How to use activin receptor type II variants

Abstract

The present invention features polypeptides comprising an extracellular ActRII variant, e.g., an ActRIIA variant, an ActRIIB variant, or an ActRII chimera. In some embodiments, the polypeptides of the invention comprise an extracellular ActRII variant fused to an Fc domain monomer or moiety. The present invention also features pharmaceutical compositions comprising the polypeptides and methods of using the polypeptides to treat diseases and conditions treatable with erythropoietin or erythropoiesis-stimulating agents.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing, which is filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on October 1, 2021, is named 51184-026WO4_Sequence_Listing_10_1_21_ST25 and is 546,175 bytes in size. [Background technology]

[0002] Erythropoietin (EPO) is a hormone primarily secreted by the kidneys, typically released in response to hypoxic levels. While known to promote red blood cell production, erythropoietin has also been found to stimulate the recruitment, proliferation, and differentiation of endothelial progenitor cells, improve gastrointestinal motility, possess broad neuroprotective and anti-inflammatory capabilities, and play a central role in tissue protection and recovery. Recombinant EPOs and EPO mimics, such as epoetin alfa and epoetin beta, are often referred to as erythropoiesis-stimulating agents (ESAs) and are currently used to treat anemia associated with chronic kidney disease and anemia associated with cancer or chemotherapy. However, recombinant EPO therapy requires intravenous administration 1-3 times per week for up to 12 weeks, which is an inconvenient treatment regimen for patients. Furthermore, high-dose EPO treatment can lead to cancer cell proliferation in cancer patients or tumor recurrence in patients with a history of cancer. Therefore, there is a need for alternative therapies to EPO. [Overview of the project]

[0003] The present invention features polypeptides comprising extracellular activin receptor type II (ActRII) variants, such as extracellular ActRIIA variants, extracellular ActRIIB variants, or extracellular ActRII chimeras. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRII variant fused to the N-terminus or C-terminus of an Fc domain monomer or portion. Such portions can be linked by amino acids or other covalent bonds, thereby increasing the stability of the polypeptide. Polypeptides comprising an extracellular ActRII variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interactions between two Fc domain monomers. The polypeptides of the present invention can be used as a substitute for EPO to treat subjects having diseases or conditions that can be treated with EPO or ESA, such as end-stage renal disease, renal failure, polycythemia, diseases associated with endothelial progenitor cell dysfunction, neurological or inflammatory brain diseases, gastrointestinal motility disorders, ischemia (e.g., central nervous system (CNS), liver, kidney, or cardiac ischemia), hypoxia, or diseases or conditions having inflammatory or autoimmune components, or to treat subjects undergoing renal dialysis, subjects scheduled for surgery, or subjects who have recently undergone stem cell transplantation. The polypeptides of the present invention can also be used to increase EPO levels and EPO receptor levels in subjects that require them. Furthermore, the polypeptides of the present invention can also be used to affect the signaling of myostatin, activin (activin A and / or activin B), and / or bone morphogenetic protein 9 (BMP9) in subjects at risk of developing or having the diseases or conditions described herein.

[0004] Exemplary embodiments of the present invention are described in the following items. E1. A method for treating a subject who has or is at risk of developing a disease or condition that can be treated with erythropoietin or an erythropoiesis-stimulating agent, the method comprising administering to the subject a therapeutically effective amount of one of the compositions in Table 10, Table 11, or Table 12.

[0005] E2. A method for treating a subject who has or is at risk of developing a disease or condition that can be treated with erythropoietin or an erythropoiesis-stimulating agent, by affecting the signaling of myostatin, activin A, activin B, and / or BMP9 (e.g., by reducing or inhibiting the binding of myostatin, activin A, activin B, and / or BMP9 to their endogenous receptors), wherein the method involves administering a therapeutically effective amount of one of the compositions in Table 10, Table 11, or Table 12 to the subject.

[0006] E3. The method according to E1 or E2, wherein the disease or condition that can be treated with erythropoietin or the erythropoiesis stimulant is anemia due to dialysis or anemia of prematurity. E4. The method according to E1 or E2, wherein the disease or condition treatable with the erythropoietin or erythropoiesis-stimulating agent is end-stage renal disease, renal failure, polycythemia, hemochromatosis, disease or condition associated with endothelial progenitor cell dysfunction, disease or condition having autoimmune or inflammatory components, neurological or inflammatory brain disease, gastrointestinal motility disorders, endocrine disorders, reproductive system disorders, aging, pregnancy (e.g., hematological abnormalities associated with pregnancy), menstrual disorders, ischemia or ischemic injury or condition, hypoxia or hypoxic injury or condition, ulcers, burns, wounds (e.g., chronic wounds), ischemia-reperfusion injury, asthma, hypertension, viral disease or infection, systemic microbial infection, gastrointestinal disease, arteriosclerosis, cancer, psychosis, genetic disorders, inflammatory diseases, graft-versus-host disease, cardiovascular disease, allergies, or arthritis.

[0007] E5. A method for increasing erythropoietin levels in a subject requiring such treatment, comprising administering a therapeutically effective amount of one of the compositions in Table 10, one in Table 11, or one in Table 12 to the subject.

[0008] E6. A method for increasing erythropoietin receptor levels in a subject requiring such treatment, comprising administering a therapeutically effective amount of one of the compositions in Table 10, one in Table 11, or one in Table 12 to the subject.

[0009] E7. The method according to E5 or E6, wherein the subject has dialysis-induced anemia or anemia of prematurity, or is at risk of developing them. E8. The method according to E5 or E6, wherein the subject has or is at risk of developing end-stage renal disease, renal failure, polycythemia, hemochromatosis, disease or condition related to endothelial progenitor cell dysfunction, disease or condition having an autoimmune or inflammatory component, neurological or inflammatory brain disease, gastrointestinal motility disorder, endocrine disorders, reproductive system disorders, aging, pregnancy (e.g., hematological abnormalities associated with pregnancy), menstrual disorders, ischemia or ischemic injury or condition, hypoxia or hypoxic injury or condition, ulcers, burns, wounds (e.g., chronic wounds), ischemia-reperfusion injury, asthma, hypertension, viral disease or infection, systemic microbial infection, gastrointestinal disease, arteriosclerosis, cancer, psychosis, genetic disorders, inflammatory diseases, graft-versus-host disease, cardiovascular disease, allergies, or arthritis.

[0010] E9. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis stimulant is ischemia, or the subject has ischemia or is at risk of developing it.

[0011] E10. The method according to E9, wherein the ischemia is central nervous system ischemia, hepatic ischemia, renal ischemia, or cardiac ischemia. E11. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis stimulant is an ischemic disorder or condition, or the subject has an ischemic disorder or condition or is at risk of developing one.

[0012] E12. The method according to E11, wherein the ischemic disorder or condition is obstructive arterial disease, chronic venous insufficiency, circulatory shock (e.g., hemorrhagic, septic, or cardiogenic shock), pulmonary embolism, myocardial infarction, ischemic stroke, acute respiratory failure, chronic heart failure, atherosclerosis, cardiac cirrhosis, macular degeneration, sleep apnea, Raynaud's disease, systemic sclerosis, nonbacterial thrombotic endocarditis, transient ischemic attack, or ischemia due to general anesthesia.

[0013] E13. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis stimulant is hypoxic injury or condition, or the subject has or is at risk of developing hypoxic injury or condition.

[0014] E14. The method according to E13, wherein the hypoxic injury or condition is a lung injury (e.g., chronic obstructive pulmonary disease), perinatal hypoxia, severe pneumonia, pulmonary edema, hyaline membrane disease, liver disease, kidney disease, cancer, or altitude sickness.

[0015] E15. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis stimulant is a viral disease or infection, or the subject has a viral disease or infection or is at risk of developing one.

[0016] E16. The method according to E15, wherein the viral disease or infection is hepatitis C virus infection or HIV infection. E17. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis stimulant is a disease or condition associated with endothelial progenitor cell dysfunction, or the subject has a disease or condition associated with endothelial progenitor cell dysfunction or is at risk of developing one.

[0017] E18. The method according to E17, wherein the disease or condition associated with the dysfunction of the endothelial progenitor cells is heart failure, angina pectoris, endotheliopathy, reticuloendotheliopathy, age-related cardiovascular disease, coronary heart disease, atherosclerosis, myocardial ischemia, hypercholesterolemia, ischemic injury of the limbs, Raynaud's disease, pre-eclampsia, pregnancy-induced hypertension, endothelial-mediated chronic inflammatory disease (e.g., inflammation of blood vessels), wound healing, chronic renal failure (chronic kidney disease), or acute renal failure (acute renal failure).

[0018] E19. The method according to E18, wherein the disease or condition associated with the dysfunction of the endothelial progenitor cells is chronic renal failure (chronic kidney disease). E20. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis stimulant is an autoimmune or inflammatory disease or condition, or the subject has an autoimmune or inflammatory disease or condition, or is at risk of developing one.

[0019] E21. The method according to E20, wherein the autoimmune or inflammatory disease or condition is acute cerebrovascular injury, acute brain injury, acute cardiovascular injury, arthritis, autoimmune disease, stroke, nerve injury, or immune-mediated inflammation.

[0020] E22. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis-stimulating agent is a neurological disorder or inflammatory brain disease, or the subject has a neurological disorder or inflammatory brain disease or is at risk of developing one.

[0021] E23. The method according to E22, wherein the neurological disorder or inflammatory brain disease is a demyelinating disease, epilepsy, spinal cord injury (e.g., acute spinal cord injury), complications after traumatic brain injury (e.g., treatment of symptoms of traumatic brain injury such as hypotension, hypoxemia, cerebral swelling, headache, neck pain, memory problems, difficulty concentrating, difficulty making decisions, fatigue, mood changes, nausea, photophobia, blurred vision, tinnitus, loss of taste, loss of smell, seizures, coma, muscle weakness, paralysis, or progressive decline in neurological function), chronic inflammatory brain disease, or neurological disorder associated with surgery (e.g., thoracoabdominal aortic surgery).

[0022] E24. The method according to E23, wherein the chronic inflammatory brain disease is a neurodegenerative disease. E25. The method according to E24, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or age-related macular degeneration (AMD).

[0023] E26. The method according to E23, wherein the demyelinating disease is multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, or transverse myelitis. E27. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis-stimulating agent is a gastrointestinal motility disorder, or the subject has a gastrointestinal motility disorder or is at risk of developing one.

[0024] E28. The method according to E27, wherein the gastrointestinal motility disorder is related to intestinal injury, abdominal trauma, intestinal inflammatory condition, intestinal infection, delayed constipation, postoperative ileus, neurodegenerative injury, neurotrauma, congenital problems, or malnutrition-malabsorption problems.

[0025] E29. The method according to E28, wherein the intestinal infection is a bacterial infection (e.g., an infection resulting in sepsis or bacteremia), peritonitis, or ascites. E30. The method according to E28, wherein the inflammatory state of the intestine is inflammatory bowel disease, Crohn's disease, or ulcerative colitis.

[0026] E31. The method according to E28, wherein the delayed constipation is chronic constipation, idiopathic constipation, constipation due to postoperative ileus, or constipation due to opioid use. E32. The method according to E28, wherein the congenital problem is gastric spondylolysis, umbilical hernia, aganglionic megacolon, Hirschsprung's disease, chronic pseudoobstruction, left microcolon syndrome, anorectal anomaly, esophageal dysplasia and atresia, ectopic anus, congenital hernia, or internal anal sphincter achalasia.

[0027] E33. The method according to E28, wherein the malnutrition-malabsorption problem is due to intestinal injury, abdominal trauma, intestinal inflammatory state, intestinal infection, constipation, postoperative ileus, neurodegenerative injury, neurotrauma, congenital problems, Gaucher disease, refeeding syndrome, extremely low birth weight, cancer cachexia, infection, cancer, spinal cord dysfunction, spinal dysplasia, spina bifida, tumor, central nervous system dysfunction, peripheral neuropathy, partial resection of the gastrointestinal tract, bleeding, liver dysfunction, celiac disease, cystic fibrosis, muscular dystrophy, or cerebral palsy.

[0028] E34. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis stimulant is end-stage renal disease, or the subject has end-stage renal disease or is at risk of developing it.

[0029] E35. The method according to E4 or E8, wherein the disease or condition treatable with erythropoietin or the erythropoiesis stimulant is polycythemia, or the subject has polycythemia or is at risk of developing it.

[0030] E36. The method according to any one of E1 to E35, wherein a composition from Table 10, Table 11, or Table 12 is administered to the subject before surgery (for example, to increase the red blood cell count before surgery), after stem cell transplantation, before or during space flight, during or after tissue or organ transplantation, to promote the proliferation of new blood vessels, for granulation tissue formation, for the treatment of trauma, or for the treatment after vascular transplantation.

[0031] E37. The method according to any one of E1 to E36, wherein the subject is undergoing renal dialysis. E38. The method according to any one of E1, E2, E4-E6, and E8-E37, wherein the subject does not have anemia.

[0032] E39. The method according to any one of E1, E2, E4-E6, and E8-E38, wherein the subject has normal hematopoiesis. E40. The method according to any one of E1 to E39, wherein the subject has low serum erythropoietin levels.

[0033] E41. A method for preparing tissue or organ for transplantation, wherein the tissue or organ (for example, in or after being removed from the tissue or organ donor) is brought into contact with a therapeutically effective amount of a composition of Table 10, Table 11, or Table 12.

[0034] E42. The method according to any one of E1 to E41, wherein the method comprises administering a therapeutically effective amount of the composition of Table 10 to the subject. E43. The method according to any one of E1 to E41, wherein the method comprises administering a therapeutically effective amount of the composition of Table 11 to the subject.

[0035] E44. The method according to any one of E1 to E41, wherein the method comprises administering a therapeutically effective amount of the composition of Table 12 to the subject. E45. The method according to any one of E1 to E44, wherein the composition is administered in an amount sufficient to increase EPO levels, increase EPO receptor levels, promote the proliferation of new blood vessels and / or replacement of damaged vascular areas, promote granulation tissue formation, reduce mononuclear cell infiltration into the brain of the subject, improve neuropathy, reduce axonal injury, reduce neuronal cell death, reduce glial cell death, affect the signaling of myostatin, activin A, activin B, and / or BMP9 in the subject, or reduce or inhibit the binding of activin A, activin B, and / or myostatin to their receptors.

[0036] E46. A method according to any one of E1 to E45, wherein the method does not cause vascular complications in the subject. E47. The method according to E46, wherein the method does not increase vascular permeability or leakage.

[0037] E48. The method according to any one of E1 to E47, wherein the subject is a human. definition To facilitate understanding of the present invention, numerous terms and phrases are defined below. The terms defined herein have meanings that are generally understood by those skilled in the art related to the present invention. Terms such as "a," "an," and "the" are not intended to refer only to singular entities but include general classes for which specific examples can be used for illustrative purposes. The terms herein are used to describe specific embodiments of the present invention, but their use does not limit the present invention except as outlined in the claims.

[0038] Where used herein, the term “approximately” means a value that is 10% above or below the stated value. When used herein, any value provided in a range of values ​​includes both the upper and lower limits, and any value that falls within the upper and lower limits.

[0039] As used herein, the terms “extracellular activin receptor type IIA (ActRIIA) variant” and “ActRIIA variant” refer to the soluble extracellular portion of the single transmembrane receptor ActiRIIA having at least one amino acid substitution relative to wild-type extracellular ActiRIIA (e.g., the bolded portion of the sequence in SEQ ID NO: 75 shown below), or peptides containing extracellular ActiRIIA having one of the sequences in SEQ ID NOs: 76-96. The sequence of the wild-type human ActiRIIA precursor protein is shown below (SEQ ID NO: 75). Here, the signal peptide is shown in italics and the extracellular portion is shown in bold.

[0040] Wild-type, human ActRIIA precursor protein (SEQ ID NO: 75):

[0041] [ka]

[0042] An extracellular ActRIIA variant may have any one sequence from SEQ ID NOs: 1 to 72. In certain embodiments, an extracellular ActRIIA variant may have any one sequence from SEQ ID NOs: 6 to 72 (Table 2). In some embodiments, an extracellular ActRIIA variant may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97%, or more) amino acid sequence identity with wild-type extracellular ActRIIA sequence (SEQ ID NO: 73).

[0043] As used herein, the terms “extracellular activin receptor type IIB (ActRIIB) variant” and “ActRIIB variant” refer to a peptide comprising the soluble extracellular portion of the single transmembrane receptor ActRIIB, having at least one amino acid substitution compared to wild-type extracellular ActRIIB (e.g., the bolded portion of the sequence in Sequence ID No. 173 shown below). The sequence of wild-type human ActRIIB is shown below (Sequence ID No. 173), with the signal peptide in italics and the extracellular portion in bold.

[0044] Wild-type human ActRIIB (SEQ ID NO: 173):

[0045] [ka]

[0046] Extracellular ActRIIB variants may have any one of the sequences of SEQ ID NOs. 157–171. In certain embodiments, extracellular ActRIIB variants may have any one of the sequences of SEQ ID NOs. 158–171 (Table 5). In some embodiments, extracellular ActRIIB variants may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence identity with wild-type extracellular ActRIIB sequence (SEQ ID NO. 74). Extracellular ActRIIB variants may also have 1–7 amino acid N-terminal truncation compared to the extracellular portion of ActRIIB.

[0047] As used herein, the terms “extracellular activin receptor type II (ActRII) chimera,” “extracellular ActRII chimera,” and “ActRII chimera” refer to a peptide comprising the soluble extracellular component of a single transmembrane receptor ActRIIB and the soluble extracellular component of a single transmembrane receptor ActRIIA. The ActRII chimeras described herein result from the conjugation of the N-terminal portion of extracellular ActRIIB to the C-terminal portion of extracellular ActRIIA to create a sequence continuity (for example, the ActRIIA sequence continues from where the ActRIIB sequence was interrupted, starting after the amino acid at the corresponding position in ActRIIA). Extracellular ActRII chimeras may also contain one or more amino acid substitutions in the chimeric portion corresponding to the ActRIIB sequence (e.g., the bolded portion of the sequence of SEQ ID NO: 173 shown above) compared to wild-type extracellular ActRIIB, and one or more amino acid substitutions in the chimeric portion corresponding to the ActRIIA sequence (e.g., the bolded portion of the sequence of SEQ ID NO: 75 shown above) compared to wild-type extracellular ActRIIA. Extracellular ActRII chimeras may also have 1 to 9 amino acid N-terminal truncations on the extracellular portion of ActRIIB or ActRIIA. The sequences of wild-type human ActRIIB (SEQ ID NO: 173) and wild-type human ActRIIA (SEQ ID NO: 75) are shown in the definition above, with the signal peptide italicized and the extracellular portion in bold. Extracellular ActRII chimeras may contain any one of the sequences from SEQ ID NOs: 174 to 216. In certain embodiments, the extracellular ActRII chimera has one of the sequences from sequence numbers 195 to 216 (Table 7).

[0048] As used herein, the term “extracellular activin receptor type II (ActRII) variant” refers to the extracellular ActRIIA variant, extracellular ActRIIB variant, or extracellular ActRII chimera as described herein.

[0049] As used herein, the term “N-terminal truncation” refers to the deletion of 1 to 7 amino acids (e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids) from the N-terminus of an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157–171 (e.g., SEQ ID NOs. 158–171)), or the deletion of 1 to 9 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) from the N-terminus of an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174–216 (e.g., SEQ ID NOs. 195–216)). N-terminal truncation allows for the removal of amino acids up to two amino acids prior to the first cysteine ​​(for example, the two amino acids prior to the first cysteine ​​(RE) are retained in the N-terminally truncated extracellular ActRIIB variant, and the two amino acids prior to the first cysteine ​​(RE or QE) are retained in the N-terminally truncated ActRII chimera).

[0050] As used herein, the term “linker” refers to a linkage between two elements, such as peptide or protein domains. Polypeptides described herein may include extracellular ActRII variants (e.g., extracellular ActRIIA variants (e.g., extracellular ActRIIA variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72))), extracellular ActRIIB variants (e.g., extracellular ActRIIB variants having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171)), or extracellular ActRII chimeras (e.g., extracellular ActRII chimeras having any one sequence of SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)) fused to a portion. This portion can enhance the stability of the polypeptide or improve its pharmacokinetic properties. This portion (e.g., Fc domain monomer, Fc domain (e.g., wild-type Fc domain), Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), albumin bonds) A peptide (such as a fibronectin domain or human serum albumin) can be fused to a polypeptide by a linker. The linker can be a covalent bond or a spacer. The term "bond" refers to a chemical bond, e.g., an amide bond or a disulfide bond, or any kind of bond resulting from a chemical reaction, e.g., a chemical conjugation. The term "spacer" refers to a portion (e.g., a polyethylene glycol (PEG) polymer) or amino acid sequence (e.g., a sequence of 1 to 200 amino acids) present between two elements, providing space and / or flexibility between them. An amino acid spacer is part of the primary sequence of a polypeptide (e.g., fused to a spaced peptide via the polypeptide backbone). For example, the formation of a disulfide bond between two hinge regions forming an Fc domain is not considered a linker.

[0051] As used herein, the term “Fc domain” refers to a dimer of two Fc domain monomers. An Fc domain is at least C H 2 domains and CH The Fc domain monomer has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity) to a human Fc domain containing three domains. H 2 and C H 3) includes. In some embodiments, the Fc domain monomer also includes a hinge domain. The Fc domain does not contain any portion of the immunoglobulin that can act as an antigen recognition region, such as a variable domain or a complementarity-determining region (CDR). In the wild-type Fc domain, the two Fc domain monomers are two C H Dimerization occurs through interactions between the three antibody constant domains and through one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domain monomers. In some embodiments, the Fc domain may be mutated to lack effector function, typical of a “dead Fc domain”. In certain embodiments, each of the Fc domain monomers in the Fc domain is mutated to reduce the interaction or binding between the Fc domain and the Fcγ receptor. H The antibody constant domain contains amino acid substitutions. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. The Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. Furthermore, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a naturally occurring Fc domain, such as a recombinant Fc domain.

[0052] As used herein, the term “albumin-binding peptide” refers to an amino acid sequence of 12 to 16 amino acids that has affinity for and the function of binding to serum albumin. Albumin-binding peptides may be of different origins, e.g., human, mouse, or rat. In some embodiments, the albumin-binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 151).

[0053] As used herein, the term “fibronectin domain” refers to a high molecular weight glycoprotein or fragment of the extracellular matrix that binds to transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain (SEQ ID NO: 152) having amino acids 610-702 of the sequence with UniProt ID number P02751. In other embodiments, the fibronectin domain is an adnectin protein.

[0054] As used herein, the term “human serum albumin” refers to the albumin protein present in human plasma. Human serum albumin is the most abundant protein in blood, accounting for approximately half of all serum proteins. In some embodiments, human serum albumin has the sequence UniProt ID number P02768 (SEQ ID NO: 153).

[0055] As used in this book, the term “endogenous” refers to molecules (e.g., polypeptides, nucleic acids, or cofactors) that are naturally found in a particular organism (e.g., a human) or in a particular location within the organism's body (e.g., organs, tissues, or cells such as human cells, e.g., human red blood cells, platelets, neutrophils, or muscle cells).

[0056] As used herein, the term “fused” is used to describe a combination or linkage of two or more elements, components, or protein domains, such as peptides or polypeptides, by means of chemical conjugation, recombination, and chemical bonding, such as amide bonding. For example, two single peptides linked in tandem can be fused by chemical conjugation, chemical bonding, peptide linkers, or any other covalent bonding to form a single continuous protein structure, such as a polypeptide. In some embodiments of the polypeptides described herein, extracellular ActRII variants (e.g., extracellular ActRIIA variants (e.g., extracellular ActRIIA variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72))), extracellular ActRIIB variants (e.g., extracellular ActRIIB variants having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171)), or extracellular ActRII chimeras (e.g., having any one sequence of SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)) The extracellular ActRII chimera can be fused in a tandem chain to the N-terminus or C-terminus of a portion (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97), an Fc domain (e.g., a wild-type Fc domain (e.g., the sequence of SEQ ID NO: 150 or SEQ ID NO: 155)), an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide (e.g., the sequence of SEQ ID NO: 151), a fibronectin domain (e.g., the sequence of SEQ ID NO: 152), or human serum albumin (e.g., the sequence of SEQ ID NO: 153)) by a linker.For example, an extracellular ActRII variant is fused by a peptide linker to a portion (e.g., an Fc domain monomer, an Fc domain (e.g., a wild-type Fc domain), an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin), the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRII variant by a chemical bond, e.g., a peptide bond, and the C-terminus of the peptide linker is fused to the N-terminus of the portion (e.g., an Fc domain monomer, an Fc domain (e.g., a wild-type Fc domain), an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) by a chemical bond, e.g., a peptide bond.

[0057] As used herein, the term “C-terminal elongation” refers to the addition of one or more amino acids to the C-terminus of an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1–70 (e.g., SEQ ID NOs. 6–70)) or at the C-terminus of an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174–216 (e.g., SEQ ID NOs. 195–216)). C-terminal elongation may consist of one or more amino acids, such as 1–6 amino acids (e.g., 1, 2, 3, 4, 5, 6, or more). C-terminal elongation may include amino acids from the corresponding position in wild-type ActRIIA (in the case of an ActRIIA variant) or from the corresponding position in wild-type ActRIIA or ActRIIB (in the case of an ActRII chimera). Exemplary C-terminal elongations are the amino acid sequence NP (2-amino acid C-terminal elongation) and the amino acid sequence NPVTPK (SEQ ID NOs. 154) (6-amino acid C-terminal elongation). Any amino acid sequence that does not interfere with the polypeptide activity can be used. Sequence ID 71, which is the sequence of Sequence ID 69 having a C-terminal extension of NP, and Sequence ID 72, which is the sequence of Sequence ID 69 having a C-terminal extension of NPVTPK, represent two possible ways in which the polypeptide of the present invention can be modified to include a C-terminal extension.

[0058] As used herein, the term “identity percentage (%)” means the percentage of amino acid (or nucleic acid) residues in a candidate sequence, e.g., an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera, that are identical to amino acid (or nucleic acid) residues in a reference sequence, e.g., wild-type extracellular ActRIIA (e.g., SEQ ID NO: 73) or wild-type extracellular ActRIIB (e.g., SEQ ID NO: 74), after aligning sequences to achieve the maximum identity percentage and introducing gaps as necessary (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment for the purpose of determining identity percentage can be achieved in various ways within the scope of the art using publicly available computer software, e.g., BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve the maximum alignment over the entire length of the sequences being compared. In some embodiments, the percentage of amino acid (or nucleic acid) sequence identity of a given candidate sequence to a given reference sequence, or to a given reference sequence (which can be rephrased as a given candidate sequence having, or containing, a specific percentage of amino acid (or nucleic acid) sequence identity to a given reference sequence) is calculated as follows: 100 × (Fraction of A / B) In the formula, A is the number of amino acid (or nucleic acid) residues evaluated as identical in the alignment of the candidate sequence and the reference sequence, and B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where the length of the candidate sequence is not equal to the length of the reference sequence, the percentage of amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence is not equal to the percentage of amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0059] In certain embodiments, the reference sequence aligned for comparison with the candidate sequence can show 50% to 100% identity over the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for the purpose of comparison can be, for example, at least 30%, such as at least 40%, such as at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. A molecule is identical at a position if the position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence.

[0060] As used herein, the term "serum half-life" refers, in the context of administering a therapeutic protein to a subject, to the time required for the plasma concentration of the protein in the subject to decrease by half. The protein can be redistributed from the bloodstream or removed, or degraded, for example, by proteolysis. Comparison of serum half-lives can be done by comparing the serum half-lives of Fc fusion proteins.

[0061] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the total strength of the non-covalent interactions between a molecule and its binding partner, such as an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera, and BMP9 or activin A. Unless otherwise indicated, binding affinity refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is usually described by the dissociation constant (K D ) or the affinity constant (K A ). Two molecules with low binding affinity for each other generally bind slowly and tend to dissociate readily, showing a large K D . Two molecules with high affinity for each other generally bind readily and tend to remain bound longer, showing a small K DThis shows the K of two interacting molecules. D This can be determined using methods and techniques well known in the art, such as surface plasmon resonance. D is, k off / k on It is calculated as a ratio of .

[0062] As used herein, the phrase "affects the signaling of myostatin, activin A, activin B, and / or BMP9" means altering the binding of myostatin, activin A, activin B, and / or BMP9 to their receptors, e.g., ActRIIA, ActRIIB, and / or BMPRII (e.g., ActRIIA or ActRIIB, e.g., endogenous ActRIIA or ActRIIB). In some embodiments, polypeptides comprising extracellular ActRIIA variants, extracellular ActRIIB variants, or extracellular ActRII chimeras described herein reduce or inhibit the binding of myostatin, activin A, activin B, and / or BMP9 to their receptors, e.g., ActRIIA, ActRIIB, and / or BMPRII (e.g., ActRIIA or ActRIIB, e.g., endogenous ActRIIA or ActRIIB).

[0063] As used herein, the terms “increase” and “decrease” refer to adjustments that result in a greater or lesser amount of function, expression, or activity of a metric compared to a reference. For example, following administration of a polypeptide of the present invention, including an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera, the amount of a metric marker described herein (e.g., EPO or EPO receptor level) can be increased in a subject relative to the amount of the marker before administration. Generally, the metric is measured after administration at a point in time when the described effect is obtained by the administration, for example, at least one week, one month, three months, or six months after the start of the treatment regimen.

[0064] As used herein, the term “anemia” refers to an abnormality in hemoglobin or red blood cells that results in a decrease in the oxygen level in the blood. Anemia may be related to an abnormal production, processing, or performance of red blood cells and / or hemoglobin. The term anemia refers to any decrease in the number of red blood cells and / or hemoglobin levels in the blood compared to normal blood levels and can be diagnosed using routine tests such as a complete blood count.

[0065] As used herein, the term “normal hematopoiesis” refers to the process by which components of blood and plasma are produced, including the formation of red blood cells (erythrocytes), white blood cells (leukocytes), including lymphocytes, neutrophils, eosinophils, basophils, and macrophages, and platelets (thrombocytes). A subject is considered to have normal hematopoiesis if the production of these cells is not impaired and the number of these cells in the blood is within a range recognized as normal by a medical professional. For example, the normal range for red blood cells (RBCs) in males is 4.7 million to 6.1 million cells per microliter (mcL), the normal range for RBCs in non-pregnant women is 4.2 million to 5.4 million mcL, and the normal range for RBCs in children is 4 million to 5.5 million mcL.

[0066] As used herein, the term “disease or condition treatable with EPO or ESA” means a disease or condition currently being treated by administering EPO, recombinant EPO, EPO mimetic, or another agent that increases the levels of EPO or EPO receptors, a disease or condition in which, based on studies conducted in cell culture conditions, animal models, or human trials, is expected to benefit from an increase in EPO or EPO receptor levels, or a disease or condition associated with low serum EPO levels. Such diseases and conditions include end-stage renal disease, renal failure, renal dialysis, spinal cord injury, iron overload (e.g., hemochromatosis), inflammatory brain disease, gastrointestinal motility disorders, ischemia, and other diseases and conditions described in U.S. Patent Nos. 5,013,718, 7,745,387, 8,466,172, 8,729,030, and 10,695,402, and U.S. Patent Application Publication Nos. US20180303903A1 and US20170312268A1 (each of which is incorporated herein by reference).

[0067] As used herein, the term “low serum erythropoietin” refers to serum erythropoietin levels below the normal range. Normal erythropoietin levels range from 4 to 26 milliunits per liter (mU / mL).

[0068] Hypercholesterolemia is characterized by elevated cholesterol levels in the blood. The most common form of primary hypercholesterolemia is polygenic hypercholesterolemia. Secondary hypercholesterolemia frequently occurs in association with diabetes mellitus, nephrotic syndrome, hypothyroidism, and liver damage.

[0069] Endothelial-mediated chronic inflammatory disorders are disorders or conditions of the human or animal body that stem from the body's and its tissues' defensive response to harmful stimuli, in which specific signaling molecules alter the properties of endothelial cells, and in conjunction with the activation of other cell types, cause leukocytes to remain attached to the endothelial cells, eventually penetrating the tissue and initiating inflammation. An example of endothelial-mediated inflammation is leukocyte vasculitis. A central role is played in the activation of endothelial-mediated inflammatory events by the transcription factor NF-κB. Another system that leads to the development of endothelial-mediated chronic inflammation is the AGE-RAGE system.

[0070] "Endotheliosis" refers to degenerative and proliferative endothelial changes associated with non-thrombocytopenic purpura. "Reticuloendotheliosis" refers to diseases of the reticulohistiocytoid system, such as reticular endotheliosis, reticulocyte disease, reticular histiocytosis, and Hand-Schuller-Christian disease.

[0071] "Myocardial ischemia" refers to a bloodless or hypoperfusion condition, which is a disorder of blood supply to the muscular wall of the heart resulting from insufficient or absent arterial blood supply. Myocardial infarction is a localized necrosis of the heart muscle, usually occurring as an acute event complicated by chronic coronary heart disease.

[0072] Coronary heart disease, or ischemic heart disease, is a degenerative coronary artery disorder that results in reduced blood supply to the heart muscle due to the constriction or closure of the coronary arteries of the heart. "Angina pectoris" refers to acute coronary insufficiency or angina pectoris that can be induced by an imbalance between oxygen supply and oxygen demand associated with coronary heart disease, coronary artery spasm, impaired blood flow, cardiac arrhythmia, hypertension, or hypotension.

[0073] Raynaud's disease refers to an ischemic condition caused by vasoconstriction, or vasospasm, which usually occurs temporarily in the arteries of the fingers. Primary Raynaud's disease is a pure dysfunction of the small blood vessels that supply nutrients to the distal parts of the limbs, while secondary Raynaud's disease has an underlying condition, such as inflammation of the blood vessels.

[0074] Pre-eclampsia is a maternal endothelial and vascular disorder, thought to be caused by the influence of endotheliotropic substances from the placenta. Pre-eclampsia is a multi-system disorder that can cause dysfunction in multiple organs and manifest in diverse symptoms. The typical impaired blood supply in this disorder is a result of increased vascular resistance and may be accompanied by localized fluctuations in severity. Endothelial dysfunction is considered to be a central component of the etiology of pre-eclampsia.

[0075] Renal failure refers to a condition in which the kidneys' ability to excrete substances normally excreted in urine is limited, and in advanced stages, the ability to regulate electrolytes, fluids, and acid-base balance is also lost. End-stage renal failure is characterized by the breakdown of the kidneys' excretory and endocrine functions.

[0076] Heart failure refers to a pathological condition also known as myocardial dysfunction or weakness. It is characterized by insufficient cardiac function, preventing the heart from efficiently delivering nutrients to meet its requirements. Heart failure can be classified according to various aspects. For example, depending on the affected part of the heart, it can be classified into right heart failure, left heart failure, and bilateral failure (global failure). Compensated and decompensated heart failure are distinguished according to the stability of equilibrium affected by physiological and therapeutic mechanisms. It can also be classified into acute and chronic heart failure based on its progression over time. Causes of heart failure include, among others, myocardial infarction, cardiomyopathy, congenital or acquired heart disease, essential or pulmonary hypertension, cardiac arrhythmias, coronary artery disease, or myocarditis.

[0077] The term "ischemia" refers to a decrease in blood flow. Ischemia is associated with a reduction in oxygen-containing nutrients delivered to tissues. Ischemia can be caused by conditions such as atherosclerosis, the formation of thrombi in arteries or veins, or occlusion of arteries or veins by embolism, or vascular occlusion due to other causes such as vasospasm. Such conditions can reduce blood flow, leading to hypoperfusion of organs and tissues, or even complete blockage of blood flow. Other conditions that can cause ischemia include tissue damage from trauma or injury, such as spinal cord injury or viral infection, which can lead to conditions such as congestive heart failure. The terms "ischemic state" and "ischemic injury" include acute ischemic states such as myocardial infarction, ischemic stroke, pulmonary embolism, perinatal hypoxia, circulatory shock including hemorrhagic, septic, and cardiogenic types, and acute respiratory failure, as well as chronic ischemic states such as atherosclerosis, chronic venous insufficiency, chronic heart failure, cardiac cirrhosis, macular degeneration, sleep apnea syndrome, Raynaud's disease, systemic sclerosis, nonbacterial thrombotic endocarditis, obstructive arterial disease, angina pectoris, TIA, and chronic alcoholic liver disease. Ischemia can occur when an individual is placed under general anesthesia and can cause tissue damage to organs ready for transplantation.

[0078] The terms "hypoxia" and "hypoxia" refer to an environment where oxygen levels are lower than normal. Hypoxia can be induced in cells by culturing them in a hypoxic environment, or by treating cells with compounds that mimic hypoxia. Determining the oxygen level that defines hypoxia in cell culture is within the scope of the skills of those skilled in the art. The terms "hypoxia" and "hypoxia injury" include ischemic injury (ischemic hypoxia) as mentioned above, hypoxia resulting from decreased circulation, chronic obstructive pulmonary disease (COPD), severe pneumonia, pulmonary edema, and lung injuries such as hyaline membrane disease (hypoxic hypoxia), and hypoxia resulting from decreased oxygenation of the blood in the lungs, liver or kidney disease, cancer or other chronic diseases, and altitude sickness.

[0079] Wound healing refers to the physiological process of regenerating damaged tissue and closing a wound, particularly the formation of new connective tissue and capillaries. Wound healing can be primary wound healing (primary healing), characterized by rapid, complication-free closure and substantially complete recovery, resulting from the minimal formation of new connective tissue between the edges of a wound with good blood supply, which, when appropriate, is an approximation of a clean wound. Wounds with further separated edges, especially collapsed or necrotic wounds, and infected wounds undergo secondary healing (secondary healing), where the tissue defect is filled with granulation tissue as a result of (non-)bacterial inflammation, and extensive scar tissue is formed. Epithelialization, beginning at the edges, terminates wound healing. Wound healing is divided into three stages called the incubation phase, the proliferative phase, and the repair phase. Conversely, the incubation period can be divided into the oxidation phase, which involves scab formation, particularly in the first few hours after wound formation, and the absorption phase, which involves catabolic autolysis, lasting 1 to 3 days after wound formation. The proliferative phase is characterized by anabolic repair accompanied by collagen production by fibroblasts and occurs 4 to 7 days after wound formation. The repair phase occurs 8 days after wound formation and is characterized by the transformation of granulation tissue into scar tissue.

[0080] A "wound" refers to a disruption of the coherence of body tissue caused by mechanical or physically induced cellular damage, with or without loss of substance. Types of wounds include mechanical wounds, thermal wounds, chemical wounds, radiation wounds, and disease-related wounds. Mechanical wounds are caused by traumatic violence and occur particularly as incisions and punctures, crushes, tears, rips and abrasions, scratches and bites, and protruding wounds. Burns occur as a result of exposure to heat or cold. Chemical wounds occur particularly as a result of the action of acids or alkalis. Radiation wounds occur, for example, as a result of exposure to chemical and ionizing radiation. Disease-related wounds include congestion-related wounds, traumatic wounds, and diabetic wounds.

[0081] As used herein, the term “inflammatory brain disease or disorder” refers to a disease or disorder of the brain caused by an acute or chronic inflammatory response in the central nervous system. Acute inflammatory responses of the brain include activation of microglia in the central nervous system, the appearance of dendritic cells, and the release of inflammatory cytokines and chemokines. Chronic inflammatory responses include prolonged activation of microglia followed by sustained release of inflammatory mediators. Such prolonged activation of microglia leads to additional microglial activation and proliferation, and further release of inflammatory factors. Examples of chronic inflammatory brain diseases or disorders include demyelinating diseases such as multiple sclerosis, and neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, amyotrophic lateral sclerosis (ALS), and age-related macular degeneration (AMD).

[0082] As used herein, the term “vascular complications” refers to any damage to a blood vessel, such as vascular disorders or damage to the vessel wall. Damage to the vessel wall can result in increased vascular permeability or leakage. The term “vascular permeability or leakage” refers to the ability of the vessel wall to allow the flow of small molecules, proteins, and cells into and out of the vessel. Increased vascular permeability or leakage can be caused by an increase in the gaps between endothelial cells lining the inside of the vessel wall (e.g., an increase in the size and / or number of gaps) and / or thinning of the vessel wall.

[0083] As used herein, the term "polypeptide" refers to a single polymer in which monomers are amino acid residues covalently linked to one another via amide bonds. Polypeptides are intended to encompass any amino acid sequence that is naturally occurring, recombinant, or synthetically produced.

[0084] As used herein, the term “homodimer” refers to a molecular construct formed by two identical macromolecules, such as proteins or nucleic acids. Two identical monomers may form a homodimer by covalent or non-covalent bonds. For example, an Fc domain can be a homodimer of two Fc domain monomers if the two Fc domain monomers contain the same sequence. In another example, polypeptides described herein, including an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera fused to an Fc domain monomer, may form a homodimer through the interaction of two Fc domain monomers that form an Fc domain in the homodimer.

[0085] As used herein, the term “heterodimer” refers to a molecular construct formed by two different macromolecules, such as proteins or nucleic acids. Two monomers may form a heterodimer by covalent or non-covalent bonds. For example, polypeptides described herein, comprising an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera fused to an Fc domain monomer, may form a heterodimer through the interaction of two Fc domain monomers, each fused to a different extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera, forming an Fc domain in the heterodimer (for example, the heterodimer may include two different extracellular ActRIIA variants, two different extracellular ActRIIB variants, or two different extracellular ActRII chimeras).

[0086] As used herein, the term “host cell” refers to a vehicle containing the necessary cellular components, such as organelles, required to express proteins from the corresponding nucleic acid. The nucleic acid is typically contained in a nucleic acid vector that can be introduced into a host cell by conventional techniques known in the art (such as transformation, transfection, electroporation, calcium phosphate precipitation, or direct microinjection). The host cell may be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a mammalian cell (e.g., a CHO cell or a HEK293 cell).

[0087] As used herein, the term “therapeutic effective dose” means an amount of the polypeptide, nucleic acid, or vector of the present invention, or a pharmaceutical composition comprising the polypeptide, nucleic acid, or vector of the present invention, that is effective in achieving a desired therapeutic effect in treating a patient who has, or is at risk of developing, a disease or condition treatable with EPO or ESA (e.g., end-stage renal disease, renal failure, renal dialysis, spinal cord injury, iron overload (such as hemochromatosis), inflammatory brain disease, ischemia, or gastrointestinal motility disorder). In particular, the therapeutic effective dose of the polypeptide, nucleic acid, or vector avoids adverse side effects.

[0088] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or pharmaceutical preparation comprising an active ingredient, as well as excipients and diluents for making the active ingredient suitable for a method of administration. The pharmaceutical compositions of the present invention comprise a polypeptide, nucleic acid, or pharmaceutically acceptable component compatible with a vector. The pharmaceutical compositions may be in the form of tablets or capsules for oral administration, or in aqueous form for intravenous or subcutaneous administration.

[0089] As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to an excipient or diluent in a pharmaceutical composition. A pharmaceutically acceptable carrier must be compatible with the other components of the formulation and must not be harmful to the recipient. In the present invention, a pharmaceutically acceptable carrier or excipient must provide adequate pharmaceutically stable to a polypeptide, nucleic acid molecule(s) encoding a polypeptide, or vector(s) containing an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera. The properties of the carrier or excipient vary depending on the method of administration. For example, aqueous carriers are generally used for intravenous administration. For oral administration, solid carriers are preferred.

[0090] As used herein, the term “to treat and / or prevent” refers to the treatment and / or prevention of a disease or condition treatable with EPO or ESA, such as end-stage renal disease, renal failure, renal dialysis, spinal cord injury, iron overload (e.g., hemochromatosis), inflammatory brain disease, ischemia, or gastrointestinal motility disorders, using the methods and compositions of the present invention. Generally, treating a disease or condition treatable with EPO or ESA is done after the subject has developed the disease or condition. Preventing a disease or condition treatable with EPO or ESA refers to a step or procedure taken when the subject is at risk of developing the disease or condition. The subjects are those who may exhibit signs or mild symptoms that a physician judges to be indicators or risk factors for developing a disease or condition treatable with EPO or ESA, who may have another disease or condition related to a disease or condition treatable with EPO or ESA, who may be receiving treatment that may cause a disease or condition treatable with EPO or ESA, or who may have a family history or genetic predisposition to developing a disease or condition treatable with EPO or ESA, but have not yet developed that disease or condition.

[0091] As used herein, the term “subject” refers to mammals, for example, preferably humans. Mammals include, but are not limited to, humans and domesticated animals and livestock such as monkeys (e.g., crab-eating macaques), mice, dogs, cats, horses, and cattle. [Brief explanation of the drawing]

[0092] [Figure 1] This is a sequence alignment showing the wild-type sequences of extracellular ActRIIA and ActRIIB, as well as the sequences of exemplary extracellular ActRIIA variants. [Figure 2] This is a sequence alignment showing the wild-type sequences of extracellular ActRIIA and ActRIIB, as well as amino acid substitutions in exemplary extracellular ActRIIB variants. [Figure 3] This is a sequence alignment showing the sequence of an exemplary extracellular ActRII chimera. [Figure 4] This is a series of graphs showing that a single 10 mg / kg dose of ActRIIA / B-mFc (SEQ ID NO: 69 linked to the mouse Fc domain), administered intraperitoneally (IP) to 11-week-old male mice, increased serum EPO levels 4, 7, and 14 days after injection compared to the vehicle. Red blood cell counts increased at each time point. Two-factor ANOVA and subsequent Sidak post-hoc tests were performed between ActRIIA / B-mFc treatment and the vehicle at each time point. *p<0.05, **p<0.01, ****p<0.0001. Data are shown as mean ± SEM. [Figure 5] This graph shows that a single dose of ActRIIA / B-mFc administered intraperitoneally (IP) to 11-week-old male mice, at a dose of 10 mg / kg, had a long-lasting effect on EPO, increasing serum EPO levels from day 4 to day 37. T-tests were performed at each time point between ActRIIA / B-mFc treatment and vehicle use: *p≦0.05;**p≦0.01;p****≦0.0001. Data are shown as mean ± SEM. [Figure 6]This series of graphs shows that a single dose of 10 mg / kg of ActRIIA / B-mFc administered by IP injection to 11-week-old male mice increased EPO receptor levels in bone marrow cells on days 7 and 14 after injection, compared to the vehicle. Error bars represent SEM. [Modes for carrying out the invention]

[0093] The present invention features polypeptides comprising extracellular activin receptor type II (ActRII) variants, such as extracellular ActRIIA variants, extracellular ActRIIB variants, or extracellular ActRII chimeras. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRII variant fused to a portion (e.g., an Fc domain monomer, an Fc domain (e.g., a wild-type Fc domain), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin). Polypeptides comprising an extracellular ActRII variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interactions between two Fc domain monomers. The ActRII variants described herein have weak or no binding affinity to bone morphogenetic protein 9 (BMP9) compared to activin (e.g., activin A and / or activin B) and myostatin. The present invention also includes a method for treating diseases and conditions treatable with EPO or ESA (for example, diseases or conditions treatable by increasing the level of EPO or EPO receptors), wherein the subject is administered a polypeptide comprising the extracellular ActRII variant described herein.

[0094] I. Extracellular activin receptor type II (ActRII) variant Activin type II receptors are single transmembrane domain receptors that regulate signaling for ligands of the transforming growth factor β (TGF-β) superfamily. Ligands of the TGF-β superfamily are involved in many physiological processes, including muscle growth, blood vessel growth, cell differentiation, homeostasis, hematopoiesis, and bone formation. Examples of TGF-β superfamily ligands include activin A, activin B, inhibin, growth and differentiation factors (GDFs) (e.g., GDF8 and GDF11, also known as myostatins), and bone morphogenetic proteins (BMPs) (e.g., BMP9).

[0095] There are two types of activin type II receptors: ActRIIA and ActRIIB. Studies have shown that BMP9 binds to ActRIIB with approximately 300 times higher binding affinity than ActRIIA (see, for example, Townson et al., J. Biol. Chem. 287:27313, 2012). ActRIIA-Fc is known to have a longer half-life than ActRIIB-Fc. The following three types of ActRII variants are described herein: 1) Extracellular ActRIIA variants constructed by introducing amino acid residues of ActRIIB into ActRIIA with the aim of conferring the physiological properties conferred by ActRIIB while maintaining the beneficial physiological and pharmacokinetic properties of ActRIIA; 2) Extracellular ActRIIB variants constructed by introducing amino acid residues of ActRIIA into ActRIIB or by introducing novel amino acid substitutions with the aim of reducing BMP9 binding in order to prevent or reduce disruption of endogenous BMP9 signaling; 3) Extracellular ActRII chimeras constructed by combining parts of extracellular ActRIIA and ActRIIB with the aim of producing proteins that bind to ActRII ligands (e.g., activin A, activin B, myostatin, and GDF11) and maintain the function of the wild-type extracellular ActRII protein.

[0096] This invention is partly based on the finding that administration of the ActRIIA variant-containing polypeptide described herein (ActRIIA variant-Fc polypeptide) increased serum EPO levels in mice in the absence of compensatory downregulation of bone marrow EPO receptors. Administration of ActRIIA variant-Fc polypeptide increased both serum EPO levels and EPO receptor levels in bone marrow progenitor cells. The increase in serum EPO was observed at 4 days post-injection and persisted for at least 14 days post-injection, while the increase in bone marrow EPO receptor expression was observed at 7 and 14 days post-injection. These data suggest that not only can ActRIIA variant-containing polypeptides be used to increase EPO and EPO receptor levels and thus as an alternative to EPO therapy, but based on the sustained increases in EPO and EPO receptor levels observed in these studies, ActRIIA variant-containing polypeptides can be administered at a lower frequency than current EPO therapies. Reducing the frequency of administration could significantly improve patient convenience and potentially reduce side effects. Therefore, polypeptides comprising the ActRIIA variants described herein can be used to treat diseases or conditions that can be treated with EPO or ESA.

[0097] Actriia variant In some embodiments, ActRII variants for use according to the methods described herein are extracellular ActRIIA variants constructed by introducing amino acid residues of ActRIIB into ActRIIA, with the aim of conferring the physiological properties conferred by ActRIIB while maintaining the beneficial physiological and pharmacokinetic properties of ActRIIA. Preferred ActRIIA variants exhibit similar or improved binding to activin and / or myostatin compared to wild-type extracellular ActRIIA, are configured to compete with the endogenous activin receptor for ligand binding, and reduce or inhibit endogenous activin receptor signaling. In some embodiments, amino acid substitutions may be introduced into the extracellular ActRIIA variant to reduce or eliminate the variant's binding affinity to BMP9. The wild-type amino acid sequences of the extracellular portions of human ActRIIA and ActRIIB are shown below.

[0098] Human ActRIIA, extracellular component (SEQ ID NO: 73): GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGC WLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS Human ActRIIB, extracellular component (SEQ ID NO: 74): GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWL DDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT The polypeptides described herein include extracellular ActRIIA variants having at least one amino acid substitution compared to wild-type extracellular ActRIIA having the sequence of SEQ ID NO: 73 or extracellular ActRIIA having any one of the sequences of SEQ ID NOs: 76-96. 27 possible amino acid substitutions at different positions can be introduced into the extracellular ActRIIA variant (Table 1). In some embodiments, the extracellular ActRIIA variant may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97%, or more) amino acid sequence identity with respect to the wild-type extracellular ActRIIA sequence (SEQ ID NO: 73). Extracellular ActRIIA variants may have one or more amino acid substitutions (e.g., 1-27, 1-25, 1-23, 1-21, 1-19, 1-17, 1-15, 1-13, 1-11, 1-9, 1-7, 1-5, 1-3, or 1-2; e.g., 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, or 27) compared to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73). In some embodiments, an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of SEQ ID NO: 1) may contain amino acid substitutions at all 27 positions listed in Table 1. In some embodiments, the extracellular ActRIIA variant may contain amino acid substitutions at multiple positions, for example, at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 of the 27 positions listed in Table 1.

[0099] Amino acid substitutions can worsen or improve the activity and / or binding affinity of the ActRIIA variant of the present invention. To maintain the function of the polypeptide, position X of the sequences shown in Tables 1 and 2 (SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72)) is required. 17 It is important that the lysine (K) group is retained. Substitution at that position can lead to a loss of activity. For example, the sequence ActRIIA variants containing GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 149) show reduced activity in vivo, which is X 17 This indicates that substitution of alanine (A) with lysine (K) is not permitted. Therefore, the ActRIIA variants of the present invention, including the variants in Tables 1 and 2 (e.g., SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72)), are at position X 17 It retains amino acid K.

[0100] The ActRIIA variant of the present invention preferably has reduced, weak, or substantially no binding to BMP9. BMP9 binding is X 23 , X 24 , X 25 , and X 26 An ActRIIA variant containing the amino acid sequence TEEN (SEQ ID NO: 374) at position X, and position X 24 Maintain amino acid K at position X 23 , X 24 , X 25 , and X 26 In variants having the amino acid sequence TKEN (SEQ ID NO: 375), the amount of BMP9 binding is reduced (for example, reduced compared to wild-type ActRIIA). Sequences TEEN (SEQ ID NO: 374) and TKEN (SEQ ID NO: 375) can be interchangeably used in the ActRIIA variants of the present invention (for example, variants of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72) in Tables 1 and 2) to provide reduced BMP9 binding.

[0101] The ActRIIA variants of the present invention may further include C-terminal elongation (e.g., additional amino acids at the C-terminus). C-terminal elongation can involve adding one or more additional amino acids (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) to the C-terminus of any of the variants shown in Tables 1 and 2 (e.g., SEQ ID NOs. 1-70 (e.g., SEQ ID NOs. 6-70)). C-terminal elongation may correspond to sequences from the same position in wild-type ActRIIA. One possible C-terminal elongation that can be included in the ActRIIA variants of the present invention is the amino acid sequence NP. For example, a sequence containing the C-terminal elongation NP is SEQ ID NOs. 71 (e.g., SEQ ID NOs. 69 with the C-terminal elongation of NP). Another exemplary C-terminal elongation that can be included in the ActRIIA variants of the present invention is the amino acid sequence NPVTPK (SEQ ID NOs. 154). For example, a sequence containing the C-terminal elongation NPVTPK is SEQ ID NOs. 72 (e.g., SEQ ID NOs. 69 with the C-terminal elongation of NPVTPK).

[0102] [Table 1-1]

[0103] [Table 1-2]

[0104] In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 or 2, X3 is E, X6 is R, and X 11 is D, X 12 is K, and X 13 is R, and X 16 is K or R, X 17 is K, and X 19 is W, and X 20 L is X 21 is D, X 22 is I or F. In some embodiments of the extracellular ActRIIA variant having the sequence of Sequence ID No. 1, X2 is Y; X4 is L; X8 is E; X9 is E; X 14is L; X 18 is K; X 23 is T; X 25 is E; X 26 is N and X 27 is Q. These substitutions in SEQ ID NO: 1 can also be made in SEQ ID NOs: 2-5. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1, X1 is F or Y; X2 is Y; X4 is L; X5 is D or E; X7 is P or R; X8 is E; X9 is E; X 10 is K or Q; X 14 is L; X 15 is F or Y; X 16 is K or R; X 18 is K; X 22 is I or F; X 23 is T; X 24 is K or E; X 25 is E; X 26 is N and X 27 is Q. In some embodiments of the extracellular ActRIIA variant having the sequence of Sequence ID No. 1, X1 is F or Y; X2 is Y; X3 is E; X4 is L; X5 is D or E; X6 is R; X7 is P or R; X8 is E; X9 is E; X 10 is K or Q; X 11 is D; X 12 is K; X 13 is R and X 14 is L; X 15 is F or Y; X 16 is K or R; X 17 is K; X 18 is K; X 19 is W; X 20 is L; X 21 is D; X 22 is I or F; X 23 is T; X 24 is K or E; X 25 is E; X 26 is N and X 27is Q. In some embodiments of extracellular ActRIIA variants having sequence number 1 or 2, X 17 is K. In some embodiments of extracellular ActRIIA variants having sequences of sequence numbers 1-3, X 17 is K, and X 23 is T, and X 24 is E, and X 25 is E, and X 26 is N. In some embodiments of extracellular ActRIIA variants having one of sequence numbers 1-5, X 17 is K, and X 23 is T, and X 24 is K, and X 25 is E, and X 26 It is N.

[0105] In some embodiments, the polypeptides described herein include an extracellular ActRIIA variant having one of the sequences of SEQ ID NOs: 6-72 (Table 2).

[0106] [Table 2-1]

[0107] [Table 2-2]

[0108] [Table 2-3]

[0109] [Table 2-4]

[0110] [Table 2-5]

[0111]

Table 2-6

[0112]

Table 2-7

[0113]

Table 2-8

[0114]

Table 2-9

[0115] In some embodiments, the polypeptide of the present invention comprising an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) has an amino acid K at position X 17 Changing the amino acid at position X 17 may reduce the activity. For example, the ActRIIA variant having the sequence GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 149) has reduced activity in vivo, indicating that substitution of A with K at X 17 is not tolerated.

[0116] In some embodiments, the polypeptide of the present invention comprising an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) having the sequence TEEN (SEQ ID NO: 374) at positions X 23 X 24 X 25 and X 26 can substitute the amino acid E at position X 24 with an amino acid K. In some embodiments, X 23 X24 and X 25 and X 26 The polypeptide of the present invention comprising an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) containing the array TKEN (array number 375) at the position of X 24 can substitute the amino acid K at position X with the amino acid E. X 23 and X 24 and X 25 and X 26 The polypeptide having the array TEEN (array number 374) or TKEN (array number 375) at the position of X has a reduced or weak binding to BMP9 (e.g., a reduced binding to BMP9 compared to the BMP9 binding of wild-type ActRIIA).

[0117] In some embodiments, the polypeptide of the present invention comprising an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)) may further comprise a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension may correspond to the sequence from the same position in wild-type ActRIIA. In some embodiments, the C-terminal extension is the amino acid sequence NP. For example, the sequence comprising the C-terminal extension NP is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 having the C-terminal extension of NP). In some embodiments, the C-terminal extension is the amino acid sequence NPVTPK (SEQ ID NO: 154). For example, the sequence comprising the C-terminal extension NPVTPK is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 having the C-terminal extension of NPVTPK). The C-terminal extension can add one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus.

[0118] In some embodiments, the polypeptide of the present invention comprising an extracellular ActRIIA variant may further comprise a moiety (e.g., an Fc domain monomer, an Fc domain (e.g., a wild-type Fc domain), an Fc domain containing amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) that can be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular ActRIIA variant by a linker or other covalent bond. The polypeptide comprising an extracellular ActRIIA variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) through interactions between two Fc domain monomers that bind to form a dimeric Fc domain.

[0119] In some embodiments, the extracellular ActRIIA variants described herein do not have any one of the sequences SEQ ID NOs. 76-96 shown in Table 3 below.

[0120] [Table 3-1]

[0121] [Table 3-2]

[0122] [Table 3-3]

[0123] Furthermore, in some embodiments, the polypeptides described herein (e.g., ActRIIA variant-Fc fusion proteins) have a serum half-life of at least 7 days in humans. Polypeptides containing extracellular ActRIIA variants have a K2 level of 10 pM or higher. DIt can bind to activin A at position X 23 , X 24 , X 25 , and X 26 It has the sequence TEEN (SEQ ID NO: 374) or TKEN (SEQ ID NO: 375). In some embodiments, polypeptides containing the extracellular ActRIIA variant do not substantially bind to human BMP9.

[0124] In some embodiments, polypeptides containing the extracellular ActRIIA variant have a K content of approximately 800 pM or less. D (For example, K values ​​of approximately 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 pM) D For example, K at approximately 800 pM to 200 pM D ) can bind to human activin A. In some embodiments, polypeptides containing the extracellular ActRIIA variant have a K of 800 pM or less. D (For example, K values ​​of approximately 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 pM) D For example, K at approximately 800 pM to 200 pM D ) can bind to human activin B. Polypeptides containing the extracellular ActRIIA variant also have a K of approximately 5 pM or greater. D(For example, K levels of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher) D It can bind to growth and differentiation factor 11 (GDF-11) in this way.

[0125] ActRIIB variant In some embodiments, ActRII variants for use according to the methods described herein are extracellular ActRIIB variants constructed by introducing amino acid residues of ActRIIA into ActRIIB, or by introducing novel amino acid substitutions into ActRIIB, with the aim of reducing BMP9 binding and preventing or mitigating disruption of endogenous BMP9 signaling. Amino acid substitutions can also confer beneficial physiological and pharmacokinetic properties of ActRIIA, such as a longer half-life as an Fc fusion protein. Preferred ActRIIB variants exhibit similar or improved binding to activin and / or myostatin compared to wild-type extracellular ActRIIB, are configured to compete with the endogenous activin receptor for ligand binding, and reduce or inhibit endogenous activin receptor signaling. In some embodiments, amino acid substitutions may be introduced into extracellular ActRIIB variants to reduce or eliminate the variant's binding affinity to BMP9. Polypeptides containing the ActRIIB variant described herein can be used to increase the levels of EPO and EPO receptors and therefore can be used as an alternative to EPO therapy. These polypeptides can also be administered at a lower frequency than current EPO therapy, which may significantly improve patient convenience and reduce side effects. Therefore, polypeptides containing the ActRIIB variant described herein can be used to treat diseases or conditions that can be treated with EPO or ESA.

[0126] The polypeptides described herein include extracellular ActRIIB variants having at least one amino acid substitution compared to wild-type extracellular ActRIIB having the sequence of SEQ ID NO: 74. Twenty-eight possible amino acid substitutions at different positions can be introduced into the extracellular ActRIIB variants (Table 4). Extracellular ActRIIB variants may have one or more amino acid substitutions (e.g., 1-28, 1-25, 1-23, 1-21, 1-19, 1-17, 1-15, 1-13, 1-11, 1-9, 1-7, 1-5, 1-3, or 1-2; e.g., 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, or 28) compared to the sequence of wild-type extracellular ActRIIB (SEQ ID NO: 74). In some embodiments, an extracellular ActRIIB variant (e.g., an extracellular ActRIIB having the sequence of SEQ ID NO: 1) may contain amino acid substitutions at all 28 positions listed in Table 4. In some embodiments, the extracellular ActRIIB variant may include amino acid substitutions at multiple positions, e.g., at positions 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 18, 20, 22, 24, 26, or 27 of the 28 positions listed in Table 4. In some embodiments, the substitution is an amino acid substitution from ActRIIA to the same position as ActRIIB. In some embodiments, the substitution is a novel change (e.g., an amino acid substitution that is not at the corresponding position in ActRIIA, e.g., S48T, I51L, Q69D, or E70T).

[0127] Amino acid substitutions can worsen or improve the activity and / or binding affinity of the ActRIIB variant of the present invention (for example, an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (for example, SEQ ID NOs. 158-171)). In some embodiments, amino acid substitutions worsen the binding affinity of the ActRIIB variant to BMP9 (e.g., the variant has reduced binding to BMP9 compared to wild-type extracellular ActRIIB, or has lower binding to BMP9 than other ActRIIB ligands (e.g., activin A or B, myostatin, or GDF-11)). In some embodiments, the ActRIIB variant has reduced, weak, or virtually no binding to BMP9. In some embodiments, amino acid substitutions improve the binding affinity of ActRIIB to myostatin, activin A or B, and / or GDF-11 (e.g., the variant has improved binding affinity compared to wild-type extracellular ActRIIB, or has lower binding affinity to myostatin, activin A or B, or GDF-11 than to BMP9). (Strongly binds to -11). In some embodiments, the amino acid substitution reduces the binding affinity of ActRIIB to myostatin, activin A or B, and / or GDF-11 (e.g., the variant has reduced binding affinity compared to wild-type extracellular ActRIIB, or binds weaker to myostatin, activin A or B, or GDF-11 than BMP9). In some embodiments, the amino acid substitution does not substantially alter the function of extracellular ActRIIB (e.g., the ActRIIB variant is functionally equivalent to wild-type extracellular ActRIIB). In some embodiments, the amino acid substitution confers ActRIIA properties or activity to the ActRIIB variant (e.g., the ActRIIB variant, as an Fc fusion protein, has a longer half-life than WT extracellular ActRIIB-Fc).Preferably, the ActRIIB variant has one, two or more, or three or more of the above characteristics (e.g., reduced BMP9 binding and improved binding to activin A or B, myostatin, and / or GDF-11, or reduced BMP9 binding and functional equivalence with wild-type ActRIIB).

[0128] The ActRIIB variant of the present invention (for example, an extracellular ActRIIB variant having any one sequence from SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171)) preferably has one or more amino acid substitutions that reduce BMP9 binding. In some embodiments, the amino acid substitution that reduces BMP9 binding is E75K (e.g., X in SEQ ID NO. 157) 24 (is K). In some embodiments, the amino acid substitutions that reduce BMP9 binding are Q69T and E70D (e.g., X in SEQ ID NO: 157). 21 is T, and X 22 (is D). In some embodiments, the amino acid substitutions that reduce BMP9 binding are Q69D and E70T (for example, X in SEQ ID NO: 157). 21 is D, X 22 ( is T). In some embodiments, amino acid substitutions that reduce BMP9 linkage are T74K, E75K, E76D, N77S, and Q79E (for example, X in SEQ ID NO: 157). 23 , X 24 , X 25 , X 26 , and X 28(These are K, K, D, S, and E, respectively). In some embodiments, the ActRIIB variant has more than one of the aforementioned amino acid substitutions that reduce BMP9 binding (e.g., substitution E75K with substitutions Q69D and E70T, or substitution E75K with substitutions Q69T and E70D). In some embodiments, the ActRIIB variant of the present invention has one or more amino acid substitutions that reduce BMP9 binding and one or more additional amino acid substitutions. The additional amino acid substitutions may confer other beneficial properties, such as altered binding to activin or myostatin or improved activity. For example, amino acid substitutions T74K, E75K, E76D, N77S, and Q79E result in decreased ActRIIB variant activity (e.g., compared to wild-type extracellular ActRIIB), but the inclusion of additional substitutions S25T and S47I, E31Y, E33D, and Q34K, or Y41F, R45K, and K56Q, enhances the effects of the ActRIIB variant. Additional amino acid substitutions may include one or more of the following substitutions: I11L, Y12F, L19K, E20D, S25T, L27V, R29P, E31Y, E33D, Q34K, L38R, Y41F, R45K, S47I, S48T, T50S, I51L, L53I, K56Q, and F63I, T74K, E76D, N77S, Q79E, or F89M.

[0129] In some embodiments, the polypeptides described herein include an extracellular ActRIIB variant having the sequence of SEQ ID NO: 157.

[0130] [Table 4]

[0131] In some embodiments, the polypeptides described herein include an extracellular ActRIIB variant having one of the sequences of SEQ ID NOs: 158-171 (Table 5).

[0132] [Table 5-1]

[0133] [Table 5-2]

[0134] In some embodiments, the extracellular ActRIIB variants described herein have an N-terminal truncation of 1 to 7 amino acids (e.g., amino acids 1, 2, 3, 4, 5, 6, or 7). The N-terminal truncation may include the removal of 1 to 7 amino acids from the N-terminus of any ActRIIB variant shown in Tables 4 and 5. The N-terminal truncation may remove up to two amino acids prior to the first cysteine ​​(e.g., the two amino acids prior to the first cysteine ​​(RE) are retained in the N-terminally truncated ActRIIB variant).

[0135] In some embodiments, the polypeptide of the present invention comprising an extracellular ActRIIB variant may further comprise a moiety (e.g., an Fc domain monomer, an Fc domain (e.g., a wild-type Fc domain), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) that can be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular ActRIIB variant by a linker or other covalent bond. The polypeptide comprising an extracellular ActRIIB variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) through interactions between two Fc domain monomers that bind to form the Fc domain of the dimer.

[0136] Furthermore, in some embodiments, the polypeptides described herein (e.g., ActRIIB variant-Fc fusion proteins) have a serum half-life of at least 7 days in humans. Polypeptides containing extracellular ActRIIB variants have a K2 level of 10 pM or higher. DIt can bind to activin A. In some embodiments, polypeptides containing the extracellular ActRIIB variant do not bind to BMP9 or activin A. In some embodiments, polypeptides containing the extracellular ActRIIB variant bind to activin A, activin B, and / or myostatin and show reduced (e.g., weak) binding to BMP9. In some embodiments, polypeptides containing the extracellular ActRIIB variant do not bind substantially to human BMP9.

[0137] In some embodiments, polypeptides containing the extracellular ActRIIB variant have a K content of approximately 800 pM or less. D (For example, K values ​​of approximately 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 pM) D For example, K at approximately 800 pM to 200 pM D ) can bind to human activin A. In some embodiments, polypeptides containing the extracellular ActRIIB variant have a K of 800 pM or less. D (For example, K values ​​of approximately 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 pM) D For example, K at approximately 800 pM to 200 pM D ) can bind to human activin B. Polypeptides containing extracellular ActRIIB variants also contain K at approximately 5 pM or higher. D (For example, K levels of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher) D It can bind to growth and differentiation factor 11 (GDF-11) in this way.

[0138] ActRII Chimera In some embodiments, ActRII variants for use according to the methods described herein are extracellular ActRII chimeras constructed by combining extracellular ActRIIA and ActRIIB portions, with the aim of producing a protein that binds to ActRII ligands (e.g., activin A, activin B, myostatin, and GDF11) and retains the function of the wild-type extracellular ActRII protein. In some embodiments, the ActRII chimeras exhibit reduced BMP9 binding compared to wild-type extracellular ActRIIB, which can prevent or reduce disruption of endogenous BMP9 signaling. In some embodiments, the chimeras possess the properties of both ActRIIA (e.g., low binding affinity to BMP9 and / or a longer serum half-life as an Fc fusion protein) and ActRIIB (e.g., strong binding affinity to activin A and B). ActRII chimeras exhibit similar or improved binding to activin (e.g., activin A and / or activin B) and / or myostatin compared to wild-type extracellular ActRIIA and / or ActRIIB, and are configured to compete with endogenous activin receptors for ligand binding, thereby reducing or inhibiting endogenous activin receptor signaling. Polypeptides containing ActRII chimeras as described herein can be used to increase EPO and EPO receptor levels and therefore can be used as an alternative to EPO therapy. These polypeptides can also be administered at lower frequencies than current EPO therapy, which may significantly improve patient convenience and reduce side effects. Therefore, polypeptides containing ActRII chimeras as described herein can be used to treat diseases or conditions that can be treated with EPO or ESA.

[0139] The polypeptides described herein include extracellular ActRII chimeras containing sequences from both the extracellular portion of ActRIIB and the extracellular portion of ActRIIA. The ActRII chimeras described herein result from ligating the N-terminal portion of extracellular ActRIIB (SEQ ID NO: 74 shown above) to the C-terminal portion of extracellular ActRIIA (SEQ ID NO: 73 shown above) to make the sequences continuous (for example, the ActRIIA sequence continues where the ActRIIB sequence was interrupted and begins with the next amino acid at the corresponding position in ActRIIA). In some embodiments, the N-terminus of the ActRII chimera contains six amino acids found at the N-terminus of extracellular ActRIIA, ligated to the fifth amino acid of extracellular ActRIIB. In some embodiments, the N-terminus of the ActRII chimera begins with the first amino acid located at the N-terminus of extracellular ActRIIB. Therefore, in some embodiments, the N-terminal portion of ActRIIB begins with amino acid (A) at the fifth position of SEQ ID NO: 74, while in other embodiments (e.g., embodiments in which the six amino acids found at the N-terminus of extracellular ActRIIA are not included in the chimera), the N-terminal portion of ActRIIB begins with amino acid (G) at the first position of SEQ ID NO: 74. In some embodiments, the N-terminus of the ActRII chimera includes the first 10 amino acids found at the N-terminus of extracellular ActRIIA linked to the ninth amino acid of extracellular ActRIIB, in which case the N-terminal portion of ActRIIB begins with amino acid (E) at the ninth position of SEQ ID NO: 74. The extracellular ActRII chimera may also include one or more amino acid substitutions in the chimeric portion corresponding to the sequence of ActRIIB compared to the wild-type extracellular ActRIIB (e.g., SEQ ID NO: 74 above), and one or more amino acid substitutions in the chimeric portion corresponding to the sequence of ActRIIA compared to the sequence of wild-type extracellular ActRIIA (e.g., SEQ ID NO: 73 above). Amino acid substitutions at nine different positions can be introduced into the extracellular ActRII chimera (Table 6).An extracellular ActRII chimera may have one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) relative to the wild-type sequence (for example, if the chimeric portion corresponds to the wild-type extracellular ActRIIB region, relative to the wild-type extracellular ActRIIB sequence (SEQ ID NO: 74), or if the chimeric portion corresponds to the wild-type extracellular ActRIIA region, relative to the wild-type extracellular ActRIIA sequence (SEQ ID NO: 73)). Table 6 lists the positions where amino acid substitutions can occur and the amino acids that can be substituted at these positions.

[0140] Amino acid substitutions can alter the activity and / or binding affinity of the extracellular ActRII chimeras of the present invention. In some embodiments, the extracellular ActRII chimeras bind to activin A, activin B, myostatin, and / or GDF11 with sufficient affinity to compete with the endogenous activin receptor for binding to one or more of these ligands. In some embodiments, the extracellular ActRII chimeras of the present invention exhibit reduced, weak, or virtually no binding to BMP9 (compared to, for example, wild-type ActRIIB). BMP9 binding may be reduced in extracellular ActRII chimeras containing the amino acid sequences TEEN (SEQ ID NO: 374) or TKEN (SEQ ID NO: 375) at positions X3, 4, X5, and X6. In some embodiments, the polypeptide of the present invention, comprising an extracellular ActRII chimera (e.g., any one of SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)) having the sequence TEEN (SEQ ID NO: 374) at positions X3, X4, X5, and X6, can have amino acid E at position X4 substituted with amino acid K. In some embodiments, the polypeptide of the present invention, comprising an extracellular ActRII chimera (e.g., any one of SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)) having the sequence TKEN (SEQ ID NO: 375) at positions X3, X4, X5, and X6, can have amino acid K at position X4 substituted with amino acid E. The sequences TEEN (SEQ ID NO: 374) and TKEN (SEQ ID NO: 375) can be used interchangeably in the extracellular ActRII chimera of the present invention (e.g., the chimeras of Tables 6 and 7, e.g., SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)).

[0141] The extracellular ActRII chimeras of the present invention may further include C-terminal elongation (e.g., additional amino acids at the C-terminus). C-terminal elongation can be achieved by adding one or more additional amino acids (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) to the C-terminus of any of the chimeras shown in Tables 6 and 7 (e.g., SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)). C-terminal elongation may correspond to sequences from the same position in wild-type ActRIIA or ActRIIB. For example, C-terminal elongations that can be included in the extracellular ActRII chimeras of the present invention are amino acid sequences NP and NPVTPK (SEQ ID NOs. 154), which correspond to sequences found at the same position in wild-type ActRIIA.

[0142] [Table 6-1]

[0143] [Table 6-2]

[0144] [Table 6-3]

[0145] In some embodiments, in the extracellular ActRII chimeras of sequence numbers 174-216 (shown in Table 6), X1 is D, X2 is I, F, or E, X3 is N or T, X4 is A or E, X5 is T or K, X6 is E or K, X7 is E or D, X8 is N or S, and X9 is E or Q.

[0146] In some embodiments, the polypeptides described herein include extracellular ActRII chimeras having any one sequence of SEQ ID NOs: 195-216 (Table 7).

[0147] [Table 7-1]

[0148] [Table 7-2]

[0149] [Table 7-3]

[0150] In some embodiments, the extracellular ActRII chimeras described herein have an N-terminal truncation of 1 to 9 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). The N-terminal truncation may involve the removal of 1 to 9 amino acids from the N-terminus of any of the chimeras shown in Tables 6 and 7 (e.g., SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)). The N-terminal truncation can remove up to two amino acids prior to the first cysteine ​​(e.g., the two amino acids prior to the first cysteine ​​(RE or QE) are retained in the N-terminally truncated ActRII chimera). Exemplary ActRII chimeras having N-terminal truncation are shown in Table 8 below.

[0151] [Table 8-1]

[0152] [Table 8-2]

[0153] [Table 8-3]

[0154] [Table 8-4]

[0155] [Table 8-5]

[0156] [Table 8-6]

[0157] [Table 8-7]

[0158] [Table 8-8]

[0159] In some embodiments, the polypeptide of the present invention, comprising an extracellular ActRII chimera, may further comprise a moiety (e.g., an Fc domain monomer, an Fc domain (e.g., a wild-type Fc domain), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) that can be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular ActRII chimera by a linker or other covalent bond. A polypeptide comprising an extracellular ActRII chimera fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) through interactions between two Fc domain monomers that bind to form a dimeric Fc domain. Exemplary polypeptides comprising the ActRII chimera, Fc domain, and linker are shown in Table 9 below. In some embodiments, terminal lysine is not found in the Fc domain amino acid sequence.

[0160] Table 9-1

[0161] Table 9-2

[0162] Table 9-3

[0163] Table 9-4

[0164] Table 9-5

[0165] Table 9-6

[0166] Table 9-7

[0167] Table 9-8

[0168] Table 9-9

[0169] Table 9-10

[0170] Table 9-11

[0171] Table 9-12

[0172] Table 9-13

[0173] Table 9-14

[0174] Table 9-15

[0175] Table 9-16

[0176] Table 9-17

[0177] Table 9-18

[0178] Table 9-19

[0179] Table 9-20

[0180] Table 9-21

[0181]

Table 9-22

[0182]

Table 9-23

[0183]

Table 9-24

[0184]

Table 9-25

[0185]

Table 9-26

[0186]

Table 9-27

[0187]

Table 9-28

[0188]

Table 9-29

[0189] Furthermore, in some embodiments, the polypeptides described herein (e.g., ActRII chimeric-Fc fusion proteins) have a serum half-life of at least 7 days in humans. Polypeptides containing extracellular ActRII chimeras have a K of 10 pM or more DIt can bind to activin A. In some embodiments, polypeptides containing extracellular ActRII chimeras bind to activin A, activin B, and / or myostatin and show reduced (e.g., weak) binding to BMP9 (e.g., compared to, for example, wild-type extracellular ActRIIB). In some embodiments, polypeptides containing extracellular ActRII chimeras with reduced or weak binding to BMP9 have the sequence TEEN (SEQ ID NO: 374) or TKEN (SEQ ID NO: 375) at positions X3, X4, X5, and X6. In some embodiments, polypeptides containing extracellular ActRII chimeras with reduced or weak binding to BMP9 have the sequence KKDS or TKDS at positions X3, X4, X5, and X6. In some embodiments, polypeptides containing extracellular ActRII chimeras do not bind substantially to human BMP9.

[0190] In some embodiments, polypeptides containing extracellular ActRII chimeras have a K content of approximately 800 pM or less. D (For example, K values ​​of approximately 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 pM) D For example, K at approximately 800 pM to 30 pM D ) can bind to human activin A. In some embodiments, polypeptides containing extracellular ActRII chimeras have a K of less than 800 pM. D (For example, K values ​​of approximately 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 pM) D For example, K at approximately 800 pM to 5 pM D ) can bind to human activin B. Polypeptides containing extracellular ActRII chimeras also contain K at approximately 5 pM or greater. D(For example, K levels of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher) D It can bind to growth and differentiation factor 11 (GDF-11) in this way.

[0191] II. Fc domain In some embodiments, the polypeptides described herein may include an extracellular ActRII variant (e.g., an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera) fused to an Fc domain monomer or fragment of an immunoglobulin in order to increase the serum half-life of the polypeptide. Polypeptides containing an extracellular ActRII variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) through interactions between two Fc domain monomers that form a dimeric Fc domain. As is conventionally known in the art, the Fc domain is a protein structure found at the C-terminus of immunoglobulins. The Fc domain is C HThe molecule contains two Fc domain monomers that are dimerized by interactions between the three antibody constant domains. The wild-type Fc domain forms a minimal structure that binds to Fc receptors, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV. In some embodiments, the Fc domain may be mutated to lack effector function, typical of a "dead" Fc domain. For example, the Fc domain may contain specific amino acid substitutions known to minimize the interaction between the Fc domain and the Fcγ receptor. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions L234A, L235A, and G237A. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions D265A, K322A, and N434A. The amino acid positions described above are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Kabat numbering of amino acid residues can be determined for a given antibody by alignment of the antibody sequence homology region with a “standard” Kabat numbering sequence. Furthermore, in some embodiments, the Fc domain does not induce any immune system-related response. For example, the Fc domain in a polypeptide dimer containing an extracellular ActRII variant fused to an Fc domain monomer can be modified to reduce the interaction or binding between the Fc domain and the Fcγ receptor. The sequence of an Fc domain monomer that can be fused to an extracellular ActRII variant (e.g., extracellular ActRIIA variant, extracellular ActRIIB variant, or extracellular ActRII chimera) is shown below (SEQ ID NO: 97): THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In some embodiments, the Fc domain is derived from an IgG1 antibody and includes amino acid substitutions L12A, L13A, and G15A relative to the sequence of SEQ ID NO: 97. In some embodiments, the Fc domain is derived from an IgG1 antibody and includes amino acid substitutions D43A, K100A, and N212A relative to the sequence of SEQ ID NO: 97. In some embodiments, terminal lysine is not found in the Fc domain monomer having the sequence of SEQ ID NO: 97. In some embodiments, the extracellular ActRII variants described herein (e.g., extracellular ActRIIA variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), extracellular ActRIIB variants having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or extracellular AcRII chimeras having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)) may be fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., SEQ ID NOs. 97) by conventional genetic or chemical means, e.g., chemical conjugation. If necessary, a linker (e.g., a spacer) may be inserted between the extracellular ActRII chimera and the Fc domain monomer. The Fc domain monomer may be fused to the N-terminus or C-terminus (e.g., C-terminus) of the extracellular ActRII variant.

[0192] In some embodiments, the polypeptides described herein may include an extracellular ActRII variant fused to the Fc domain (e.g., an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an ActRII chimera). In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. In some embodiments, the Fc domain includes a hinge domain. The Fc domain may be any immunoglobulin antibody isotype, IgG, IgE, IgM, IgA, or IgD. Furthermore, the Fc domain may be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a naturally occurring Fc domain, such as a recombinant Fc domain.

[0193] Methods for manipulating Fc domains with reduced dimerization are known in the art. In some embodiments, one or more amino acids having large side chains (e.g., tyrosine or tryptophan) are used in C H 3-C H 3. It may be introduced at the dimer interface to prevent dimer formation by steric collision. In other embodiments, one or more amino acids having small side chains (e.g., alanine, valine, or threonine) are added to eliminate the preferred interaction. H 3-C H It can be introduced into the 3-dimer interface. H Methods for introducing amino acids having large or small side chains into three domains are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication No. 2006 / 0074225, U.S. Patents No. 8,216,805 and 5,731,168, Ridgway et al. (Protein Eng. 9:617-612, 1996), Atwell et al. (J Mol Biol. 270:26-35, 1997), and Merchant et al. (Nat Biotechnol. 16:677-681, 1998), all of which are incorporated herein by reference in their entirety.

[0194] In yet another embodiment, C between two Fc domains H 3-C H C that constitutes the 3 interface H One or more amino acid residues within the 3 domains are substituted with positively charged amino acid residues (e.g., lysine, arginine, or histidine) or negatively charged amino acid residues (e.g., aspartic acid or glutamic acid), so that the interaction becomes electrostatically unfavorable depending on the specific charged amino acid introduced. H Methods for introducing charged amino acids into three domains to discourage or prevent dimerization are described, for example, by Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publications 2006 / 0074225, 2012 / 0244578, and 2014 / 0024111, all of which are incorporated herein by reference in their entirety.

[0195] In some embodiments of the present invention, the Fc domain comprises one or more of the following amino acid substitutions: relative to the human IgG1 sequence, T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S 364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I. In some embodiments, terminal lysine is not found in the Fc domain amino acid sequence. In one particular embodiment, the Fc domain contains the amino acid substitution T366W relative to the human IgG1 sequence. The sequence of the wild-type Fc domain is shown below in SEQ ID NO: 150: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK An exemplary sequence of the wild-type Fc domain lacking terminal lysine is provided below (SEQ ID NO: 155): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG III. Albumin-binding peptides In some embodiments, the polypeptides described herein may include extracellular ActRII variants (e.g., extracellular ActRIIA variants, extracellular ActRIIB variants, or ActRII chimeras) fused to serum protein-binding peptides. Binding to serum protein peptides can improve the pharmacokinetics of protein pharmaceuticals.

[0196] As an example, albumin-binding peptides that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the albumin-binding peptide comprises the sequence DICLPRWGCLW (SEQ ID NO: 151).

[0197] In the present invention, albumin-binding peptides can be bound to the N-terminus or C-terminus (e.g., C-terminus) of an extracellular ActRII variant described herein (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or an ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)) to increase the serum half-life of the extracellular ActRII variant. In some embodiments, the albumin-binding peptide is bound to the N-terminus or C-terminus of the extracellular ActRII variant directly or via a linker.

[0198] In some embodiments, the extracellular ActRII variants described herein (e.g., extracellular ActRIIA variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), extracellular ActRIIB variants having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or extracellular AcRII chimeras having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)) may be fused to the N-terminus or C-terminus of an albumin-binding peptide (e.g., SEQ ID NO. 151) via conventional genetic or chemical means, e.g., chemical conjugation. A linker (e.g., a spacer) may be inserted between the extracellular ActRII variant and the albumin-binding peptide as needed. While not wishing to be bound by theory, it is anticipated that including an albumin-binding peptide in the extracellular ActRII variants described herein may extend the retention of the therapeutic protein through binding to serum albumin.

[0199] IV. Fibronectin Domain In some embodiments, the polypeptides described herein may include extracellular ActRII variants fused to a fibronectin domain (e.g., extracellular ActRIIA variants, extracellular ActRIIB variants, or ActRII chimeras). Binding to a fibronectin domain can improve the pharmacokinetics of protein pharmaceuticals.

[0200] A fibronectin domain is a high molecular weight glycoprotein or fragment of the extracellular matrix that binds to transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments of the present invention, a fibronectin domain can be bound to the N-terminus or C-terminus (e.g., C-terminus) of an extracellular ActRII variant described herein (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or an ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)) to increase the serum half-life of the extracellular ActRII variant. The fibronectin domain may be bound to the N-terminus or C-terminus of the extracellular ActRII variant directly or via a linker.

[0201] As an example, the fibronectin domains that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain (SEQ ID NO: 152) having amino acids 610-702 of the sequence of UniProt ID number P02751. GPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTST In another embodiment, the fibronectin domain is an adnectin protein.

[0202] In some embodiments, the extracellular ActRII variants described herein (e.g., extracellular ActRIIA variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), extracellular ActRIIB variants having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or extracellular AcRII chimeras having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)) may be fused to the N-terminus or C-terminus of a fibronectin domain (e.g., SEQ ID NOs. 152) by conventional genetic or chemical means, e.g., chemical conjugation. If necessary, a linker (e.g., a spacer) may be inserted between the extracellular ActRII variant and the fibronectin domain. While not wishing to be bound by theory, it is anticipated that including a fibronectin domain in the extracellular ActRII variants described herein may extend the retention of therapeutic proteins through binding to integrins and extracellular matrices such as collagen and fibrin.

[0203] V. Serum albumin In some embodiments, the polypeptides described herein may include extracellular ActRII variants fused to serum albumin (e.g., extracellular ActRIIA variants, extracellular ActRIIB variants, or ActRII chimeras). Binding to serum albumin can improve the pharmacokinetics of protein pharmaceuticals.

[0204] Serum albumin is a globular protein that is the most abundant blood protein in mammals. Produced in the liver, serum albumin makes up about half of the serum proteins. It is a monomer and soluble in blood. The most important functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining the osmotic pressure necessary for the proper distribution of body fluids between blood vessels and tissues. In preferred embodiments, serum albumin is human serum albumin. In some embodiments of the present invention, human serum albumin can increase the serum half-life of an extracellular ActRII variant by binding to the N-terminus or C-terminus (e.g., C-terminus) of an extracellular ActRII variant described herein (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or an ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216). Human serum albumin may bind to the N-terminus or C-terminus of an extracellular ActRII variant directly or via a linker.

[0205] As an example, human serum albumins that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the serum albumin comprises the sequence UniProt ID number P02768 (SEQ ID NO: 153, shown below). MKWVTFISLLFFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLERTYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCE KPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL In some embodiments, the extracellular ActRII variants described herein (e.g., extracellular ActRIIA variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), extracellular ActRIIB variants having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or extracellular AcRII chimeras having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)) may be fused to the N-terminus or C-terminus of human serum albumin (e.g., SEQ ID NOs. 153) by conventional genetic or chemical means, e.g., chemical conjugation. A linker (e.g., a spacer) may be inserted between the extracellular ActRII variant and human serum albumin as needed. While we do not wish to be bound by theory, it is anticipated that including human serum albumin in the extracellular ActRII variants described herein may extend the retention of the therapeutic protein.

[0206] VI. Linker The polypeptides described herein may include extracellular ActRII variants partially fused by a linker (e.g., extracellular ActRIIA variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), extracellular ActRIIB variants having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or extracellular ActRII chimeras having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)). In some embodiments, the portion enhances the stability of the polypeptide. Exemplary portions include Fc domain monomers, Fc domains (e.g., wild-type Fc domains), Fc domains having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), albumin-binding peptides, fibronectin domains, or human serum albumin. In the present invention, the linker between a portion (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., SEQ ID NO: 150 or SEQ ID NO: 155), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide (e.g., SEQ ID NO: 151), a fibronectin domain (e.g., SEQ ID NO: 152), or human serum albumin (e.g., SEQ ID NO: 153)) and an extracellular ActRII chimera (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs: 157-171 (e.g., SEQ ID NOs: 158-171), or an extracellular ActRII chimera having any one sequence of SEQ ID NOs: 174-216 (e.g., 195-216)) may be an amino acid spacer of 1-200 amino acids. Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine.In some embodiments, the spacer may include multiple or repeating motifs, such as GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 98), GGGS (SEQ ID NO: 99), GGGG (SEQ ID NO: 100), GGGGA (SEQ ID NO: 101), GGGGS (SEQ ID NO: 102), GGGGG (SEQ ID NO: 103), GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), AGGG (SEQ ID NO: 106), or SGGG (SEQ ID NO: 107). In some embodiments, the spacer may include 2 to 12 amino acids containing a GA or GS motif, for example, GA, GS, GAGA (SEQ ID NO: 108), GSGS (SEQ ID NO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA (SEQ ID NO: 112), GSGSGSGS (SEQ ID NO: 113), GAGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), and GSGSGSGSGSGS (SEQ ID NO: 117). In some embodiments, the spacer may include 3 to 12 amino acids containing a GGA or GGS motif, for example, GGA, GGS, GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), and GGSGGSGGSGGS (SEQ ID NO: 123). In some further embodiments, the spacer may include 4 to 12 amino acids containing the motifs of GGAG (SEQ ID NO: 104) and GGSG (SEQ ID NO: 105), for example, GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), GGAGGGAG (SEQ ID NO: 124), GGSGGGSG (SEQ ID NO: 125), GGAGGGAGGGAG (SEQ ID NO: 126), and GGSGGGSGGGSG (SEQ ID NO: 127). In some embodiments, the spacer may also include the motifs of GGGGA (SEQ ID NO: 101) or GGGGS (SEQ ID NO: 102), for example, GGGGAGGGGAGGGGA (SEQ ID NO: 128) and GGGGSGGGGSGGGGS (SEQ ID NO: 129).In some embodiments of the present invention, the amino acid spacer between a portion (e.g., an Fc domain monomer, an Fc domain (e.g., a wild-type Fc domain), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) and an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)) may be GGG, GGGA (SEQ ID NOs. 98), GGGG (SEQ ID NOs. 100), GGGAG (SEQ ID NOs. 130), GGGAGG (SEQ ID NOs. 131), or GGGAGGG (SEQ ID NOs. 132).

[0207] In some embodiments, the spacer may also include amino acids other than glycine, alanine, and serine, such as AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 138), KESGSVSSEQLAQFRSLD (SEQ ID NO: 139), GENLYFQSGG (SEQ ID NO: 140), SACYCELS (SEQ ID NO: 141), RSIAT (SEQ ID NO: 142), RPACKIPNDLKQKVMNH (SEQ ID NO: 143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 144), AAANSSIDLISVPVDSR (SEQ ID NO: 145), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 146). In some embodiments, the spacer may include motifs such as multiple motifs or repeating motifs of EAAAK (SEQ ID NO: 147). In some embodiments, the spacer is (XP) nThe formula may include a proline-rich sequence of (wherein X may be any amino acid (e.g., A, K, or E), and n is 1 to 5), and multiple motifs or repeating motifs of PAPAP (SEQ ID NO: 148).

[0208] The length of the peptide spacer and the amino acids used can be adjusted depending on the two proteins involved and the desired degree of flexibility in the final protein fusion polypeptide. The spacer length can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0209] In some embodiments, the linker between a portion (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97), an Fc domain (e.g., a wild-type Fc domain (e.g., SEQ ID NO: 150 or SEQ ID NO: 155)), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide (e.g., SEQ ID NO: 151), a fibronectin domain (e.g., SEQ ID NO: 152), or human serum albumin (e.g., SEQ ID NO: 153)) and an extracellular ActRII variant described herein (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs: 157-171 (e.g., SEQ ID NOs: 158-171), or an extracellular ActRII chimera having any one sequence of SEQ ID NOs: 174-216 (e.g., 195-216)) is an amino acid spacer having the sequence GGG. For example, the polypeptide of the present invention may include an extracellular ActRIIA variant (e.g., any one of Sequence IDs 6-72) fused to an Fc domain (e.g., Sequence ID 155) by a GGG linker. An exemplary polypeptide comprising the ActRIIA variant of Sequence ID 69, a GGG linker, and an Fc domain lacking terminal lysine (Sequence ID 155) is presented below (Sequence ID 156): GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG In another example, the polypeptide of the present invention may include an extracellular ActRIIB variant (e.g., one of SEQ ID NOs. 158-171) fused to an Fc domain (e.g., SEQ ID NOs. 150 or 155) by a GGG linker. An exemplary polypeptide comprising the ActRIIB variant of SEQ ID NOs. 171, a GGG linker, and an Fc domain (SEQ ID NOs. 150) is presented below (SEQ ID NOs. 172): GRGEAETRECLYYNANWELERTNQSGVERCEGEKDKRLHCYASWRNSSGSLEIVKKGCWLDDFNCYDRDTCVATKENPQVYFCCCEGNMCNERFTHLPEAGGPEVTYEPPPTAPTGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK VII. Vectors, host cells, and protein production The polypeptides of the present invention can be produced from host cells. A host cell refers to a vehicle containing cellular components, such as organelles, necessary for expressing the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. The nucleic acids may be contained in nucleic acid vectors that can be introduced into host cells by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). The selection of the nucleic acid vector depends in part on the host cell used. Generally, preferred host cells are either of eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0210] nucleic acid vector constructs and host cells The nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present invention can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. The nucleic acid molecules encoding the polypeptides of the present invention can be obtained using standard techniques, such as gene synthesis. Alternatively, nucleic acid molecules encoding wild-type extracellular ActRIIA or extracellular ActRIIB can be mutated to include specific amino acid substitutions using standard techniques in the art, such as QuikChange® mutagenesis. The nucleic acid molecules can be synthesized using nucleotide synthesizers or PCR techniques.

[0211] The nucleic acid sequence encoding the polypeptide of the present invention can be inserted into a vector capable of replicating and expressing the nucleic acid molecule in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector may contain various components that can be tuned and optimized for compatibility with a particular host cell. For example, vector components may include, but are not limited to, a replication origin, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding the protein of interest, and a transcription termination sequence.

[0212] In some embodiments, mammalian cells can be used as host cells of the present invention. Examples of mammalian cell types include, but are not limited to, human fetal kidney (HEK) (e.g., HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coli cells can also be used as hosts of the present invention. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ 1776 (ATCC® 31,537), E. coli BL21 (DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608). Different host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. By selecting an appropriate cell line or host system, the correct modification and processing of the expressed polypeptide can be ensured. The expression vectors described above can be introduced into appropriate host cells using conventional techniques in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vector is introduced into host cells for protein production, the host cells are cultured in a conventional nutrient medium modified to be suitable for promoter induction, transformant selection, or amplification of the gene encoding the desired sequence.Methods for expressing therapeutic proteins are known in the art; see, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2012.

[0213] Protein production, recovery, and purification The host cells used to produce the polypeptides of the present invention can be grown in media known in the art and suitable for culturing selected host cells. Examples of media suitable for mammalian host cells include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Expi293™ expression medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of media suitable for bacterial host cells include Luria culture medium (LB) containing necessary supplements such as selective agents such as ampicillin. The host cells are cultured at a suitable temperature, such as about 20°C to about 39°C, for example 25°C to about 37°C, preferably 37°C, and at a CO2 level of 5 to 10%. The pH of the medium is generally about 6.8 to 7.4, for example 7.0, depending mainly on the host organism. When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for promoter activation.

[0214] In some embodiments, depending on the expression vector and host cell used, the expressed protein may be secreted from the host cell (e.g., a mammalian host cell) into a cell culture medium. Protein recovery may involve filtering the cell culture medium to remove cellular debris. The protein can be further purified. The polypeptides of the present invention may be purified by any method known in the art of protein purification, for example, by chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, absorptiometry differential solubility, or any other standard technique for protein purification. For example, the protein may be isolated and purified by appropriately selecting and combining affinity columns such as Protein A columns (e.g., POROS Protein A chromatography) and chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting out, and dialysis procedures.

[0215] In other embodiments, the expressed protein can be recovered by destroying the host cells, for example, by osmotic shock, sonication, or lysis. Once the cells are destroyed, the cellular debris can be removed by centrifugation or filtration. In some cases, polypeptides can be conjugated to marker sequences, such as peptides, to facilitate purification. An example of a marker amino acid sequence is the hexahistidine peptide (His tag), which binds to a nickel-functionalized agarose affinity column at micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).

[0216] Alternatively, the polypeptide of the present invention can be produced by cells of a subject (e.g., human) by administering a vector (e.g., viral vector (e.g., retroviral vector, adenovirus vector, poxvirus vector (e.g., vaccinia virus vector such as modified vaccinia ankara (MVA), adeno-associated virus vector, and alphavirus vector))) containing a nucleic acid molecule encoding the polypeptide of the present invention, for example in the context of gene therapy. Once the vector enters the target cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), it promotes polypeptide expression and is subsequently secreted from the cells. If the treatment of a disease or disorder is the desired outcome, no further action may be necessary. If protein extraction is desired, blood can be collected from the subject and the protein can be purified from the blood by methods known in the art.

[0217] VIII. Pharmaceutical Compositions and Preparations The present invention is characterized by a pharmaceutical composition comprising a polypeptide described herein (for example, a polypeptide comprising an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157 to 171 (e.g., SEQ ID NOs. 158 to 171), or an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174 to 216 (e.g., 195 to 216)). In some embodiments, the pharmaceutical composition of the present invention comprises a polypeptide comprising an extracellular ActRII variant having a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) (for example, an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1 to 70 (e.g., SEQ ID NOs. 6 to 70), or an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174 to 216 (e.g., 195 to 216)) as a therapeutic protein. In some embodiments, the pharmaceutical composition of the present invention comprises a polypeptide as a therapeutic protein comprising an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)) fused to a portion (e.g., an Fc domain monomer or dimer thereof, an Fc domain (e.g., a wild-type Fc domain), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin). In some embodiments, the pharmaceutical composition of the present invention comprising the polypeptide of the present invention may be used in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions in therapeutic use. In addition to a therapeutically effective amount of polypeptide, the pharmaceutical composition may include one or more pharmaceutically acceptable carriers or excipients, which can be formulated by methods known to those skilled in the art.In some embodiments, the pharmaceutical composition of the present invention comprises a nucleic acid molecule (DNA or RNA, e.g., mRNA) encoding the polypeptide of the present invention, or a vector containing such a nucleic acid molecule.

[0218] Acceptable carriers and excipients in the pharmaceutical composition are non-toxic to the recipient at the dosage and concentration used. Acceptable carriers and excipients include buffers such as phosphates, citrates, HEPES, and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol, and benzalkonium chloride; proteins such as human serum albumin, gelatin, dextran, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine, arginine, and lysine; and carbohydrates such as glucose, mannose, sucrose, and sorbitol. The pharmaceutical composition of the present invention can be administered parenterally in the form of an injectable formulation. The injectable pharmaceutical composition can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco's modified Eagle medium (DMEM), α-modified Eagle medium (α-MEM), F-12 medium). Preparation methods are known in the art. See, for example, Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd ed.) Taylor & Francis Group, CRC Press (2015).

[0219] The pharmaceutical compositions of the present invention can be prepared in microcapsules such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. The pharmaceutical compositions of the present invention can also be formulated in other drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Such methods are described in Remington: The Science and Practice of Pharmacy 22. nd This is described in edition (2012). Pharmaceutical compositions used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0220] The pharmaceutical compositions of the present invention can also be prepared as sustained-release formulations. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptides of the present invention. Examples of sustained-release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and L-gamma-ethyl glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT®, and poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations allow for the release of molecules over several months, for example, 1 to 6 months, while others release the pharmaceutical compositions of the present invention over shorter periods, for example, several days to several weeks.

[0221] The pharmaceutical composition may be formed in unit dose form as needed. The amount of the active ingredient contained in the pharmaceutical formulation, for example, the polypeptide of the present invention, is such that an appropriate dose is provided within a specified range (for example, a dose in the range of 0.01 to 100 mg per kg of body weight).

[0222] The pharmaceutical composition for gene therapy may be present in an acceptable diluent or may contain a sustained-release matrix into which a gene delivery vehicle is embedded. When hydrodynamic injection is used as the delivery method, the pharmaceutical composition comprising a nucleic acid molecule encoding the polypeptide described herein or a vector containing a nucleic acid molecule (e.g., a viral vector) is rapidly delivered intravenously in a large volume of liquid. Vectors that can be used as in vivo gene delivery vehicles include, but are not limited to, retroviral vectors, adenovirus vectors, poxvirus vectors (e.g., vaccinia virus vectors such as modified vaccinia ankara), adeno-associated virus vectors, and alphavirus vectors.

[0223] IX. Route, Dosage, and Administration A pharmaceutical composition containing the polypeptide of the present invention as a therapeutic protein can be formulated, for example, for intravenous, parenteral, subcutaneous, intramuscular, intra-arterial, intrathecal, or intraperitoneal administration. The pharmaceutical composition can also be formulated or administered orally, nasally, as a spray, aerosol, rectally, or vaginally. For injectable formulations, various effective pharmaceutical carriers are known in the art. See, for example, the ASHP Handbook on Injectable Drugs, Toissel, 18th ed. (2014).

[0224] In some embodiments, a pharmaceutical composition comprising a nucleic acid molecule encoding the polypeptide of the present invention or a vector containing such a nucleic acid molecule may be administered by gene delivery. Methods of gene delivery are well known to those skilled in the art. Vectors that can be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenovirus vectors, poxvirus vectors (e.g., vaccinia virus vectors such as modified vaccinia ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, the mRNA molecule encoding the polypeptide of the present invention may be administered directly to the target.

[0225] In some embodiments of the present invention, nucleic acid molecules encoding the polypeptides described herein, or vectors containing such nucleic acid molecules, may be administered using a hydrodynamic injection platform. In hydrodynamic injection, the nucleic acid molecules encoding the polypeptides described herein are placed under the control of a potent promoter within an engineered plasmid (e.g., a viral plasmid). The plasmid is often rapidly delivered intravenously in a large volume of fluid. Hydrodynamic injection uses controlled hydrodynamic pressure in the vein to enhance cellular permeability, and the pressure increase due to the rapid injection of a large volume of fluid causes extravasation of the fluid and plasmid from the vein. The expression of the nucleic acid molecule is primarily driven by the liver. In mice, hydrodynamic injection is often performed by injecting the plasmid into the tail vein. In certain embodiments, mRNA molecules encoding the polypeptides described herein may be administered using hydrodynamic injection.

[0226] The dosage of the pharmaceutical composition of the present invention depends on the route of administration, the disease being treated, and factors including physical characteristics such as the subject's age, weight, and overall health status. The pharmaceutical compositions of the present invention may include dosages of the polypeptide of the present invention ranging from 0.01 to 500 mg / kg (for example, 0.01, 0.1, 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg), in more specific embodiments, from about 0.1 to about 30 mg / kg, and in more specific embodiments, from about 0.3 to about 30 mg / kg. The dosage may be adjusted by a physician according to conventional factors such as the severity of the disease and different parameters of the patient.

[0227] The pharmaceutical composition is administered in a manner suitable for the administered formulation, in an amount therapeutically effective to bring about improvement or restoration of symptoms. The pharmaceutical composition is administered in various dosage forms, e.g., intravenous, subcutaneous, and oral dosage forms (e.g., ingestible solutions, drug-release capsules). Generally, therapeutic proteins are administered at doses of 0.1 to 100 mg / kg, e.g., 0.5 to 50 mg / kg. The pharmaceutical composition containing the polypeptide of the present invention may be administered to subjects requiring it, for example, daily, weekly, every two weeks, every four weeks, monthly, every eight weeks, every two months, every twelve weeks, quarterly, every sixteen weeks, every sixteen months, annually or more (e.g., 1 to 10 times or more), or as medically required. In some embodiments, the pharmaceutical composition containing the polypeptide of the present invention may be administered to subjects requiring it weekly, every two weeks, every four weeks, monthly, every eight weeks, every two months, every twelve weeks, quarterly, or every sixteen weeks. Dosages may be provided in single or multiple administration regimens. The interval between doses may shorten as the patient's condition improves, or lengthen as the patient's health deteriorates.

[0228] X. Treatment method The ActRII variants described herein have improved properties compared to endogenous ActRIIA and ActRIIB. For example, an ActRIIA variant produced by introducing ActRIIB residues into ActRIIA retains the beneficial properties of ActRIIA, such as low binding affinity to BMP9 and a longer serum half-life as an Fc fusion protein, while acquiring the beneficial properties of ActRIIB, such as increased binding to activin A and B. An ActRIIB variant produced by introducing ActRIIA residues into ActRIIB can retain the beneficial properties of ActRIIB, such as high binding affinity to activin A and B, while acquiring some of the beneficial properties of ActRIIA, such as low binding affinity to BMP9 and a longer serum half-life as an Fc fusion protein. Furthermore, ActRII chimeras, generated by combining the extracellular components of ActRIIA and ActRIIB, may possess beneficial properties of both ActRIIB (e.g., strong binding affinity to activin A and B) and ActRIIA (e.g., decreased binding affinity to BMP9 and / or a longer serum half-life as an Fc fusion protein (e.g., compared to ActRIIB-Fc)). The properties of these ActRIIA variants, ActRIIB variants, and ActRII chimeras enable useful therapeutics that can compete with endogenous activin receptors for ligand binding. Because ActRIIA variants, ActRIIB variants, and ActRII chimeras contain the extracellular components of the receptor, they are soluble and can bind to and sequester ligands (e.g., activin A and B, myostatin, GDF11) without activating intracellular signaling pathways.Based on the finding that administration of polypeptides containing the ActRIIA variants described herein results in an increase in serum EPO levels and bone marrow EPO receptor levels at least 7 to 14 days after injection, polypeptides containing the ActRII variants described herein (e.g., ActRIIA variant, ActRIIB variant, or ActRII chimera) can be used therapeutically as a substitute for recombinant EPO or EPO mimetic and can be used to treat any disease or condition that would benefit from an increase in EPO and / or EPO receptor levels.

[0229] In some embodiments, polypeptides described herein (e.g., polypeptides comprising an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)), e.g., an effective amount of ActRIIA variant, ActRIIB variant, or ActRII chimera) can be administered to increase EPO levels (e.g., serum EPO levels) and / or EPO receptor levels (e.g., EPO receptor levels in bone marrow cells) in subjects requiring it (e.g., subjects with low serum EPO levels). The present invention also includes a method for treating (e.g., treating, delaying, and / or preventing) a subject who has or is at risk of developing a disease or condition treatable with EPO or ESA (e.g., a disease or condition treatable by increasing levels of EPO or EPO receptors), by administering to the subject an effective amount of a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), a polypeptide comprising an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or a polypeptide comprising an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)), e.g., an effective amount of an ActRIIA variant, an ActRIIB variant, or an ActRII chimera).Diseases and conditions that can be treated by increasing levels of EPO or EPO receptors include end-stage renal disease, renal failure, polycythemia, dialysis-related anemia, early anemia in premature infants, iron overload (e.g., hemochromatosis), disease states, disorders, and hematological abnormalities such as pregnancy, menstrual disorders, and space flight, ischemia (CNS ischemia, hepatic ischemia, renal ischemia, or cardiac ischemia), ulcers, burns, wounds (e.g., chronic wounds), ischemia-reperfusion injury (e.g., ischemia-reperfusion injury associated with surgery or organ transplantation), ischemic disorders or conditions (e.g., myocardial infarction, ischemic stroke, obstructive arterial disease, chronic venous insufficiency, pulmonary embolism, etc.). Examples of conditions that may be considered hazardous include hemorrhagic shock, septic shock, circulatory shock such as cardiogenic shock, acute respiratory failure, chronic heart failure, atherosclerosis, cardiac cirrhosis, macular degeneration, sleep apnea, Raynaud's disease, systemic sclerosis, nonbacterial thrombotic endocarditis, angina pectoris, transient ischemic attack, chronic alcoholic liver disease, or ischemia due to general anesthesia), hypoxia (e.g., hypoxic conditions or disorders such as perinatal hypoxia or pulmonary injury (e.g., hypoxic hypoxia such as COPD), severe pneumonia, pulmonary edema, hyaline membrane disease, liver or kidney disease, cancer or other chronic diseases, and altitude sickness), and aging. The polypeptides described herein may also be used to treat subjects undergoing renal dialysis, subjects who have recently undergone stem cell transplantation, to increase the red blood cell count of a subject before surgery, or as pre-treatment or further treatment of tissue or organ to be transplanted (e.g., treatment of tissue or organ pre-transplant (e.g., immediately before), during transplantation, or immediately after transplantation).

[0230] Given that EPOs have been shown to stimulate the recruitment, proliferation, migration, and differentiation of endothelial progenitor cells, the polypeptides described herein can also be used to treat diseases associated with endothelial progenitor cell dysfunction. Such diseases include heart failure, angina pectoris, endotheliopathy (e.g., reticuloendotheliopathy), age-related cardiovascular disease, coronary heart disease, atherosclerosis, myocardial ischemia, hypercholesterolemia, ischemic injuries of the limbs, Raynaud's disease, pre-eclampsia, pregnancy-induced hypertension, endothelial-mediated chronic inflammatory diseases (such as inflammation of blood vessels), wound healing, and chronic or acute renal failure (also known as chronic kidney disease and acute renal failure, respectively). Since EPO has been shown to have mitotic and chemotactic effects on vascular endothelial cells, it can also be used with ActRII variant polypeptides (e.g., extracellular ActRIIA variant, ActRIIB variant, or ActRII chimera) to promote the proliferation of new blood vessels (angiogenesis) and / or the replacement of damaged vascular areas, such as collateral coronary vessels (e.g., those that may occur after myocardial infarction), for granulation tissue formation (e.g., damaged tissue, wounds, and ulcers), for trauma treatment, for post-vascular transplantation treatment, and for the manufacture of artificial blood vessels such as heart valves.

[0231] EPO has also been found to have anti-inflammatory and neuroprotective effects. Therefore, the polypeptides described herein are useful in treating diseases or conditions with inflammatory or autoimmune components, such as acute cerebrovascular injury, acute brain injury, acute cardiovascular injury, arthritis, autoimmune diseases, stroke, nerve injury, and immune-mediated inflammation, as well as neurological and / or inflammatory brain diseases, such as demyelinating diseases (e.g., multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, transverse myelitis), epilepsy, spinal cord injury (e.g., acute spinal cord injury), and complications after traumatic brain injury (e.g., hypotension, hypoxemia, cerebral swelling, headache, neck pain, memory). Polypeptides can also be used to treat symptoms of traumatic brain injury such as difficulty concentrating, difficulty making decisions, fatigue, mood swings, nausea, photophobia, blurred vision, tinnitus, loss of taste, loss of smell, seizures, coma, muscle weakness, paralysis, or progressively debilitating neurological function; chronic inflammatory brain diseases (e.g., neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, amyotrophic lateral sclerosis (ALS), or age-related macular degeneration (AMD)); or neurological disorders associated with surgery such as thoracoabdominal aortic surgery. Polypeptides can treat neurological disorders or inflammatory brain diseases by reducing mononuclear cell infiltration into the target brain, improving neurological disorders, and / or reducing axonal damage and / or neuronal cell death and / or glial cell death in at least one region of the brain of a subject directly or indirectly affected by the disease, disorder, or condition.

[0232] Gastrointestinal motility disorders can also be treated with EPO. Therefore, the polypeptides described herein can be used to treat intestinal injury, abdominal trauma, intestinal inflammatory conditions (e.g., inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis), intestinal infections (e.g., bacterial infections such as infections causing sepsis or bacteremia, localized infections such as peritonitis or ascites), delayed constipation (e.g., chronic constipation, idiopathic constipation, constipation due to postoperative ileus, or constipation due to opioid use), postoperative ileus, neurodegenerative injury, neurotraumatic injury, congenital problems (e.g., gastric spondylolysis, umbilical hernia, aganglionic megacolon, Hirschsprung's disease, chronic intestinal pseudoobstruction, small left colon syndrome, anorectal anomalies, esophageal dysplasia and It can be used to treat gastrointestinal motility disorders due to atresia, ectopic anus, congenital hernia, achalasia of the internal anal sphincter, or malnutrition-malabsorption problems (e.g., intestinal injury, abdominal trauma, intestinal inflammatory conditions, intestinal infections, constipation (e.g., constipation due to opioid use), postoperative ileus, neurodegenerative injury, neurotrauma, congenital diseases, Gaucher disease, refeeding syndrome, extremely low birth weight infants, cancerous cachexia, infections, cancer, spinal cord dysfunction, spinal dysplasia, spina bifida, tumors, central nervous system dysfunction, peripheral neuropathy, partial resection of the gastrointestinal tract, bleeding, liver dysfunction, celiac disease, cystic fibrosis, muscular dystrophy, or cerebral palsy).

[0233] Polypeptides described herein can also be used to treat chronic or recurrent diseases such as asthma, viral diseases or infections (e.g., HIV infection or HCV infection), hypertension, systemic microbial infections, cancer, endocrine disorders, reproductive disorders, psychosis, genetic disorders, allergies, gastrointestinal disorders, arteriosclerosis, cardiovascular diseases, graft-versus-host diseases, or inflammatory diseases. Polypeptides containing ActRII variants (e.g., ActRIIA variant, ActRIIB variant, or ActRII chimera) can also be used to enhance athletic performance, improve athletic ability, and promote or enhance aerobic conditioning. Such methods can be used, for example, by athletes to facilitate training or by soldiers to improve endurance and stamina. In some embodiments, the methods described herein aim to affect the signaling of myostatin, activin A, activin B, and / or BMP9 in subjects having a disease or condition that can be treated with EPO or ESA (e.g., reducing or inhibiting the binding of activin A, activin B, myostatin, and / or BMP9 to their endogenous receptors, e.g., ActRIIA, ActRIIB, and / or BMPRII). In some embodiments, the methods described herein increase EPO levels (e.g., serum EPO levels) and / or EPO receptor levels (e.g., bone marrow EPO receptor levels) compared to measurements obtained before treatment or compared to measurements obtained from untreated subjects or subjects having the same disease or condition treated with controls.

[0234] In some embodiments, the methods described herein (for example, methods for treating any of the diseases or conditions described herein) do not cause any vascular complications, such as increased vascular permeability or leakage.

[0235] In any of the methods described herein, a polypeptide comprising an extracellular ActRII variant (e.g., an extracellular ActRII variant having any one sequence of SEQ ID NOs. 1-71 (e.g., SEQ ID NOs. 6-71), or an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)), further comprising a C-terminal extension of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids), can be used as a therapeutic protein. In any of the methods described herein, a dimer (e.g., a homodimer or heterodimer) formed by the interaction of two fc domain monomers fused to a polypeptide containing an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., 195-216)) can be used as a therapeutic protein. In any of the methods described herein, an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having any one sequence of any of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72), an extracellular ActRIIB variant having any one sequence of any of SEQ ID NOs: 157-171 (e.g., SEQ ID NOs: 158-171), or an extracellular ActRII chimera having any one sequence of any of SEQ ID NOs: 174-216 (e.g., 195-216)) fused to a portion (e.g., an Fc domain monomer, an Fc domain (e.g., a wild-type Fc domain), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) can be used as a therapeutic protein. Nucleic acids encoding the polypeptides described herein, or vectors containing such nucleic acids, can also be administered according to any of the methods described herein.In any of the methods described herein, polypeptides, nucleic acids, or vectors can be administered as part of a pharmaceutical composition. Compositions that can be administered to a subject according to the methods described herein are shown in Tables 10, 11, and 12 below.

[0236] [Table 10-1]

[0237] [Table 10-2]

[0238] [Table 10-3]

[0239] [Table 10-4]

[0240] [Table 10-5]

[0241] [Table 10-6]

[0242] [Table 10-7]

[0243] [Table 11-1]

[0244] [Table 11-2]

[0245] Table 11-3

[0246] Table 11-4

[0247] Table 11-5

[0248] Table 11-6

[0249] Table 11-7

[0250] Table 12-1

[0251] Table 12-2

[0252] Table 12-3

[0253] Table 12-4

[0254] Table 12-5

[0255] [Table 12-6]

[0256] [Table 12-7]

[0257] [Table 12-8]

[0258] [Table 12-9]

[0259] [Table 12-10]

[0260] [Table 12-11]

[0261] [Table 12-12]

[0262] [Table 12-13]

[0263] [Table 12-14] [Examples]

[0264] The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention. Their exemplary nature will lead to the understanding that other procedures, methods, or techniques known to those skilled in the art may be used instead.

[0265] Example 1 - Effects of ActRIIA / B-Fc on erythropoietin and erythropoietin receptor levels Evaluation of serum erythropoietin levels Eleven-week-old male mice (received from Taconic Biosciences) were treated with a single dose of 10 mg / kg of ActRIIA / B-mFc (SEQ ID NO: 69 linked to the mouse Fc domain) via intraperitoneal (IP) injection or with a vehicle. Mice were sacrificed on days 2, 4, 7, 14, 37, and 51 post-treatment. Blood was sampled from submandibular / cheek hemorrhages into EDTA tubes and stored at 4°C until RBC count analysis was performed using IDEXX BioAnalytics. At necropsy, whole blood was collected from the heart and collected in Microvette tubes (Sarstedt). The tubes were incubated at room temperature for 2 hours and then centrifuged at 6000 RPM for 10 minutes. Serum was collected and stored at -80°C until analysis.

[0266] Erythropoietin (EPO) levels were measured using a mouse erythropoietin immunoassay (Quantikine ELISA kit) according to the manufacturer's instructions for use. ELISA plate colorimetric readings were measured at 450 nm using a SpectraMax M5 microplate reader, with background subtracted from the 540 nm reading. Erythropoietin concentrations were converted by creating a four-parameter logistic curve using GraphPad Prism software. As shown in Figure 4, EPO levels increased in response to ActRIIA / B-mFc treatment, even when RBC levels increased. Two-factor ANOVA between ActRIIA / B-mFc treatment and vehicle, followed by a Sidak post-hoc test, were performed at each time point: *p<0.05, **p<0.01, ****p<0.0001. Data are shown as mean ± SEM. Figure 5 shows that EPO levels increased from day 4 to day 37 in mice treated with ActRIIA / B-mFc compared with vehicles-treated mice. t-tests were performed at each time point between ActRIIA / B-mFc treatment and vehicle treatment (*p≦0.05;**p≦0.01;p****≦0.0001). Data are shown as mean ± SEM.

[0267] Evaluation of erythropoietin receptor expression Eleven-week-old male mice (received from Taconic Biosciences) were treated with a single dose of 10 mg / kg of ActRIIA / B-mFc via IP injection or with a vehicle. Mice were sacrificed on days 4, 7, and 14 post-treatment. Bone marrow cells were isolated by washing the bone marrow from the femur. Red blood cells were removed by lysis using ammonium chloride solution (Stem Cell Technologies). One million cells were lysed with Trizol reagent, and RNA was isolated using the Direct-zol RNA MicroPrep (Zymo Research) kit. cDNA was synthesized using a reverse transcription kit (Qiagen), and EpoR expression was measured by qPCR using the ViiA 7 real-time PCR system (Applied Biosystems). Primers used: EpoR forward: TTCAGCGGATTCTGGAGTGCCT (SEQ ID NO: 376) and EpoR reverse: AGCAACAGCGAGATGAGGACCA (SEQ ID NO: 377). β-actin was used as a housekeeping gene. Primers used were: β-actin forward: CATCGTGGGCCGCCCTA (SEQ ID NO: 378) and β-actin reverse: CACCCACATAGGAGTCCTTCTG (SEQ ID NO: 379). Relative gene expression was determined using the ΔΔCT method, and the graph was plotted in GraphPad Prism. Error bars represent SEM. Statistical analysis was performed using Student's t-test. As shown in Figure 6, EPO receptor expression increased after ActRIIA / B-mFc treatment.

[0268] Example 2 - Treatment of end-stage renal disease by administration of extracellular ActRII variant According to the methods disclosed herein, a physician skilled in the art can treat a subject with end-stage renal disease, such as a human patient, to increase EPO levels. To treat the subject, a physician skilled in the art may administer to the subject a composition comprising a polypeptide containing an extracellular ActRII variant (e.g., a polypeptide containing an extracellular ActRIIA variant having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), a polypeptide containing an extracellular ActRIIB variant having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or a polypeptide containing an extracellular ActRII chimera having any one sequence of SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216), such as an extracellular ActRIIa variant linked to an Fc domain, an extracellular ActRIIB variant, or an extracellular ActRII chimera). The composition containing the extracellular ActRII variant may be administered to the subject, for example, by parenteral injection (e.g., intravenous or subcutaneous injection) to treat end-stage renal disease. Polypeptides containing extracellular ActRII variants (e.g., extracellular ActRIIA variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72), extracellular ActRIIB variants having any one sequence of SEQ ID NOs. 157-171 (e.g., SEQ ID NOs. 158-171), or extracellular ActRII chimeras having any one sequence of SEQ ID NOs. 174-216 (e.g., SEQ ID NOs. 195-216)) are 0.01-500m It is administered in therapeutically effective doses such as g / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg).In some embodiments, the extracellular ActRII variant is administered once every 16 weeks, quarterly, every 12 weeks, every two months, every eight weeks, every month, every four weeks, every two weeks, or at least once a week or more (e.g., once, two, three, four, five, six, or seven times or more per week). The extracellular ActRII variant is administered in a dose sufficient to increase EPO levels, increase EPO receptor levels, and / or slow disease progression.

[0269] After administering the composition to a patient, a physician in the art can monitor the patient's improvement in response to the treatment in various ways. For example, a physician can monitor the patient's EPO levels using blood tests and measure renal function using blood tests, urine tests, and imaging tests. Findings that the patient's EPO levels increase or that the disease progresses more slowly after administration of the composition, compared to test results before administration of the composition, indicate that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.

[0270] Other Embodiments While the present invention is described in relation to its specific embodiments, it should be understood that the invention is subject to further modifications, and this application is intended to encompass any variations, uses, or adaptations of the invention, including any deviations from the present disclosure that are generally in accordance with the principles of the invention, are known or practice, fall within the scope of the art to which the invention relates, and can be applied to the essential features described above.

[0271] All publications, patents, and patent applications are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated so as if it were incorporated herein by reference in whole.

[0272] Other embodiments are described in the claims.

Claims

1. A pharmaceutical composition for increasing erythropoietin levels or erythropoietin receptor levels in a subject requiring such increase, comprising a therapeutically effective amount of polypeptide containing an extracellular activin receptor type IIA (ActRIIA) variant, wherein the ActRIIA variant comprises the amino acid sequence of SEQ ID NO:

69.

2. The pharmaceutical composition according to claim 1, wherein the subject has dialysis-induced anemia or anemia of prematurity, or is at risk of developing either of these conditions.

3. The pharmaceutical composition according to claim 1 or 2, wherein the subject has or is at risk of developing end-stage renal disease, renal failure, polycythemia, hemochromatosis, diseases or conditions related to endothelial progenitor cell dysfunction, diseases or conditions having autoimmune or inflammatory components, neurological disorders or inflammatory brain diseases, gastrointestinal motility disorders, endocrine disorders, reproductive system disorders, aging, pregnancy, menstrual disorders, ischemia or ischemic disorders or conditions, hypoxia or hypoxic disorders or conditions, ulcers, burns, wounds, ischemia-reperfusion injury, asthma, hypertension, viral diseases or infections, systemic microbial infections, gastrointestinal diseases, arteriosclerosis, cancer, psychosis, genetic disorders, inflammatory diseases, graft-versus-host diseases, cardiovascular diseases, allergies, or arthritis.

4. (a) Whether the ischemia is central nervous system ischemia, hepatic ischemia, renal ischemia, or cardiac ischemia, (b) The pharmaceutical composition according to claim 3, wherein the ischemic disorder or condition is obstructive arterial disease, chronic venous insufficiency, circulatory shock, pulmonary embolism, myocardial infarction, ischemic stroke, acute respiratory failure, chronic heart failure, atherosclerosis, cardiac cirrhosis, macular degeneration, sleep apnea, Raynaud's disease, systemic sclerosis, nonbacterial thrombotic endocarditis, transient ischemic attack, or ischemia due to general anesthesia.

5. The pharmaceutical composition according to claim 3, wherein the hypoxic injury or condition is lung injury, severe pneumonia, pulmonary edema, hyaline membrane disease, liver disease, kidney disease, cancer, or altitude sickness.

6. The pharmaceutical composition according to claim 3, wherein the disease or condition associated with the dysfunction of the endothelial progenitor cells is heart failure, angina pectoris, endotheliopathy, reticuloendotheliopathy, age-related cardiovascular disease, coronary heart disease, atherosclerosis, myocardial ischemia, hypercholesterolemia, ischemic injury of the limbs, Raynaud's disease, pre-eclampsia, pregnancy-induced hypertension, endothelial-mediated chronic inflammatory disease, wound healing, chronic renal failure, or acute renal failure.

7. The pharmaceutical composition according to claim 3, wherein the autoimmune or inflammatory disease or condition is acute cerebrovascular injury, acute brain injury, acute cardiovascular injury, arthritis, autoimmune disease, stroke, nerve injury, or immune-mediated inflammation.

8. The pharmaceutical composition according to claim 3, wherein the neurological disorder or inflammatory brain disease is a demyelinating disease, epilepsy, spinal cord injury, complications after traumatic brain injury, chronic inflammatory brain disease, or a neurological disorder related to surgery.

9. The pharmaceutical composition according to claim 8, wherein the chronic inflammatory brain disease is a neurodegenerative disease.

10. The pharmaceutical composition according to claim 3, wherein the gastrointestinal motility disorder is related to intestinal injury, abdominal trauma, intestinal inflammatory condition, intestinal infection, delayed constipation, postoperative ileus, neurodegenerative injury, neurotrauma, congenital problems, or malnutrition-malabsorption problems.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the polypeptide is administered to the subject before surgery, after stem cell transplantation, before or during space flight, during or after tissue or organ transplantation, to promote the proliferation of new blood vessels, for granulation tissue formation, for the treatment of trauma, or for the treatment after vascular transplantation.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the subject is undergoing renal dialysis.

13. The pharmaceutical composition according to any one of claims 1 or 3 to 12, wherein the subject does not have anemia.

14. The pharmaceutical composition according to any one of claims 1 or 3 to 13, wherein the subject has normal hematopoiesis.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the subject has low serum erythropoietin levels.

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