recombinant nucleic acids of the disclosure contain one or more regulatory The regulatory nucleotide sequence can be operably linked to a regulatory nucleotide sequence. Suitable for the host cell used for expression. Many types of suitable expression vectors and suitable regulatory sequences are known in the art and may be used. Typically, the one or more regulatory nucleotide sequences include a promoter sequence. tRNA sequence, leader or signal sequence, ribosome binding site, transcription initiation sequence and and transcription termination sequences, translation initiation and termination sequences, and enhancer or These sequences may include, but are not limited to, constitutive activator sequences known in the art. Alternatively, inducible promoters are contemplated by the present disclosure. existing promoters or high-quality promoters that combine elements of more than one promoter The expression construct can be either an epigenetic promoter or a hybrid promoter. The expression construct may be present in the cell on a chromosome or may be inserted into a chromosome. In one embodiment, the expression vector comprises a sequence encoding the nucleotide sequence of the target gene, to allow for selection of transformed host cells. Selectable marker genes are well known in the art. , and may vary depending on the host cell used.

[0185] In certain embodiments, the subject nucleic acids disclosed herein are ActRII and / or or ALK4 polypeptide and operably linked to at least one regulatory sequence. The regulatory sequence is provided in an expression vector comprising the nucleotide sequence linked to the It is recognized in the art that expression of ActRII and / or ALK4 polypeptides can be achieved by Thus, the term regulatory sequence includes promoters, enhancers, and Exemplary regulatory sequences include those listed in Goeddel; ene Expression Technology:Methods in Enz ymology, Academic Press, San Diego, CA (1990 For example, a wide range of genes that control the expression of DNA sequences when operably linked are described in Any of a wide variety of expression control sequences may be used to encode the ActRII and / or ALK4 polypeptides. These vectors can be used to express DNA sequences encoding the genes. Such useful expression control sequences include, for example, the early and late promoters of SV40. -, tet promoter, adenovirus or cytomegalovirus immediate early promoter promoter, RSV promoter, lac system, trp system, TAC or TRC system, a T7 promoter whose expression is driven by T7 RNA polymerase; The major operator and promoter region of phage lambda, controlling the fd coat protein region, promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatase promoters of phospholipase A (e.g., Pho5), yeast α-mating factor (mating factor promoter, the baculovirus polyhedrin promoter, and Controlling the expression of genes in prokaryotic or eukaryotic cells or their viruses and other sequences known to those skilled in the art, as well as various combinations thereof. The design of the gene may depend on the choice of host cell to be transformed and / or the type of gene desired to be expressed. It should be understood that the amount of protein consumed may depend on factors such as the type of protein. the copy number of the vector, the ability to control that copy number, and any other Expression of proteins (e.g., antibiotic markers) should also be considered.

[0186] Recombinant nucleic acids of the disclosure include those derived from cloned genes or portions thereof, which are expressed in prokaryotic cells, Expression in either or both eukaryotic cells (yeast, avian, insect, or mammalian) Recombinant ActRI can be produced by ligating it into an appropriate vector to effect the transformation of ActRI. Expression vehicles for production of the I and / or ALK4 polypeptides include plasmids. For example, suitable vectors include those of the following types: Plasmids include: for expression in prokaryotic cells (e.g., E. coli) , pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids pBTac-derived plasmids and pUC-derived plasmids.

[0187] Some mammalian expression vectors contain prokaryotic vectors to facilitate propagation of the vector in bacteria. A sequence of an organism and one or more eukaryotic transcription units that are expressed in a eukaryotic cell. Contains both: pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2 gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg-derived vectors are useful in the transfection of eukaryotic cells. are examples of mammalian expression vectors suitable for transfection. Some of these vectors are , which facilitates replication and drug resistance selection in both prokaryotic and eukaryotic cells For this purpose, the vector is modified with sequences from a bacterial plasmid (e.g., pBR322). or bovine papillomavirus (BPV-1) or Epstein-Barr virus (HBV) Derivatives of viruses such as EBo, pREP-derived and p205 have been shown to be effective in eukaryotic cells. It can be used for transient expression of proteins in other viruses (including retroviruses). Examples of expression systems (including plasmids) can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of vectors and in the transformation of host organisms are well known in the art. Other suitable expression systems for both prokaryotic and eukaryotic cells, as well as Common recombinant procedures, such as those described in Molecular Cloning A Laboratory ory Manual, 3rd Ed., Sambrook, Fritsch and Ma edited by Niatis (Cold Spring Harbor Laboratory Pr. ess, 2001). In some cases, recombinant DNA technology has been developed using baculovirus expression systems. It may be desirable to express the polypeptide in a baculovirus-derived form. Examples of systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pAcUW-derived vectors (e.g., pAcUWl) and pBlu Examples include eBac-derived vectors (e.g., pBlueBac III containing β-gal). can be done.

[0188] In a preferred embodiment, the vector expresses the subject ActRII and and / or designed for the production of ALK4 polypeptides (e.g., Pcmv-Scri pt vector (Stratagene, La Jolla, Calif.), pcDNA 4 vector (Invitrogen, Carlsbad, Calif.) and pCI- neo vector (Promega, Madison, Wisconsin). The subject genetic constructs can be used to express, for example, proteins (fusion proteins or variant proteins). For the production of proteins (including proteins), the present invention is carried out in cells grown in culture for purification. The subject ActRII polypeptides can be used to induce expression of the polypeptides.

[0189] The present disclosure also provides a method for the production of one or more of the subject ActRII and / or ALK4 polypeptides. The present invention relates to a host cell transfected with a recombinant gene containing a coding sequence for the nucleotide sequence of the host. The cell can be any prokaryotic or eukaryotic cell, for example, an ActR cell of the present disclosure. II and / or ALK4 polypeptides can be expressed in bacterial cells such as E. coli, insect cells, or the like. (e.g., using a baculovirus expression system), yeast cells or mammalian cells [e.g., Other suitable hosts include the Chinese hamster ovary (CHO) cell line. Cells are known to those skilled in the art.

[0190] Thus, the present disclosure further provides a method for the detection of ActRII and / or ALK4 polypeptides of interest. The present invention relates to methods for producing ActRII and / or ALK4 polypeptides. Host cells transfected with an expression vector encoding ActRII and / or Alternatively, it may be cultured under appropriate conditions that allow expression of the ALK4 polypeptide to occur. The polypeptide can be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the ActRII and / or ALK4 polypeptides are retained in the cytoplasm. or in collected and lysed membrane fractions or cells and isolated proteins. A cell culture comprises host cells, media, and other by-products. A suitable medium for cell culture is: The polypeptide of interest may be purified using techniques well known in the art for purifying proteins. Techniques known in the art include ion exchange chromatography, gel filtration chromatography, and the like. , ultrafiltration, electrophoresis, specific epitopes of ActRII and / or ALK4 polypeptides Immunoaffinity purification with topope-specific antibodies and ActRII polypeptides Affinity purification with agents that bind to the domain fused to the domain (e.g., protein ActRII-Fc and / or ALK4-Fc fusion proteins were analyzed using column A. The cell culture medium, host cells or these can be purified using techniques including In some embodiments, ActRII and / or A The LK4 polypeptide is a fusion protein containing a domain that facilitates its purification. .

[0191] In some embodiments, the purification includes, for example, three or more of the following, in any order: This is achieved through a series of column chromatography steps: Protein A chromatograph Q Sepharose chromatography, phenyl Sepharose chromatography, Size exclusion chromatography and cation exchange chromatography. Purification was performed using viral This can be accomplished by filtration and buffer exchange. The K4 fusion protein was determined by size exclusion chromatography to be >90%, 95%, >96%, >98% or >99%, as determined by SDS PAGE Purification can be performed to >90%, >95%, >96%, >98% or >99% purity. The target level of purity is consistent with mammalian systems, particularly non-human primates, rodents (mice) and The level should be sufficient to achieve the desired result in humans.

[0192] In another embodiment, a purification leader sequence (e.g., recombinant ActRII and / or Poly-(His) / Enterokinae located at the N-terminus of the desired portion of the ALK4 polypeptide The fusion gene encoding the ribosomal enzyme cleavage site sequence was 2+ Affinity chromium using metal resin This allows for the purification of the expressed fusion protein by chromatography. The leader sequence was subsequently removed by treatment with enterokinase, and the purified Ac tRII and / or ALK4 polypeptides can be provided. See, for example, Hochuli et al. , (1987) J. Chromatography 411:177; and Ja See Inknecht et al. (1991) PNAS USA 88:8972.

[0193] Techniques for creating fusion genes are well known. Essentially, a gene is a gene that expresses a polypeptide sequence different from the polypeptide sequence of the present invention. The joining of various DNA fragments encoding the nucleotides can be carried out using blunt ends for ligation or If staggered ends are required, restriction enzyme digestion is required to provide the appropriate ends. Filling-in of sticky ends according to the need, avoiding unwanted bonding conventional methods using alkaline phosphatase treatment for cleavage and enzymatic ligation In another embodiment, the fusion gene is synthesized by automated DNA synthesis. Alternatively, PCR amplification of the gene fragment can be performed by conventional techniques. This results in complementary overhangs between the consecutive gene fragments. These fragments can then be used to generate chimeric genes. They can be annealed to produce a child sequence. ls in Molecular Biology, edited by Ausubel et al., John W See iley & Sons:1992.

[0194] 4. Antibody antagonists In certain embodiments, the GDF used in accordance with the methods and uses disclosed herein The GDF / BMP antagonist is an antibody (GDF / BMP antagonist antibody) or a combination of antibodies. The GDF / BMP antagonist antibody or combination of antibodies may be, for example, one or or multiple ActRII ligands (e.g., activin, GDF8, GDF11, BMP 6, BMP15, BMP10 and / or GDF3), ActRII receptor (ActR IIA and / or ActRIIB), type I receptors (ALK4, ALK5 and / or can bind to ALK7) and / or co-receptors. and GDF / BMP antagonist antibodies, either alone or in combination with one or more supportive therapies. or in combination with an active agent to treat or prevent pulmonary hypertension (PH); or reducing the rate of progression and / or severity thereof, in particular one or more P Treating, preventing, or reducing the rate of progression and / or severity of H-related complications It is possible.

[0195] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least At least activin (e.g., activin A, activin B, activin C, activin E) , activin AB, activin AC, activin BC, activin AE and / or activin Thus, in some embodiments, the antibody inhibits GDF / BM. The P antagonist antibody or combination of antibodies binds to at least activin. As used herein, an activin antibody (or anti-activin antibody) generally refers to an antibody that: The present invention provides a method for the preparation of a compound having sufficient affinity to be useful as a diagnostic and / or therapeutic agent targeting activin. In certain embodiments, an activin antibody binds to activin with high affinity. The extent to which the body binds to unrelated, non-activin proteins can be determined, for example, by radioimmunoassay. (RIA), Biacore, or other protein interaction or binding affinity - assay, approximately 10%, 9%, 8%, and less than 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1%. In this context, activin antibodies are directed against activin proteins that are conserved among activins from different species. In certain preferred embodiments, the anti-activin antibody binds to an epitope of activin. In some embodiments, the activin antibody binds to human activin, where activin is type I. and / or type II receptors (e.g., ActRIIA, ActRIIB, ALK4, A LK5 and / or ALK7), thus inhibiting activin-mediated Signal transduction (e.g., Smad signaling) can be inhibited. In this state, activin antibodies inhibit activin binding to the ActRII co-receptor. , thus inhibiting activin-mediated signaling (e.g., Smad signaling). Activin A has similar sequence homology to activin B, and therefore Antibodies that bind to activin A may, in some instances, also bind to and / or inhibit activin B. It should be noted that this also applies to anti-activin B antibodies. In some embodiments, the present disclosure provides a method for the treatment of inflammatory bowel disease, comprising administering to a subject a therapeutically effective amount of a steroid hormone, such as steroid hormone, a steroid hormone, or a steroid hormone. Additional GDF / BMP ligands [e.g., GDF11, GDF8, GDF3, BMP 15, BMP10 and BMP6], one or more type I receptors and / or I Type I receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5 and / or or ALK7) and / or one or more co-receptors (e.g., bispecific antibodies) and uses thereof. A multispecific antibody that binds to BMP9 does not bind or does not substantially bind to BMP9 (e.g., 1×10 -7 K is larger than M D binds to BMP9 with relatively low binding capacity , for example, about 1 × 10 -8 M or approximately 1 x 10 -9 In some embodiments, A multispecific antibody that binds to activin does not bind or substantially binds to activin A. (e.g., 1×10 -7 K is larger than M D binds to activin A at has a relatively low binding affinity, e.g., about 1 × 10 -8 M or approximately 1 x 10 -9 M). In some embodiments, the present disclosure relates to antibody combinations and uses thereof, wherein the antibody combinations are and, e.g., one or more additional GDF / BMP superfamily members. Myri ligands [e.g., GDF8, GDF11, GDF3, BMP6 and BMP15] ], one or more type I receptors and / or type II receptors (e.g., ActRI IA, ActRIIB, ALK4, ALK5 and / or ALK7), and / or It contains one or more additional antibodies that bind to one or more co-receptors. In some embodiments, the antibody combination that includes an activin antibody does not include a BMP9 antibody. In embodiments, the antibody combination that includes an activin antibody does not include an activin A antibody.

[0196] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, antibodies that inhibit GDF / activin B. The BMP antagonist antibody or combination of antibodies binds to at least activin B. As used herein, an activin B antibody (or anti-activin B antibody) generally refers to an activin B antibody. The antibodies are believed to be useful as diagnostic and / or therapeutic agents targeting activin B. In certain embodiments, the term "antibody" refers to an antibody that binds to activin B with sufficient affinity to bind to activin B. The extent to which an activin B antibody binds to an unrelated, non-activin B protein may be determined, e.g., by measuring the binding of the antibody to a non-activin B protein. Genomic immunoassay (RIA), Biacore, or other protein interaction or is approximately 1 / 2 the binding affinity of the antibody to activin as measured by binding affinity assay. 0%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, less than 2%, or less than about 1% In certain embodiments, the activin B antibody is capable of binding to activin B from different species. In certain preferred embodiments, the activin B epitope is conserved in In some embodiments, the anti-activin B antibody binds to human activin B. Activin B antibodies are antibodies that bind to type I and / or type II receptors (e.g., ActRII). A, ActRIIB, ALK4, ALK5 and / or ALK7) impairs activin B-mediated signaling (e.g., Smad signaling) In some embodiments, the activin B antibody can inhibit activin B co-receptor. inhibits activin B-mediated signal transduction (e.g., S Activin B inhibits mad signaling. Due to sequence homology, antibodies that bind to activin B may, in some instances, bind to activin A. Note that in some embodiments, the ATP may bind to and / or inhibit the In some embodiments, the present disclosure provides a method for the production of antibodies that bind to activin B and further comprise, for example, one or more additional GDF / BMP ligands [e.g., GDF11, GDF8, GDF3, BMP15, BM P10 and BMP6], one or more type I receptors and / or type II receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5, and / or ALK 7) and / or multispecific antibodies that bind to one or more co-receptors (e.g., In some embodiments, the present invention relates to a bispecific antibody that binds to activin B and uses thereof. The multispecific antibody does not bind or does not substantially bind to BMP9 (e.g., 1× 10 -7 K is larger than M D bind to BMP9 with high affinity or with relatively low affinity, e.g. Approximately 1×10 -8 M or approximately 1 x 10 -9 In some embodiments, the active ingredient Multispecific antibodies that bind to activin B do not or substantially do not bind to activin A. (e.g., 1×10 -7 K is larger than M D Binding to activin A or comparison relatively low binding, e.g., about 1×10 -8 M or approximately 1 x 10 -9 Some real In embodiments, the present disclosure relates to antibody combinations and uses thereof, the antibody combinations comprising: and, for example, one or more additional GDF / BMP ligands [e.g., , GDF8, GDF11, GDF3, BMP6, BMP10 and BMP15], as well as or multiple type I and / or type II receptors (e.g., ActRIIA, Ac tRIIB, ALK4, ALK5 and / or ALK7) and / or one or In some embodiments, the antibody comprises one or more additional antibodies that bind to multiple co-receptors. The antibody combination comprising an activin B antibody does not include a BMP9 antibody. , an antibody combination that includes an activin B antibody does not include an activin A antibody.

[0197] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, the antibody inhibits GDF / BM. The P antagonist antibody or combination of antibodies binds to at least GDF8. As used herein, a GDF8 antibody (or anti-GDF8 antibody) generally refers to an antibody that binds to a GDF Sufficient affinity to be useful as a diagnostic and / or therapeutic agent targeting 8 In certain embodiments, the GDF8 antibody binds to an unrelated GDF8 antibody. The extent of binding to non-GDF8 proteins can be measured, for example, by radioimmunoassay (RIA), B iacore, or other protein interaction or binding affinity assays The binding of the antibody to GDF8 was measured by , 4%, 3%, 2%, or less than about 1%. In certain embodiments, GDF8 The antibody binds to an epitope of GDF8 that is conserved among GDF8 from different species In certain preferred embodiments, the anti-GDF8 antibody binds to human GDF8. In some embodiments, the GDF8 antibody is an antibody that binds to type I and / or type II receptors (e.g., For example, ActRIIA, ActRIIB, ALK4, ALK5, and / or ALK7) inhibits GDF8-mediated signal transduction (e.g., Smad signal transduction) from binding to In some embodiments, the GDF8 antibody can inhibit GDF8 from binding to its co-receptor, thus inhibiting GDF8-mediated signaling (e.g., GDF8 has high sequence similarity with GDF11. Because they have the same sex, antibodies that bind to GDF8 may also, in some instances, bind to GDF11. Note that in some embodiments, the present invention may also inhibit and / or inhibit the The antibodies shown bind to GDF8 and further bind, for example, one or more additional GDF / BMP receptors. Gands [e.g., activins (e.g., activin A, activin B, activin C, activin B] Activin E, Activin AB, Activin AC, Activin BC, Activin AE, Activin thibin BE), GDF11, GDF3, BMP15, BMP10 and BMP6], one or multiple type I receptors and / or type II receptors (e.g., ActRIIA, A ctRIIB, ALK4, ALK5 and / or ALK7) and / or one or more Multispecific antibodies (e.g., bispecific antibodies) that bind to multiple co-receptors and their uses In some embodiments, the multispecific antibody that binds to GDF8 binds to BMP9. No or substantially no binding (e.g., 1×10 -7 K is larger than M D BM binds to P9 or has relatively low binding, e.g., about 1×10 -8 M or approx. 1 x 1 0 -9 In some embodiments, the multispecific antibody that binds to GDF8 has Does not bind or does not substantially bind to thiamine A (e.g., 1 x 10 -7 Larger than M NaK D or binds to activin A with relatively low binding, e.g., about 1 x 10 -8 M Or about 1 x 10 -9 In some embodiments, the present disclosure provides antibody combinations and and uses thereof, the antibody combination may comprise a GDF8 antibody and, e.g., one or more Additional GDF / BMP ligands [e.g., activins (e.g., activin A, activin B], Activin B, Activin C, Activin E, Activin AB, Activin AC, Activin BC, activin AE, activin BE), GDF11, GDF3, BMP6, BMP1 0 and BMP15], one or more type I receptors and / or type II receptors ( For example, ActRIIA, ActRIIB, ALK4, ALK5 and / or ALK7 ), and / or one or more additional antibodies that bind to one or more co-receptors. In some embodiments, the antibody combination comprising a GDF8 antibody also comprises a BMP9 antibody. In some embodiments, the antibody combination comprising a GDF8 antibody does not include an activin A antibody. Does not include.

[0198] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, antibodies that inhibit GDF / B The MP antagonist antibody or antibody combination binds to at least GDF11. As used herein, a GDF11 antibody (or anti-GDF11 antibody) generally refers to an antibody that and (iii) a GDF11-targeting antibody that is sufficiently active to be useful as a diagnostic and / or therapeutic agent. In certain embodiments, the antibody binds to GDF11 with high affinity. The extent to which the antibody binds to unrelated, non-GDF11 proteins can be determined, for example, by radioimmunoassay. RIA, Biacore, or other protein interaction or binding affinity Approximately 10%, 9%, and 8% of the antibody's binding to GDF11 was measured by a ELISA. , 7%, 6%, 5%, 4%, 3%, less than 2%, or less than about 1%. In terms of morphology, GDF11 antibodies are GDF11 antibodies that are conserved among GDF11 from different species. In certain preferred embodiments, the anti-GDF11 antibody binds to an epitope of GDF11. and binds to human GDF11. In some embodiments, the GDF11 antibody binds to human GDF11. Type I and / or type II receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5 and / or ALK7), thus inhibiting GDF11-mediated Signal transduction (e.g., Smad signaling) can be inhibited. In this state, GDF11 antibodies inhibit GDF11 from binding to its co-receptor, thus It can inhibit GDF11-mediated signaling (e.g., Smad signaling). GDF11 shares high sequence homology with GDF8, so antibodies that bind to GDF11 It has been noted that in some cases, it may also bind to and / or inhibit GDF8. Note that in some embodiments, the present disclosure provides a method for the treatment of GDF11-associated leukemia, e.g., leukemia, encephalopathy, rheumatoid arthritis, and rheumatoid arthritis. one or more additional GDF / BMP ligands [e.g., activin (e.g., activin Activin A, Activin B, Activin C, Activin E, Activin AB, Activin A C, activin BC, activin AE, activin BE), GDF8, GDF3, BMP 15, BMP10 and BMP6], one or more type I receptors and / or I Type I receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5 and / or or ALK7) and / or one or more co-receptors (e.g., bispecific antibodies) and uses thereof. A multispecific antibody that binds to BMP9 does not bind or does not substantially bind to BMP9 (e.g., 1×10 -7 K is larger than M D binds to BMP9 with relatively low binding capacity , for example, about 1 × 10 -8 M or approximately 1 x 10 -9 In some embodiments, Multispecific antibodies that bind to GDF11 do not bind or substantially bind to activin A. (e.g., 1×10 -7 K is larger than M D binds to activin A at has a relatively low binding affinity, e.g., about 1 × 10 -8 M or approximately 1 x 10 -9 M). In some embodiments, the present disclosure relates to antibody combinations and uses thereof, wherein the antibody combinations are DF11 antibody and, e.g., one or more additional GDF / BMP ligands [e.g., For example, activins (e.g., activin A, activin B, activin C, activin E) , Activin AB, Activin AC, Activin BC, Activin AE, Activin BE ), GDF8, GDF3, BMP6, BMP10 and BMP15], one or more Type I receptors and / or type II receptors (e.g., ActRIIA, ActRIIB) , ALK4, ALK5 and / or ALK7) and / or one or more co-receptors In some embodiments, the antibody comprises one or more additional antibodies that bind to the receptor. In some embodiments, the antibody combination does not include a BMP9 antibody. The antibody combination comprising the antibody does not include an activin A antibody.

[0199] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, the antibody inhibits GDF / BM P6. The P antagonist antibody or combination of antibodies binds to at least BMP6. As used herein, a BMP6 antibody (or anti-BMP6 antibody) generally refers to an antibody that binds to BMP Sufficient affinity to be useful as a diagnostic and / or therapeutic agent targeting 6 In certain embodiments, the BMP6 antibody The extent to which the antibody binds to unrelated, non-BMP6 proteins can be determined, for example, by radioimmunoassay ( RIA), Biacore, or other protein interaction or binding affinity Approximately 10%, 9%, 8%, and 7% of the antibody's binding to BMP6 as measured by the assay , 6%, 5%, 4%, 3%, 2%, or less than about 1%. The BMP6 antibody targets the BMP6 epitope that is conserved among BMP6 derived from different species. In certain preferred embodiments, the anti-BMP6 antibody binds to a human BMP6 In some embodiments, the BMP6 antibody binds to type I and / or type II BMP6. type receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5 and / or inhibits binding to ALK7) and thus inhibits BMP6-mediated signaling (e.g., In some embodiments, the BMP6 antibody can inhibit IL-16 signaling. , inhibiting BMP6 from binding to its co-receptor and thus inhibiting BMP6-mediated signal transduction In some embodiments, the present invention can inhibit the expression of steroid hormones (e.g., Smad signaling). The antibodies shown bind to BMP6 and further bind to, for example, one or more additional GDF / BMP receptors. Gands [e.g., activins (e.g., activin A, activin B, activin C, activin B] Activin E, Activin AB, Activin AC, Activin BC, Activin AE, Activin Thibin BE), GDF8, GDF3, BMP15, BMP10 and GDF11], one or multiple type I receptors and / or type II receptors (e.g., ActRIIA, A ctRIIB, ALK4, ALK5 and / or ALK7) and / or one or more Multispecific antibodies (e.g., bispecific antibodies) that bind to multiple co-receptors and their uses In some embodiments, the multispecific antibody that binds to BMP6 is No or substantially no binding (e.g., 1×10 -7 K is larger than MD BM binds to P9 or has relatively low binding, e.g., about 1×10 -8 M or approx. 1 x 1 0 -9 In some embodiments, the multispecific antibody that binds to BMP6 has Does not bind or does not substantially bind to thiamine A (e.g., 1 x 10 -7 Larger than M NaK D or binds to activin A with relatively low binding, e.g., about 1 x 10 -8 M Or about 1 x 10 -9 In some embodiments, the present disclosure provides antibody combinations and and uses thereof, the antibody combination may comprise a BMP6 antibody and, e.g., one or more Additional GDF / BMP ligands [e.g., activins (e.g., activin A, activin B], Activin B, Activin C, Activin E, Activin AB, Activin AC, Activin BC, activin AE, activin BE), GDF8, GDF11, GDF3, BMP1 0 and BMP15], one or more type I receptors and / or type II receptors ( For example, ActRIIA, ActRIIB, ALK4, ALK5 and / or ALK7 ), and / or one or more additional antibodies that bind to one or more co-receptors. In some embodiments, the antibody combination comprising a BMP6 antibody also comprises a BMP9 antibody. In some embodiments, the antibody combination comprising a BMP6 antibody does not include an activin A antibody. Does not include.

[0200] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, GDF / BM The P antagonist antibody or combination of antibodies binds to at least GDF3. As used herein, a GDF3 antibody (or anti-GDF3 antibody) generally refers to an antibody that binds to a GDF Sufficient affinity to be useful as a diagnostic and / or therapeutic agent targeting 3 In certain embodiments, a GDF3 antibody is an antibody capable of binding to GDF3. The extent to which the antibody binds to unrelated non-GDF3 proteins can be determined, for example, by radioimmunoassay ( RIA), Biacore, or other protein interaction or binding affinity Approximately 10%, 9%, 8%, and 7% of the antibody's binding to GDF3 as measured by the assay , 6%, 5%, 4%, 3%, 2%, or less than about 1%. GDF3 antibodies target the GDF3 epitope that is conserved among GDF3 from different species. In certain preferred embodiments, the anti-GDF3 antibody binds to a human GDF3 In some embodiments, the GDF3 antibody binds to type I and / or type II GDF3. type receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5 and / or inhibits binding to ALK7) and thus inhibits GDF3-mediated signaling (e.g. In some embodiments, the GDF3 antibody can inhibit GDF3 signaling. inhibits GDF3 from binding to its co-receptors, thus inhibiting GDF3-mediated signal transduction. In some embodiments, the present invention can inhibit signal transduction (e.g., Smad signaling). The disclosure provides compounds that bind to GDF3 and further bind to, for example, one or more additional GDFs / BMPs. Ligands [e.g., activins (e.g., activin A, activin B, activin C, Activin E, Activin AB, Activin AC, Activin BC, Activin AE, Activin Cutibin BE), GDF8, BMP6, BMP15, BMP10 and GDF11], 1 one or more type I receptors and / or type II receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5 and / or ALK7) and / or any one of or multispecific antibodies (e.g., bispecific antibodies) that bind to multiple co-receptors, and In some embodiments, the multispecific antibody that binds to GDF3 is No or substantially no binding (e.g., 1×10 -7 K is larger than M D DeB binds to MP9 or has relatively low binding, e.g., about 1×10 -8 M or approx. 1 x 10 -9 In some embodiments, the multispecific antibody that binds to GDF3 has Does not bind or does not substantially bind to activin A (e.g., 1 x 10 -7 Larger than M Kina K D or binds to activin A with relatively low binding, e.g., about 1 x 10 -8 M or approximately 1 x 10 -9 In some embodiments, the present disclosure provides a combination of antibodies. For purposes of this and related uses, the antibody combination may comprise a GDF3 antibody and, for example, one or more A number of additional GDF / BMP ligands [e.g., activins (e.g., activin A, activin B], Activin B, Activin C, Activin E, Activin AB, Activin AC, Activin Activin BC, Activin AE, Activin BE), GDF8, GDF11, BMP6, BMP 10 and BMP15], one or more type I receptors and / or type II receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5, and / or ALK 7), and / or one or more additional co-receptors that bind to one or more co-receptors. In some embodiments, the antibody combination comprising a GDF3 antibody comprises a BMP9 antibody. In some embodiments, the antibody combination comprising a GDF3 antibody does not include an activin A antibody. Does not include.

[0201] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, antibodies that inhibit GDF / B The BMP antagonist antibody or antibody combination binds to at least BMP15. As used herein, a BMP15 antibody (or anti-BMP15 antibody) generally refers to an antibody that and (iii) a compound having sufficient activity to be useful as a diagnostic and / or therapeutic agent targeting BMP15. In certain embodiments, the term "antibody" refers to an antibody that can bind to BMP15 with high affinity. The extent to which BMP15 antibodies bind to unrelated, non-BMP15 proteins can be determined by, for example, radioimmunoassays. Immunoassay (RIA), Biacore, or other protein interaction or binding assays Approximately 10% of the binding of the antibody to BMP15 as measured by a combined affinity assay , 9%, 8%, 7%, 6%, 5%, 4%, 3%, less than 2%, or less than about 1%. In certain embodiments, the BMP15 antibody is a BMP15 antibody that is conserved among BMP15 from different species. In certain preferred embodiments, the anti-BM The P15 antibody binds to human BMP15. In some embodiments, the BMP15 antibody MP15 binds to type I and / or type II receptors (e.g., ActRIIA, ActRIIB) , ALK4, ALK5 and / or ALK7), thus inhibiting B Can inhibit MP15-mediated signaling (e.g., Smad signaling) In some embodiments, the BMP15 antibody inhibits BMP15 from binding to a co-receptor. and thus inhibiting BMP15-mediated signaling (e.g., Smad signaling) In some embodiments, the present disclosure provides a method for the treatment of BMP15-associated leukemia, e.g., rheumatoid arthritis ... and rheumatoid arthritis. For example, one or more additional GDF / BMP ligands [e.g., activin (e.g., Activin A, Activin B, Activin C, Activin E, Activin AB, Activin activin AC, activin BC, activin AE and activin BE), GDF8, GDF1 1, GDF3, BMP10 and BMP6], one or more type I receptors and / or or type II receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5, and and / or ALK7) and / or one or more co-receptors. In some embodiments, B Multispecific antibodies that bind to MP15 do not bind or substantially do not bind to BMP9. (e.g., 1×10 -7 K is larger than M D binds to BMP9 at a relatively low low binding capacity, e.g., about 1×10 -8 M or approximately 1 x 10 -9 Some implementations In some embodiments, the multispecific antibody that binds to BMP15 does not bind to or does not bind to activin A. Qualitatively non-binding (e.g., 1 × 10 -7 K is larger than M D binds to activin A at or relatively low binding, e.g., about 1×10 -8 M or approximately 1 x 10 -9 Having M In some embodiments, the present disclosure relates to antibody combinations and uses thereof. The method includes the step of administering a BMP15 antibody and, for example, one or more additional GDF / BMP ligases. and [e.g., activin (e.g., activin A, activin B, activin C, activin Activin E, Activin AB, Activin AC, Activin BC, Activin AE and Activin cutin BE), GDF8, GDF3, BMP6, BMP10 and GDF11], one or multiple type I receptors and / or type II receptors (e.g., ActRIIA, A ctRIIB, ALK4, ALK5 and / or ALK7), and / or one or more of In some embodiments, the antibody comprises one or more additional antibodies that bind to multiple co-receptors. In some embodiments, the antibody combination comprising a BMP15 antibody does not include a BMP9 antibody. The antibody combination including the BMP15 antibody does not include an activin A antibody.

[0202] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, antibodies that inhibit GDF / B The BMP antagonist antibody or antibody combination binds to at least BMP10. As used herein, a BMP10 antibody (or anti-BMP10 antibody) generally refers to an antibody that and (iii) a compound having sufficient activity to be useful as a diagnostic and / or therapeutic agent targeting BMP10. In certain embodiments, the term "antibody" refers to an antibody that can bind to BMP10 with high affinity. The extent to which BMP10 antibodies bind to unrelated, non-BMP10 proteins can be determined by, for example, radioimmunoassays. Immunoassay (RIA), Biacore, or other protein interaction or binding assays Approximately 10% of the binding of the antibody to BMP10 as measured by a combined affinity assay , 9%, 8%, 7%, 6%, 5%, 4%, 3%, less than 2%, or less than about 1%. In certain embodiments, the BMP10 antibody is a BMP10 antibody that is conserved among BMP10 from different species. In certain preferred embodiments, the anti-BMPC binds to an epitope of BMP10 that is known to be a BMP10 antibody. The P10 antibody binds to human BMP10. In some embodiments, the BMP10 antibody MP10 binds to type I and / or type II receptors (e.g., ActRIIA, ActRIIB) , ALK4, ALK5 and / or ALK7), thereby inhibiting BM P10-mediated signaling (eg, Smad signaling) can be inhibited. In some embodiments, the BMP10 antibody inhibits BMP10 from binding to a co-receptor. , thus inhibiting BMP10-mediated signaling (e.g., Smad signaling). In some embodiments, the present disclosure provides a method for the treatment of cancer, comprising administering to a subject a therapeutic agent that binds to BMP10 and further comprises administering to a subject a therapeutic agent, e.g., one or more additional GDF / BMP ligands [e.g., activin (e.g., activin Activin A, Activin B, Activin C, Activin E, Activin AB, Activin AC, activin BC, activin AE, and activin BE), GDF8, GDF11 , GDF3 and BMP6], one or more type I receptors and / or type II receptors encephalopathy (e.g., ActRIIA, ActRIIB, ALK4, ALK5 and / or A LK7) and / or multispecific antibodies that bind to one or more co-receptors (e.g., In some embodiments, the present invention relates to a method for the production of antibodies that bind to BMP10, such as a bispecific antibody, and uses thereof. A multispecific antibody that binds to BMP9 does not or does not substantially bind to BMP9 (e.g., x10 -7 K is larger than M D bind to BMP9 with high affinity or with relatively low affinity, e.g. Approximately 1 x 10 -8 M or approximately 1 x 10 -9 In some embodiments, BMP Multispecific antibodies that bind to 10 do not bind or substantially do not bind to activin A. (e.g., 1×10 -7 K is larger than M D Binding to activin A or comparison relatively low binding, e.g., about 1×10 -8 M or approximately 1 x 10 -9 Some real In embodiments, the present disclosure relates to antibody combinations and uses thereof, the antibody combinations being directed against BMP1 0 antibody, and, for example, one or more additional GDF / BMP ligands [e.g., Activins (e.g., activin A, activin B, activin C, activin E, activin activin AB, activin AC, activin BC, activin AE and activin BE) , GDF8, GDF3 BMP6, BMP10 and GDF11], one or more Type I receptors and / or type II receptors (e.g., ActRIIA, ActRIIB, ALK4, ALK5 and / or ALK7) and / or one or more co-receptors In some embodiments, the antibody comprises one or more additional antibodies that bind to the BMP10 antibody. In some embodiments, the antibody combination does not include a BMP9 antibody. The antibody combination containing the body does not include an activin A antibody.

[0203] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, the antibody inhibits at least ActRIIB. The BMP antagonist antibody or antibody combination binds to at least ActRIIB. As used herein, an ActRIIB antibody (anti-ActRIIB antibody) generally refers to an ActRIIB antibody. In addition, the antibodies are useful as diagnostic and / or therapeutic agents targeting ActRIIB. In certain embodiments, the term refers to an antibody that binds to ActRIIB with sufficient affinity to The extent to which anti-ActRIIB antibodies bind to unrelated, non-ActRIIB proteins is unknown, e.g. For example, radioimmunoassay (RIA), Biacore, or other protein-protein interactions Antibodies to ActRIIB as measured by action or binding affinity assays Less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the binding capacity of In certain embodiments, the anti-ActRIIB antibodies are derived from different species. It binds to an epitope of ActRIIB that is conserved among ActRIIBs. In certain preferred embodiments, the anti-ActRIIB antibody binds to human ActRIIB. In some embodiments, the anti-ActRIIB antibody is a fusion protein comprising one or more GDF / BMP ligands. GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and activin BE), GDF11, BMP6, GDF3, BMP10 and BMP1 5] can inhibit the binding of the anti-ActRIIB. ActRIIB antibodies bind to ActRIIB and one or more GDF / BMP receptors. Gands [e.g., GDF11, GDF8, activins (e.g., activin A, activin activin B, activin C, activin E, activin AB, activin AC), GDF3, B MP6 and BMP10], type I receptors (e.g., ALK4, ALK5 and / or A LK7), co-receptors, and / or additional type II receptors (e.g., ActRIIA) In some embodiments, the antibody is a multispecific antibody (e.g., a bispecific antibody) that binds to The disclosure relates to antibody combinations and uses thereof, the antibody combinations comprising an anti-ActRIIB antibody, and, for example, one or more GDF / BMP ligands [e.g., GDF11, GD F8, activin (e.g., activin A, activin B, activin C, activin E) , activin AB, activin AC, activin BC, activin AE and activin BE), BMP6, GDF3 and BMP10], co-receptors, type I receptors (e.g., AL K4, ALK5 and / or ALK7) and / or additional type II receptors (e.g. and one or more additional antibodies that bind to ActRIIB. Because of its sequence similarity to ActRIIA, antibodies that bind to ActRIIB are Note that in some cases, the compounds may bind to and / or inhibit ActRIIA. I want to be done that.

[0204] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, the antibody inhibits at least ActRIIA. The BMP antagonist antibody or antibody combination binds to at least ActRIIA. As used herein, an ActRIIA antibody (anti-ActRIIA antibody) generally refers to an ActRIIA antibody. In addition, the antibodies may be useful as diagnostic and / or therapeutic agents targeting ActRIIA. In certain embodiments, the term "antibody" refers to an antibody that binds to ActRIIA with sufficient affinity to bind to ActRIIA. The extent to which anti-ActRIIA antibodies bind to unrelated, non-ActRIIA proteins, e.g. For example, radioimmunoassay (RIA), Biacore, or other protein-protein interactions Antibodies to ActRIIA as measured by action or binding affinity assays Less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the binding capacity of In certain embodiments, the anti-ActRIIA antibodies are derived from different species. It binds to an epitope of ActRIIA that is conserved among ActRIIA. In certain preferred embodiments, the anti-ActRIIA antibody binds to human ActRIIA. In some embodiments, the anti-ActRIIA antibody binds one or more GDF / BMP ligands. GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and activin BE), GDF11, BMP6, GDF3, BMP10 and BMP1 5] can inhibit the binding of the anti-ActRIIA. ActRIIA antibodies are directed against ActRIIA and one or more GDF / BMP receptors. Gands [e.g., GDF11, GDF8, activins (e.g., activin A, activin activin B, activin C, activin E, activin AB, activin AC), GDF3, B MP6 and BMP10], type I receptors (e.g., ALK4, ALK5 and / or A LK7), co-receptors, and / or additional type II receptors (e.g., ActRIIB) In some embodiments, the antibody is a multispecific antibody (e.g., a bispecific antibody) that binds to The disclosure relates to antibody combinations and uses thereof, the antibody combinations comprising anti-ActRIIA antibodies, and, for example, one or more GDF / BMP ligands [e.g., GDF11, GD F8, activin (e.g., activin A, activin B, activin C, activin E) , activin AB, activin AC, activin BC, activin AE and activin BE), BMP6 and BMP10], co-receptors, type I receptors (e.g., ALK4, AL K5 and / or ALK7), and / or additional type II receptors (e.g., Act ActRIIA comprises one or more additional antibodies that bind to Act Because of its sequence similarity to RIIB, antibodies that bind to ActRIIA may, in some instances, Note that it may also bind to and / or inhibit ActRIIB. .

[0205] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, the antibody inhibits GDF / BM. The P antagonist antibody or combination of antibodies binds to at least ALK4. As used herein, an ALK4 antibody (anti-ALK4 antibody) generally refers to an antibody that targets ALK4. with sufficient affinity to be useful as a diagnostic and / or therapeutic agent targeting A In certain embodiments, the anti-ALK4 antibody is an antibody that binds to an unrelated non-ALK4 antibody. The degree of binding to the LK4 protein can be measured by, for example, radioimmunoassay (RIA), Bia core, or other protein-protein interaction or binding affinity assays The binding of the antibody to ALK4 was measured at approximately 10%, 9%, 8%, 7%, 6%, 5%, less than 4%, 3%, 2%, or less than about 1%. In certain embodiments, the anti-ALK4 The antibody binds to an epitope on ALK4 that is conserved among ALK4 from different species In certain preferred embodiments, the anti-ALK4 antibody binds to human ALK4. In some embodiments, the anti-ALK4 antibody is administered in combination with one or more GDF / BMP ligands, e.g. For example, GDF8, activin (e.g., activin A, activin B, activin C, activin B), Activin E, Activin AB, Activin AC, Activin BC, Activin AE and activin BE), GDF11, BMP6, GDF3, BMP10 and BMP15] In some embodiments, the anti-ALK4 antibody can inhibit binding to ALK4. The present invention relates to the binding of ALK4 and one or more GDF / BMP ligands [e.g., GDF1 1, GDF8, activin (e.g., activin A, activin B, activin C, activin B) activin E, activin AB, activin AC), GDF3, BMP6 and BMP10] , type II receptors (e.g., ActRIIA and / or ActRIIB), co-receptors, and / or binds to additional type I receptors (e.g., ALK5 and / or ALK7) In some embodiments, the present disclosure provides a multispecific antibody (e.g., a bispecific antibody) that The antibody combination includes an anti-ALK4 antibody and, for example, For example, one or more GDF / BMP ligands [e.g., GDF11, GDF8, actin activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE, and activin BE), BM P6 and BMP10], co-receptors, type II receptors (e.g., ActRIIA and / or or ActRIIB), and / or additional type I receptors (e.g., ALK5 and / or or ALK7).

[0206] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, the antibody inhibits GDF / BM. The P antagonist antibody or combination of antibodies binds to at least ALK5. As used herein, an ALK5 antibody (anti-ALK5 antibody) generally refers to an antibody that targets ALK5. with sufficient affinity to be useful as a diagnostic and / or therapeutic agent targeting A In certain embodiments, the anti-ALK5 antibody is an antibody that binds to an unrelated non-ALK5 antibody. The degree of binding to the LK5 protein can be measured by, for example, radioimmunoassay (RIA), Bia core, or other protein-protein interaction or binding affinity assays The binding of the antibody to ALK5 was measured at approximately 10%, 9%, 8%, 7%, 6%, 5%, less than 4%, 3%, 2%, or less than about 1%. In certain embodiments, the anti-ALK5 The antibody binds to an epitope of ALK5 that is conserved among ALK5 from different species In certain preferred embodiments, the anti-ALK5 antibody binds to human ALK5. In some embodiments, the anti-ALK5 antibody is administered in combination with one or more GDF / BMP ligands [e.g., For example, GDF8, activin (e.g., activin A, activin B, activin C, activin B), Activin E, Activin AB, Activin AC, Activin BC, Activin AE and activin BE), GDF11, BMP6, GDF3, BMP10 and BMP15] In some embodiments, the anti-ALK5 antibody can inhibit binding to ALK5. The present invention relates to the binding of ALK5 and one or more GDF / BMP ligands [e.g., GDF1 1, GDF8, activin (e.g., activin A, activin B, activin C, activin B) activin E, activin AB, activin AC), GDF3, BMP6 and BMP10] , type II receptors (e.g., ActRIIA and / or ActRIIB), co-receptors, and / or binds to additional type I receptors (e.g., ALK4 and / or ALK7) In some embodiments, the present disclosure provides a multispecific antibody (e.g., a bispecific antibody) that The antibody combination includes an anti-ALK5 antibody and, for example, For example, one or more GDF / BMP ligands [e.g., GDF11, GDF8, actin activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE, and activin BE), BM P6 and BMP10], co-receptors, type II receptors (e.g., ActRIIA and / or or ActRIIB), and / or additional type I receptors (e.g., ALK4 and / or or ALK7).

[0207] In certain embodiments, the GDF / BMP antagonist antibody or antibody combination comprises at least Thus, in some embodiments, the antibody inhibits GDF / BM. The P antagonist antibody or combination of antibodies binds to at least ALK7. As used herein, an ALK7 antibody (anti-ALK7 antibody) generally refers to an antibody that targets ALK7. with sufficient affinity to be useful as a diagnostic and / or therapeutic agent targeting A In certain embodiments, the anti-ALK7 antibody is an antibody that binds to an unrelated non-ALK7 antibody. The degree of binding to the LK7 protein can be measured by, for example, radioimmunoassay (RIA), Bia core, or other protein-protein interaction or binding affinity assays The binding of the antibody to ALK7 was measured at approximately 10%, 9%, 8%, 7%, 6%, 5%, In certain embodiments, the anti-ALK7 The antibody binds to an epitope on ALK7 that is conserved among ALK7 from different species In certain preferred embodiments, the anti-ALK7 antibody binds to human ALK7. In some embodiments, the anti-ALK7 antibody is administered in combination with one or more GDF / BMP ligands [e.g., For example, GDF8, activin (e.g., activin A, activin B, activin C, activin B), Activin E, Activin AB, Activin AC, Activin BC, Activin AE and activin BE), GDF11, BMP6, GDF3, BMP10 and BMP15] In some embodiments, the anti-ALK7 antibody can inhibit binding to ALK7. The present invention relates to the binding of ALK7 and one or more GDF / BMP ligands [e.g., GDF1 1, GDF8, activin (e.g., activin A, activin B, activin C, activin B) activin E, activin AB, activin AC), GDF3, BMP6 and BMP10] , type II receptors (e.g., ActRIIA and / or ActRIIB), co-receptors, and / or binds to additional type I receptors (e.g., ALK4 and / or ALK5) In some embodiments, the present disclosure provides a multispecific antibody (e.g., a bispecific antibody) that The antibody combination includes an anti-ALK7 antibody and, for example, For example, one or more GDF / BMP ligands [e.g., GDF11, GDF8, actin activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE, and activin BE), BM P6 and BMP10], co-receptors, type II receptors (e.g., ActRIIA and / or or ActRIIB), and / or additional type I receptors (e.g., ALK4 and / or or ALK5).

[0208] The term "antibody" is used herein in the broadest sense to refer to any antibody having the desired antigen-binding activity. As long as they exhibit the above properties, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., Various antibodies, including but not limited to, bispecific antibodies, and antibody fragments Antibody fragments contain antibodies that bind to the same antigen as an intact antibody. Refers to a molecule other than an intact antibody that contains a portion of an intact antibody. Examples of antibody fragments Examples include Fv, Fab, Fab', Fab'-SH, and F(ab')2; diabodies; formed from linear antibodies; single-chain antibody molecules (e.g., scFv); and antibody fragments Multispecific antibodies [see, e.g., Hudson et al. (2003) Nat. Med. 9:12 pp. 9-134; Pluckthun, The Pharmacology of Mon. oclonal Antibodies, Vol. 113, Rosenburg and Moor e, (Springer-Verlag, New York), p...

Claims

[Claim 1] The invention described in the specification.

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