Methods of treating myostatin-related or activin a-related disorders using novel hybrid actriib ligand trap proteins

Abstract

The present disclosure describes novel hybrid soluble ActRIIB-ECD polypeptides which fully retain binding affinity for myostatin and activin A but demonstrate significantly reduced binding to BMPs, especially BMP-9. The novel compositions described herein can be used to prepare novel hybrid ActRIIB ligand trap proteins, which can be used for modulating the growth of muscle, bone, cartilage, fat, fibroblast, blood and neuronal tissue to counteract muscle wasting, bone loss, inflammation and fibrosis in a therapeutically meaningful manner. Because these novel next generation myostatin / activin inhibitors are safer and more effective molecules than the currently available myostatin inhibitors, they are useful in a wide variety of clinical indications.

Description

METHODS OF TREATING MYOSTATIN-RELATED OR ACTIVIN A-RELATED DISORDERS USING NOVEL HYBRID ACTRIIB LIGAND TRAP PROTEINSRELATED PATENT APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 558,391 , filed on February 27, 2024, incorporated in its entirety by reference herein.SEQUENCE LISTINGS

[0002] The contents of the electronic sequence listing (ALIVEGEN-0011 P1 a1 b.xml; Size: 13 Kilobytes; Date of Initial Creation: February 12, 2025; Production Date: February 18, 2025, is herein incorporated by reference in its entirety.BACKGROUND

[0003] Muscle wasting is a debilitating and life-threatening disease state, which has been associated with the development of a number of chronic, neurological, genetic, inflammatory, fibrotic or infectious pathologies, including, e.g, muscular dystrophies, spinal muscular atrophy, amyotrophic lateral sclerosis, myositis, denervation muscle atrophies, neurodegenerative diseases such as Alzheimer’s disease, Parkinson's disease and Huntington’s disease, cancers, chronic kidney disease, heart failure, chronic obstructive pulmonary disease, pulmonary hypertension, pulmonary fibrosis, trauma, rheumatoid arthritis, osteoarthritis, diabetes, sarcopenic obesity, age-related sarcopenia, androgen deprivation, corticosteroid myopathy, inflammatory bowel disease, nonalcoholic fatty liver disease, liver cirrhosis, anorexia-cachexia syndrome, and HIV infection. Other conditions said to cause muscle wasting include chronic lower back pain, advanced age, damage to central nervous system, peripheral nerve injury, chemical injury, extended burns, hip / knee replacement, disuse atrophy, exposure to microgravity, and longterm hospitalization.

[0004] Bone disease is considered any affliction that involves the skeletal system. Bone diseases can be very serious and require prompt and effective treatment. Bone diseases can be very painful and can rob the patient of mobility and independence. While the causative factors vary by disease, many bone diseases are caused by, e.g., genetic factors, viral infection,chemical abnormalities, lack of bone collagen, injuries, fractures, damage to blood vessel, excessive use of alcohol, or the long-term use of certain medications. Examples of bone disease include osteoporosis, osteomalacia, osteogenesis imperfecta, fibrous dysplasia, ossificans progressiva, renal osteodystrophy, corticosteroid-induced bone loss, bone fracture, bone metastasis and Paget’s disease of the bone.

[0005] There have been important advancements in the treatment of such disorders, but there still exists a critical need to provide novel therapeutics, which are both highly effective and safe, for the treatment of muscle wasting and / or bone disease associated with the development of a number of chronic, neurological, genetic, inflammatory, fibrotic or infectious pathologies.

[0006] Activin 11 A receptor (ActRIIA) and Activin 11 B receptor (ActRIIB) are type II receptors for a subset of TGF- family member ligands, including, e.g., activin A, myostatin (also known as GDF-8), growth differentiation factor-11 (GDF-11 ), and various other bone morphogenetic proteins (BMPs) such as BMP-3, BMP-6, BMP-9 (also known as GDF-2) and BMP-10. ActRIIA and ActRIIB have been identified as the type II receptors for activins, including activin A, activin B and activin AB. ActRIIB is a high affinity receptor for myostatin, a key negative regulator of muscle growth, and thus plays central role in controlling muscle mass. The binding of these ligands to ActRIIA and / or ActRIIB can regulate cell differentiation, apoptosis, protein synthesis and degradation, mineralization, hematopoiesis, angiogenesis, steroid synthesis, adhesion, migration, extracellular matrix production and fibrogenesis. The specific response depends upon the types and levels of the TGF-p ligands and receptors as well as the cellular state and environment. The ActRIIB signaling pathway mediates cellular responses via Smad2 / 3 transcription factors, and activation of the ActRIIB signaling pathway has been implicated in pathogenesis and progression of many diseases including muscle wasting, bone loss, fibrosis and inflammation. Several members of the TGF-p family, including myostatin, activins and GDF11 , mediate Smad2 / 3 activation by coupling to ActRIIB.

[0007] The present inventors have previously designed a class of novel, nextgeneration, selective hybrid ActRIIB ligand traps in the form of an Fc fusion protein consisting of a modified extracellular domain (ECD) of the human Activin Type 2B Receptor (ActRIIB-ECD) fused to a stabilized human lgG4 Fc (see, e.g., PCTWO2016171948). On such ligand trap, ALG- 801 (referred to as “AG0027” in PCTWO2016171948) has been designed to selectively block Smad2 / 3 signaling mediated by a subset of TGF-p superfamily members, including myostatin, activin A, activin B and GDF11 , while completely sparing the BMP9-mediated Smad1 / 5 / 8 signaling. In several preclinical disease models, including experimentally induced androgendeficiency, ALG-801 has been shown to promote muscle growth and enhancing bone formation and thereby reverse muscle and bone loss. Based on these preclinical data, the present inventors are evaluating the efficacy of ALG-801 for the treatment of muscle wasting and / or bone disease associated with the development of a number of chronic, neurological, genetic, inflammatory, fibrotic or infectious pathologies.

[0008] The present inventors’ patent documents PCT WO2016171948; PCT WO2018075747; US 10,913,782; US 1 1 ,292,826; US 17 / 682,277 and US 17 / 544,579 are herein specifically incorporated by reference for all teachings.DISCLOSURE OF THE INVENTION

[0009] In one aspect, the present invention provides a method of treating myostatin- related or activin A-related disorders in a subject, comprising administering to said subject a therapeutically effective amount of an isolated protein comprising a hybrid soluble activin I IB receptor-extracellular domain (ActRIIB-ECD) polypeptide fused to a human Fc domain, wherein said myostatin-related or activin A-related disorder is selected from the group consisting of: a bone disorder, a muscle wasting disease, a cardiovascular disease, a metabolic disorder, a renal disease, an inflammatory / autoimmune disease, a fibrosis disease, and aging, and treating cancer cells.

[0010] In various embodiments, the myostatin-related or activin A-related disorder is a muscle wasting disease selected from the group consisting of muscular dystrophy, spinal muscular atrophy, myositis, denervation atrophy, muscle atrophy associated with chronic heart failure, chronic obstruct pulmonary disease, chronic kidney disease, inflammation, or cancer, and disuse muscle atrophy associated with prolonged bed rest.

[0011] In various embodiments, the myostatin-related or activin A-related disorder is a bone disorder selected from the group consisting of osteopenia, bone fragility, fracture, osteogenesis imperfecta, bone loss resulting from androgen deprivation or menopause, and osteoporosis.

[0012] In various embodiments, the myostatin-related or activin A-related disorder is a cardiovascular disease selected from the group consisting of congestive heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), pulmonary arterial hypertension (PAH), hypertension, myocardial infarction (Ml),cardiac fibrosis, cardiomyopathy, cardiac arrythmia, cancer / chemotherapy / immunotherapy induced myocardial dysfunction, and vascular calcification.

[0013] In various embodiments, the myostatin-related or activin A-related disorder is a metabolic disorder selected from the group consisting of obesity, insulin resistance, sarcopenic obesity, diabetes, prediabetes, and metabolic syndrome.

[0014] In various embodiments, the myostatin-related or activin A-related disorder is a renal disease selected from the group consisting of chronic kidney disease, diabetic nephropathy, mineral bone disease, vascular calcification, polycystic kidney disease, and renal fibrosis.

[0015] In various embodiments, the myostatin-related or activin A-related disorder is a a pulmonary disease selected from the group consisting of chronic obstructive pulmonary disease, asthma, severe acute respiratory syndrome, pulmonary fibrosis, and pulmonary hypertension.

[0016] In various embodiments, the hybrid ActRIIB ligand trap proteins comprise hybrid soluble ActRIIB-ECD polypeptides having the amino acid sequence set forth in SEQ ID NO: 3, wherein the hybrid ActRIIB-ECD polypeptide is capable of binding myostatin and activin A, but demonstrates a decreased binding affinity for BMP9 relative to a wild-type ActRIIB-ECD polypeptide. In various embodiments, the hybrid soluble ActRIIB polypeptides are hybrid soluble ActRIIB polypeptides having an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, wherein the hybrid ActRIIB-ECD polypeptide is capable of binding myostatin and activin A, but demonstrates a decreased binding affinity for BMP9 relative to a wild-type ActRIIB-ECD polypeptide.

[0017] In various embodiments, the Fc domain is selected from the group consisting of the Fc domain of a human IgG 1 , the Fc domain of a human lgG2, and the Fc domain of a human lgG4. In various embodiments, the Fc domain is selected from the group consisting of a human Fc domain having the amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

[0018] In various embodiments, the Fc domain is attached to the hybrid soluble ActRIIB- ECD polypeptide by a linker and / or a hinge linker peptide. The linker or hinge linker may be an artificial sequence of between 5, 10, 15, 20, 30, 40 or more amino acids that are relatively free of secondary structure. In various embodiments, the linker is rich in G / S content (e.g., at least about 60%, 70%, 80%, 90%, or more of the amino acids in the linker are G or S. In various embodiments, the linker has a (GGGGS (SEQ ID NO: 10))nmotif, wherein n = 1 -6.

[0019] In various embodiments, a linker having the amino acid sequence set forth in SEQ ID NO: 10 is used with a hinge linker having the amino acid sequence set forth in SEQ ID NO: 11 to link a human lgG4 Fc (SEQ ID NO: 9), to a hybrid soluble ActRIIB-ECD polypeptide (e.g., SEQ ID NO: 3) of the present disclosure.

[0020] In various embodiments, a hybrid soluble ActRIIB-ECD polypeptide having the amino acid sequence of SEQ ID NO: 3, is attached to a human Fc domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. In various embodiments, a hybrid soluble ActRIIB-ECD polypeptide having an amino acid sequence of SEQ ID NO: 3 is attached to a human Fc domain having an amino acid sequence of SEQ ID NO: 9.

[0021] In various embodiments, a hybrid soluble ActRIIB-ECD polypeptide having the amino acid sequence of SEQ ID NO: 3 is fused to a peptide linker having the amino acid sequence set forth in SEQ ID NO: 10, the peptide linker is fused to a hinge linker having the amino acid sequence set forth in SEQ ID NO: 11 , and the hinge linker is fused to an Fc domain having an amino acid sequence of SEQ ID NO: 9 (hereinafter “ALG-801 ”).

[0022] In various embodiments, an exemplary, non-limiting daily dosing range for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure can be 0.001 to 100 mg / kg, 0.001 to 90 mg / kg, 0.001 to 80 mg / kg, 0.001 to 70 mg / kg, 0.001 to 60 mg / kg, 0.001 to 50 mg / kg, 0.001 to 40 mg / kg, 0.001 to 30 mg / kg, 0.001 to 20 mg / kg, 0.001 to 10 mg / kg, 0.001 to 5 mg / kg, 0.001 to 4 mg / kg, 0.001 to 3 mg / kg, 0.001 to 2 mg / kg, 0.001 to 1 mg / kg, 0.010 to 50 mg / kg, 0.010 to 40 mg / kg, 0.010 to 30 mg / kg, 0.010 to 20 mg / kg, 0.010 to 10 mg / kg, 0.010 to 5 mg / kg, 0.010 to 4 mg / kg, 0.010 to 3 mg / kg, 0.010 to 2 mg / kg, 0.010 to 1 mg / kg, 0.1 to 50 mg / kg, 0.1 to 40 mg / kg, 0.1 to 30 mg / kg, 0.1 to 20 mg / kg, 0.1 to 10 mg / kg, 0.1 to 5 mg / kg, 0.1 to 4 mg / kg, 0.1 to 3 mg / kg, 0.1 to 2 mg / kg, 0.1 to 1 mg / kg, 1 to 50 mg / kg, 1 to 40 mg / kg, 1 to 30 mg / kg, 1 to 20 mg / kg, 1 to 10 mg / kg, 1 to 5 mg / kg, 1 to 4 mg / kg, 1 to 3 mg / kg, 1 to 2 mg / kg, or 1 to 1 mg / kg body weight. It is to be noted that dosage values may vary with the type and severity of the conditions to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0023] In various embodiments, the individual dosing range for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 0.3 mg / kg to 4.5 mg / kg administered subcutaneously under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 0.3 mg / kg administered subcutaneously under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 1 mg / kg administered subcutaneously under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 3 mg / kg administered subcutaneously under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 4.5 mg / kg administered subcutaneously under fasting conditions.

[0024] In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 1 mg / kg administered subcutaneously weekly for 4 weeks under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 3 mg / kg administered subcutaneously weekly for 4 weeks under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 4.5 mg / kg administered subcutaneously weekly for 4 weeks under fasting conditions.

[0025] In various embodiments, the total dose administered will achieve a plasma antibody concentration in the range of, e.g., about 1 to 1000 pg / ml, about 1 to 750 pg / ml, about 1 to 500 pg / ml, about 1 to 250 pg / ml, about 10 to 1000 pg / ml, about 10 to 750 pg / ml, about 10 to 500 pg / ml, about 10 to 250 pg / ml, about 20 to 1000 pg / ml, about 20 to 750 pg / ml, about 20 to 500 pg / ml, about 20 to 250 pg / ml, about 30 to 1000 pg / ml, about 30 to 750 pg / ml, about 30 to 500 pg / ml, about 30 to 250 pg / ml.BRIEF DESCRIPTION OF THE FIGURES

[0026] FIG. 1 is a line graph depicting free activin levels as measured using ELISA. Data indicates that subcutaneous administration of ALG-801 resulted in a dose-dependent and long-lasting decrease in free activin A levels in clinical serum samples from community dwelling,healthy older men, thus demonstrating the ability of ALG-801 to sequester the target ligand activin A.

[0027] FIG. 2 is a line graph depicting free BMP9 levels as measured by ELISA. Data indicates that SC administration of ALG-801 had no effect on free BMP9 levels in clinical serum samples from community dwelling, healthy older men, thus demonstrating the ability of ALG-801 to spare BMP9.

[0028] FIGS. 3A is a line graph depicting that a single subcutaneous administration of ALG-801 resulted in dose dependent decrease in bone resorption biomarker CTX in community dwelling, healthy older men. FIGS. 3B is a line graph depicting that a single subcutaneous administration of ALG-801 resulted in an increase in bone formation biomarker BSAP in community dwelling, healthy older men.

[0029] FIG. 4 is a line graph depicting that a single subcutaneous administration of ALG- 801 resulted in dose dependent decrease in BUN biomarker in community dwelling, healthy older men, suggesting anabolic and anti-catabolic effect on protein metabolism.

[0030] FIG. 5A is a line graph depicting the percent change from baseline of right anterior thigh muscle volume and FIG. 5B is a line graph depicting the percent change from baseline of left anterior thigh muscle volume in healthy postmenopausal volunteer subjects in the Phase 1 b study who received 4 weekly SC injections of ALG-801 at 1 .Omg / kg, 3.0mg / kg, and 4.5mg / kg, respectively, compared to placebo control. Thigh muscle volumes were measured by MRI imaging at baseline (pre-dose), Day 29 and Day 43. ALG-801 administration led to dose-dependent and statistically significant increase in thigh muscle volume in postmenopausal women.

[0031] FIG. 6 is a line graph depicting the percent change from baseline of abdominal subcutaneous fat in healthy postmenopausal volunteer subjects in the Phase 1 b study who received 4 weekly SC injections of ALG-801 at 1 .0mg / kg, 3. Omg / kg, and 4.5mg / kg, respectively, compared to placebo control. Abdominal subcutaneous fat volumes were measured by MRI imaging at baseline (pre-dose), Day 29 and Day 43. ALG-801 administration resulted in dosedependent and statistically significant reduction of body fat in postmenopausal women.

[0032] FIG. 7 is a line graph depicting the percent change from baseline of leptin-to- adiponectin ratio in healthy postmenopausal volunteer subjects in the Phase 1 b study who received 4 weekly SC injections at 1 .Omg / kg, 3. Omg / kg, and 4.5mg / kg, respectively, compared to placebo control. ALG-801 administration significantly reduced leptin-to-adiponectin ratio (LAR), an acute index of insulin resistance in postmenopausal women.MODE(S) FOR CARRYING OUT THE INVENTION

[0033] The present disclosure provides for methods of treating myostatin-related or activin A-related disorders in a subject, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a novel isolated hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier. The novel isolated hybrid ActRIIB ligand trap proteins genetically engineered as fusion proteins which function as a multi-cytokine antagonist designed to selectively block the actions of multiple cachectic (atrophy-inducing) cytokines without affecting the signaling of nonmuscle related cytokines. In various embodiments, the hybrid ActRIIB ligand trap proteins comprise isolated hybrid soluble ActRIIB-ECD polypeptides which are capable of binding myostatin and activin A but demonstrate a decreased binding affinity for BMP9 (i.e., retain myostatin- and activin A-neutralizing activities, but demonstrate dramatically reduced BMP9- neutralization) relative to a wild-type ActRIIB-ECD polypeptide.Definitions

[0034] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those commonly used and well known in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0035] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of two or more amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The terms “antibody” and “antibodies” are used interchangeably herein and refer to a polypeptide capable of interacting with and / or binding to another molecule, often referred to as an antigen. Antibodies can include, for example “antigen-binding polypeptides” or “target-molecule binding polypeptides.” Antigens of the present invention can include for example any polypeptides described in the present invention.

[0036] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gammacarboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. All single letters used in the present invention to represent amino acids are used according to recognized amino acid symbols routinely used in the field, e.g., A means Alanine, C means Cysteine, etc. An amino acid is represented by a single letter before and after the relevant position to reflect the change from original amino acid (before the position) to changed amino acid (after position). For example, A19T means that amino acid alanine at position 19 is changed to threonine.

[0037] The terms "polypeptide variant" and “polypeptide mutant” as used herein refers to a polypeptide that comprises an amino acid sequence wherein one or more amino acidresidues are inserted into, deleted from and / or substituted into the amino acid sequence relative to another polypeptide sequence. In certain embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length. Variants of the present disclosure include fusion proteins.

[0038] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

[0039] The term "% sequence identity" is used interchangeably herein with the term "% identity" and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same thing as 80% sequence identity determined by a defined algorithm and means that a given sequence is at least 80% identical to another length of another sequence. In certain embodiments, the % identity is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In certain embodiments, the % identity is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0040] The term "% sequence homology" is used interchangeably herein with the term "% homology" and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same thing as 80% sequence homology determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence homology over a length of the given sequence. In certain embodiments, the % homology is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence homology to a given sequence. In certain embodiments, the % homology is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0041] Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet at the NCBI website. See also Altschul et aL, 1990, J. Mol. Biol. 215:403-10 (with special reference to the published default setting, i.e., parameters w=4, t=17) and Altschul et aL, 1997, Nucleic Acids Res., 25:3389-3402. Sequence searches are typically carried out using the BLASTP program when evaluating a given amino acid sequence relative to amino acid sequences in the GenBank Protein Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 1 1 .0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (Id).

[0042] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is, e.g., less than about 0.1 , less than about 0.01 , or less than about 0.001 .

[0043] The term "Fc region" as used herein defines the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. The Fc portion of an antibody mediates several important effector functions e.g. cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life / clearance rate of antibody and antigen-antibody complexes (e.g., the neonatal FcR (FcRn) binds to the Fc region of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG). Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (see, e.g., Winter et aL, U.S. Patent No. 5,648,260 and 5,624,821 ).

[0044] The term "isolated molecule" (where the molecule is, for example, a polypeptide, a polynucleotide, or an antibody) is a molecule that by virtue of its origin or source of derivation(1 ) is not associated with naturally associated components that accompany it in its native state,(2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be "isolated" from its naturally associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0045] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60% to 75% of a sample exhibits a single species of polypeptide. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W / W of a protein sample, more usually about 95%, and e.g., will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0046] "Linker" refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thus joining two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded or otherwise severed to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers may also be cleaved by environmental cues, such as, for example, changes in temperature, pH, salt concentration, etc.

[0047] The terms "label" or "labeled" as used herein refers to incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In anotherembodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,3H,14C,15N,35S,90Y, "Tc,111In,125l,1311), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0048] A "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal or human. A pharmaceutical composition comprises a pharmacologically and / or therapeutically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and carriers, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21 st Ed. 2005, Mack Publishing Co, Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0049] The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and / or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats.Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.

[0050] "Pharmaceutically acceptable excipient "means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0051] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0052] "Dosage unit" refers to physically discrete units suited as unitary dosages for the individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0053] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In an embodiment, the mammal is a human. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g. mouse, rat, etc.

[0054] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease, and / or symptoms of the disease. “Treatment,” as used herein, covers any treatment of any virus infection or exposure in a mammal, particularly in a human, and includes: (a) preventing the infection; (b) inhibiting the infection, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of infection.

[0055] The phrase “determining the treatment efficacy” and variants thereof can include any methods for determining that a treatment is providing a benefit to a subject. The term“treatment efficacy” and variants thereof are generally indicated by alleviation of one or more signs or symptoms associated with the disease and can be readily determined by one skilled in the art. “Treatment efficacy” may also refer to the prevention or amelioration of signs and symptoms of toxicities typically associated with standard or non-standard treatments of a disease. Determination of treatment efficacy is usually indication and disease specific and can include any methods known or available in the art for determining that a treatment is providing a beneficial effect to a patient. For example, evidence of treatment efficacy can include but is not limited to remission of the disease or indication. Further, treatment efficacy can also include general improvements in the overall health of the subject, such as but not limited to enhancement of patient life quality, increase in predicted subject survival rate, decrease in depression or decrease in rate of recurrence of the indication (increase in remission time).(See, e.g., Physicians' Desk Reference (2010)).

[0056] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.

[0057] It is understood that aspect and embodiments of the disclosure described herein include “consisting” and / or “consisting essentially of” aspects and embodiments.

[0058] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0059] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variations of the disclosure described herein include "consisting" and / or "consisting essentially of" aspects and variations.Soluble Hybrid ActRIIB Polypeptides

[0060] The methods of the present disclosure utilize novel hybrid soluble ActRIIB-ECD polypeptides that are derived from wild-type ActRIIB-ECD and wild-type ActRIIA-ECD. The hybrid soluble ActRIIB polypeptides are specifically engineered by replacing one or more aminoacids of a truncated wild-type ActRIIB-ECD with the amino acids from a truncated wild-type ActRIIA-ECD at corresponding positions based on sequence alignment between the two truncated ActRII ECD domains at the amino acid level. The one or more amino acid replacements are specifically selected for purposes of providing hybrid soluble ActRIIB-ECD polypeptides which demonstrate a marked reduction of BMP9-neutralization as compared to wild-type ActRIIB-ECD polypeptide, while fully retaining strong myostatin- and activin A- neutralization. The present inventors have previously designed a class of novel, nextgeneration, selective hybrid ActRIIB ligand traps in the form of an Fc fusion protein consisting of a modified extracellular domain (ECD) of the human Activin Type 2B Receptor (ActRIIB-ECD) fused to a stabilized human lgG4 Fc (see, e.g., PCTWO2016171948, incorporated by reference in its entirety).

[0061] In various embodiments, the hybrid ActRIIB ligand trap proteins comprise a hybrid soluble ActRIIB-ECD polypeptide having the amino acid sequence set forth in SEQ ID NO: 3, wherein the hybrid ActRIIB-ECD polypeptide is capable of binding myostatin and activin A, but demonstrates a decreased binding affinity for BMP9 relative to a wild-type ActRIIB-ECD polypeptide. In various embodiments, the hybrid soluble ActRIIB polypeptides are hybrid soluble ActRIIB polypeptides having an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 3, wherein the hybrid ActRIIB- ECD polypeptide is capable of binding myostatin and activin A, but demonstrates a decreased binding affinity for BMP9 relative to a wild-type ActRIIB-ECD polypeptide.Fc Domains

[0062] In another aspect, the hybrid ActRII ligand traps comprise a hybrid soluble ActRIIB-ECD polypeptide and at least one Fc domain attached to the ActRIIB-ECD polypeptide either directly or through a linker sequence to form a fusion protein referred to herein as hybrid ActRIIB ligand trap. As used herein the term "fusion protein" refers to a protein having a Fc domain attached via recombinant DNA techniques. In various embodiments, the Fc domain is a human IgG Fc domain. In various embodiments, the Fc domain is derived from the human IgG 1 heavy chain constant domain sequence set forth in SEQ ID NO: 4. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO: 5. In various embodiments, the Fc domain is derived from the human lgG2 heavy chain constant domain sequence set forth in SEQ ID NO: 6. In various embodiments, the Fc domain is an Fcdomain having the amino acid sequence set forth in SEQ ID NO: 7. In various embodiments, the Fc domain is derived from the human lgG4 heavy chain constant domain sequence set forth in SEQ ID NO: 8. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO: 9.Linkers

[0063] The hybrid ActRIIB hybrid traps can optionally further comprise a "linker" or “hinge linker” sequence. Linkers serve primarily as a spacer between a hybrid soluble ActRIIB- ECD polypeptide a Fc domain or other type of fusion or between two or more hybrid soluble ActRIIB-ECD polypeptides. In various embodiments, the Fc domain is attached to the hybrid soluble ActRIIB-ECD polypeptide by a linker or a hinge linker peptide. The linker and / or hinge linker may be an artificial sequence of between 5, 10, 15, 20, 30, 40 or more amino acids that are relatively free of secondary structure. In various embodiments, the linkers comprise amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In various embodiments, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine, and are polyglycines (particularly (Gly)s, (Gly)s, poly(Gly-Ala), and polyalanines. In various embodiments, the linker is rich in G / S content (e.g., at least about 60%, 70%, 80%, 90%, or more of the amino acids in the linker are G or S. In various embodiments, the linker has a (GGGGS (SEQ ID NO: 10))nmotif, wherein n = 1 -6. Such linkers and hinge linkers have been described extensively in art (see, e.g., US 8,410,043 (Sun et al), incorporated by reference herein for the purposes of teaching such linkers).

[0064] Linkers may also be non-peptide linkers. For example, alkyl linkers such as -NH- -(CH2)S-C(O)-, wherein s = 2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., Ci-Ce) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc.Molecular Configurations for the Hybrid ActRIIB ligand trap proteins

[0065] It is understood that the different elements of the hybrid ActRIIB ligand trap may be arranged in any manner that is consistent with the desired functionality. For example, an Fc domain may be placed C-terminal to a hybrid soluble ActRIIB-ECD polypeptide, or alternatively the hybrid soluble ActRIIB-ECD polypeptide may be placed C-terminal to a Fc domain. The hybridsoluble ActRII-ECD polypeptide domain and the Fc domain need not be adjacent, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains (i.e. include a linker described herein).

[0066] In various embodiments, the Fc domain is attached to the hybrid soluble ActRIIB- ECD polypeptide by a linker and / or a hinge linker peptide. The linker or hinge linker may be an artificial sequence of between 5, 10, 15, 20, 30, 40 or more amino acids that are relatively free of secondary structure. In various embodiments, the linker is rich in G / S content (e.g., at least about 60%, 70%, 80%, 90%, or more of the amino acids in the linker are G or S. In various embodiments, the linker has a (GGGGS (SEQ ID NO: 10))nmotif, wherein n = 1 -6.

[0067] In various embodiments, a linker having the amino acid sequence set forth in SEQ ID NO: 10 and a hinge linker having the amino acid sequence set forth in SEQ ID NO: 11 is used to link a human lgG1 Fc (SEQ ID NO: 5) or a human lgG4 Fc (SEQ ID NO: 9) to a hybrid soluble ActRIIB-ECD polypeptide of the present disclosure.

[0068] In various embodiments, a linker having the amino acid sequence set forth in SEQ ID NO: 10 is used with a hinge linker having the amino acid sequence set forth in SEQ ID NO: 11 to link a human lgG4 Fc (SEQ ID NO: 9) to a hybrid soluble ActRIIB-ECD polypeptide (e.g., SEQ ID NO: 3) of the present disclosure.

[0069] In various embodiments, a hybrid soluble ActRIIB-ECD polypeptide having the amino acid sequence of SEQ ID NO: 3 is attached to a human Fc domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. In various embodiments, a hybrid soluble ActRIIB-ECD polypeptide having an amino acid sequence of SEQ ID NO: 3 is attached to a human Fc domain having an amino acid sequence of SEQ ID NO: 9.

[0070] In various embodiments, a hybrid soluble ActRIIB-ECD polypeptide having the amino acid sequence of SEQ ID NO: 3 is fused to a peptide linker having the amino acid sequence set forth in SEQ ID NO: 10, the peptide linker is fused to a hinge linker having the amino acid sequence set forth in SEQ ID NO: 11 , and the hinge linker is fused to an Fc domain having an amino acid sequence of SEQ ID NO: 9 (hereinafter “ALG-801 ”).Clinical Indications-Theraoeutic Uses

[0071] The in vitro and in vivo activities of ALG-801 have been extensively characterized. Compared to WT-ActRIIB-Fc, a benchmark Fc fusion molecule containing thewild-type human ActRIIB ECD, ALG-801 demonstrates equally potent binding affinity to myostatin or activin A but shows significantly reduced binding activity to BMP9 (David L, et aL, Circ Res., 102(8): 914-922 (2008); Wooderchak-Donahue WL et aL, J Hum Genet., 93(3): 530-7, 2013). In cell-based assays, ALG-801 strongly inhibits Smad2 / 3 signaling mediated by myostatin, activin A, activin B and GDF11 with equal potency as WT-ActRIIB-Fc; however, in contrast to WT-ActRIIB-Fc which potently binds to BMP9 and inhibits BMP9-mediated Smad1 / 5 / 8 signaling, ALG-801 spares BMP9 and does not inhibit BMP9-mediated Smad1 / 5 / 8 signaling in cell-based assays.

[0072] In vivo pharmacology studies demonstrate that ALG-801 promotes muscle growth, enhances bone formation and thereby reverses muscle and bone loss in several preclinical disease models. In orchiectomized mice (ORX mice), subcutaneous administration of ALG-801 rapidly and completely reversed the marked loss of muscle and bone resulting from androgen deprivation. In MDX mice, a model of Duchenne muscular dystrophy, subcutaneous administration of ALG-801 significantly increased muscle mass, enhanced grip strength, improved bone density and markedly reduced serum creatine kinase (CK) levels. In Oim / Oim mice, a model of Type III Osteogenesis Imperfecta, subcutaneous administration of ALG-801 significantly attenuated the profound loss of bone and muscle and normalized body length. These data demonstrate that ALG-801 is a novel muscle-bone dual anabolic agent with promising therapeutic potential for the treatment of muscle atrophy and bone fragility associated with a wide range of catabolic and degenerative conditions.

[0073] Following a single subcutaneous injection of ALG-801 in rats at 3, 15 and 70 mg / kg, the mean Cmax was 0.851 pg / mL, 9.90 pg / mL and 46.4 pg / mL, respectively; the mean AUCo- last was 156 hr*pg / mL, 1740 hr*pg / mL and 5920 hr*pg / mL, respectively; and the mean T1 / 2 was 115 hr, 72.9 hr and 79.9 hr, respectively. Following a single subcutaneous injection of ALG- 801 in monkeys at 2 mg / kg, 10 mg / kg and 50 mg / kg, mean Cmax was 1 .06 pg / mL, 8.69 pg / mL and 108 pg / mL, respectively; mean AUCo-iast was 121 hr*pg / mL, 894 hr* pg / mL and 6800 hr*pg / mL, respectively; and the mean T 1 / 2 was 75.0 hr, 76.3 hr and 106 hr, respectively. The results indicate that Cmax and AUCo-iast are increased with increasing dose level.

[0074] In addition, in monkeys, a single intravenous injection of ALG-801 at 50 mg / kg was evaluated. The mean Tmax was 0.0830 hr and 0.0830 hr in male and female monkeys; mean Co was 1150 pg / mL and 801 pg / mL in male and female monkeys; and mean AUCo-iast was 15200 hr*pg / mL and 10100 hr*pg / mL in male and female monkeys, respectively. The resultsindicate that ALG-801 has no relative gender difference (within 2-fold difference) in exposure to ALG-801 after single intravenous injection at 50 mg / kg ALG-801 .

[0075] GLP toxicological studies of up to 4 weeks have been conducted in rats and cynomolgus monkeys. Dose levels of 0, 2, 10, or 50 mg / kg / dose ALG-801 , administered via subcutaneous injection once weekly for 4 weeks to male and female cynomolgus monkeys, did not result in any adverse effects. The only test article-related effect included increased red blood cell count in animals administered 50 mg / kg / dose, which was reversible during the recovery (4 weeks) phase. Thus, the no observed adverse effect level (NOAEL) was 50 mg / kg / dose in the cynomolgus monkey.

[0076] In one aspect, the present disclosure provides a method for treating myostatin- related or activin A-related disorders in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier. Importantly, the pharmaceutical compositions of the present disclosure can be used to increase lean muscle mass as a percentage of body weight and decrease fat mass as percentage of body weight, while avoiding safety concerns reported for existing ActRIIB-Fc fusion proteinbased therapeutics.

[0077] In one aspect, the present disclosure provides a method of treating or preventing a muscle wasting in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in admixture with a pharmaceutically acceptable carrier, wherein such administration attenuates the loss of muscle mass and / or loss of muscle function. In various embodiments, the muscle wasting is associated with a disease selected from the group consisting of: muscular dystrophies (such as DMD, Becker MD, Limb-Girdle MD, Myotonic MD and FSHD), myositis (such as Dermatomyositis, Polymyositis and Inclusion body myositis), myopathy (including inherited myopathy as well as acquired myopathy such as myopathy induced by androgen-deprivation therapy, corticosteroids or statins), motoneuron disease (such as Lou Gehrig's Disease or ALS), spinal muscular atrophy (including Infantile progressive spinal muscular atrophy, Intermediate spinal muscular atrophy, Juvenile spinal muscular atrophy and Adult spinal muscular atrophy), neuromuscular junction disease (such as Myasthenia gravis, Lambert-Eaton syndrome and Botulism), peripheral nerve disease (such as Charcot-Marie tooth disease, Dejerine-Sottas disease and Friedreich's ataxia), spinal cord injury, stroke, neurodegenerative disease (including Parkinson’s disease, Huntington’s disease, Alzheimer’sdisease and Creutzfeldt-Jakob disease), cancer (such as lung cancer, pancreatic cancer, gastric cancer, colon cancer, prostate cancer, breast cancer, esophageal cancer, head and neck cancer, ovarian cancer, rhabdomyosarcoma, glioma, neuroblastoma, lymphoma, and multiple myeloma, skin cancer, and blood cancer), organ failure (such as heart failure, renal failure and liver failure, trauma (such as burns or motorcycle accident), disuse (such as longterm bed-rest, hospitalization, and spaceflight), infection (such as HIV, Polio and Sepsis), chronic obstructive pulmonary disease (COPD), and aging (such as sarcopenia, sarcopenic obesity and osteroarthritis).

[0078] In another aspect, the present disclosure provides a method of treating or preventing bone disease in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in admixture with a pharmaceutically acceptable carrier. In various embodiments, the bone disease is selected from the group consisting of: osteoporosis, renal osteodystrophy, osteogenesis imperfecta, fibrodysplasia ossificans progressiva, corticosteroid-induced bone loss, androgen-depriviation therapy-induced bone loss, hip fracture, cancer-induced bone loss, renal osteodystrophy, bone metastasis, Paget's disease, Rickets, osteomalacia, Perthes' disease and fibrous dysplasia.

[0079] In another aspect, the present disclosure provides a method of treating or preventing a metabolic disorder in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in admixture with a pharmaceutically acceptable carrier. In various embodiments, the metabolic disorder is selected from the group consisting of: metabolic syndrome, obesity, dyslipidemia, sarcopenic obesity, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis (NASH), alcoholic fatty liver disease, insulin resistance, diabetes as well as diabetic myopathy, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, and hemochromatosis.

[0080] In another aspect, the present disclosure provides a method of treating or preventing fibrosis in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in admixture with a pharmaceutically acceptable carrier. In various embodiments, the fibrosis is selected from the group consisting of: interstitial lung disease, idiotypic pulmonary fibrosis, cystic fibrosis, liver fibrosis, cirrhosis, biliary atresia, myocardial infarction, cardiac fibrosis, renal fibrosis, myelofibrosis, idiopathic retroperitonealfibrosis, nephrogenic fibrosing dermopathy, inflammatory bowel disease or Crohn's disease, keloid, scleroderma, retroperitoneal fibrosis, and arthrofibrosis.

[0081] In another aspect, the present disclosure provides a method of treating or preventing muscle atrophy and bone loss associated with an autoimmune / inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in admixture with a pharmaceutically acceptable carrier. In various embodiments, the disease is selected from the group consisting of: autoimmune / inflammatory disorders including rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, diabetes (type-1 ), glomerulonephritis, myasthenia gravis, psoriasis, systemic lupus erythematosus, polymyositis, Crohn's disease, ulcerative colitis, and primary biliary cirrhosis, arthritis, asthma, and sepsis.

[0082] In another aspect, the present disclosure provides a method of treating cardiovascular disease in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in admixture with a pharmaceutically acceptable carrier. In various embodiments, the cardiovascular disease is selected from the group consisting of: heart failure, cardiac atrophy, pulmonary hypertension, myocarditis, coronary artery disease, myocardial infarction, cardiac arrhythmias, heart valve disease, cardiomyopathy, pericardial disease, aorta disease, Marfan syndrome and cardiac transplant.

[0083] In another aspect, the present disclosure provides for a method of treating cardiac dysfunction or heart failure in a subject comprising administering an effective amount of a hybrid ActRIIB ligand trap into the subject. The modulation may improve cardiac function of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. The improvement of cardiac function can be evaluated by echocardiography to measure 1) cardiac pump functions focusing on the ejected blood volume and the efficiency of ejection and 2) myocardial functions focusing on the strength of myocardial contraction.

[0084] In another aspect, the present disclosure provides for a method of treating cancer cells in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of a cancer cell. Specifically, a hybridActRIIB ligand trap of the present disclosure is useful in treating disorders characterized as cancer. Such disorders include, but are not limited to solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases, lymphomas, sarcomas, multiple myeloma and leukemia. Examples of breast cancer include, but are not limited to invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of cancers of the respiratory tract include, but are not limited to small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancers include, but are not limited to brain stem and hypophthalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumor. Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus. Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, smallintestine, and salivary gland cancers. Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, and urethral cancers. Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma. Examples of liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head-and-neck cancers include, but are not limited to nasopharyngeal cancer, and lip and oral cavity cancer. Lymphomas include, but are not limited to AIDS-related lymphoma, nonHodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of the central nervous system. Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. In certain embodiments, the cancer will be a cancer with high expression of TGF-p family member, such as activin A, myostatin, TGF-|3 and GDF15, e.g., pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma leukemia, lung cancer, prostate cancer, brain cancer, bladder cancer, and head-neck cancer.

[0085] In another aspect, the present disclosure provides for a method of treating chronic kidney disease (CKD) in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier, wherein such administration attenuates the loss of muscle mass and / or loss of muscle function or inhibits kidney fibrosis. Specifically, a hybrid ActRIIB ligand trap of the present disclosure is useful in treating CKD including renal failure, interstitial fibrosis, and kidney dialysis as well as protein energy wasting (PEW) associated with CKD. The modulation may improve CKD or PEW of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. The improvement of renal function can be evaluated by measuring protein / creatinine ratio (PCR) in the urine and glomerular filtration rate (GFR). Improvement of PEW can be evaluated by measuring serum levels of albumin and inflammatory cytokines, rate of protein synthesis and degradation, body mass, muscle mass, physical activity and nutritional outcomes.

[0086] In another aspect, the present disclosure provides for methods for treating arthritis in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier. Specifically, a hybrid ActRIIB ligand trap of the present disclosure is useful in treating an arthritis selected from rheumatoid arthritis and osteoarthritis.

[0087] In another aspect, the present disclosure provides for methods for treating anorexia in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier. Specifically, a hybrid ActRIIB ligand trap of the present disclosure is useful in treating an anorexia selected from anorexia nervosa and anorexia-cachexia syndrome.

[0088] In another aspect, the present disclosure provides for methods for treating liver disease in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier. Specifically, a hybrid ActRIIB ligand trap of the present disclosure is useful in treating a liver disease selected from non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic fatty liver disease, liver cirrhosis, liver failure, autoimmune hepatitis and hepatocellular carcinoma.

[0089] In another aspect, the present disclosure provides for methods for organ or tissue transplantation in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier. Specifically, a hybrid ActRIIB ligand trap of the present disclosure is useful in treating a transplantation selected from organ transplantations of the heart, kidneys, liver, lungs, pancreas, intestine and thymus or from tissues transplantations of the bones, tendons, cornea, skin, heart valves, nerves and veins.

[0090] In another aspect, the present disclosure provides methods of treating pain in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the pain is selected from neuropathic pain, inflammatory pain, or cancer pain.

[0091] In another aspect, the present disclosure provides a method of treating aging in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier. In various embodiments, the aging condition is selected from the group consisting of: frailty of the elderly, age-related sarcopenia, and osteoarthritis.

[0092] In another aspect, the present disclosure provides methods of inducing stem cell growth for tissue repair or organ regeneration in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a hybrid ActRIIB ligand trap of the present disclosure in pharmaceutically acceptable carrier. In various embodiments, the stem cell is selected from the group consisting of: muscle stem (satellite) cell, cardiac stem cell, bone marrow-derived mesynchymal stem cell and pluripotent stem cell.

[0093] In various embodiments, the present disclosure provides for a method of inhibiting loss of muscle mass and / or muscle function in a subject comprising administering an effective amount of a hybrid ActRIIB ligand trap into the subject. The modulation may attenuate the loss of the muscle mass and / or function of the subject by at least 5%, 10%, at least 25%, at least 50%, at least 75%, or at least 90%. The inhibition of loss of muscle mass and function canbe evaluated by using imaging techniques and physical strength tests. Examples of imaging techniques for muscle mass evaluation include Dual-Energy X-Ray Absorptiometry (DEXA), Magnetic Resonance Imaging (MRI), and Computed Tomography (CT). Examples of muscle function tests include grip strength test, stair climbing test, short physical performance battery (SPPB) and 6-minute walk, as well as maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP) that are used to measure respiratory muscle strength.

[0094] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses (multiple or repeat or maintenance) can be administered over time and the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure will be dictated primarily by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0095] Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a subject may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the subject. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a subject in practicing the present disclosure.

[0096] It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Further, the dosage regimen with the compositions of this disclosure may bebased on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the subject, the severity of the condition, the route of administration, and the particular antibody employed. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Thus, the present disclosure encompasses intra-subject dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.

[0097] An exemplary, non-limiting daily dosing range for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure can be 0.001 to 100 mg / kg, 0.001 to 90 mg / kg, 0.001 to 80 mg / kg, 0.001 to 70 mg / kg, 0.001 to 60 mg / kg, 0.001 to 50 mg / kg, 0.001 to 40 mg / kg, 0.001 to 30 mg / kg, 0.001 to 20 mg / kg, 0.001 to 10 mg / kg, 0.001 to 5 mg / kg, 0.001 to 4 mg / kg, 0.001 to 3 mg / kg, 0.001 to 2 mg / kg, 0.001 to 1 mg / kg, 0.010 to 50 mg / kg, 0.010 to 40 mg / kg, 0.010 to 30 mg / kg, 0.010 to 20 mg / kg, 0.010 to 10 mg / kg, 0.010 to 5 mg / kg, 0.010 to 4 mg / kg, 0.010 to 3 mg / kg, 0.010 to 2 mg / kg, 0.010 to 1 mg / kg, 0.1 to 50 mg / kg, 0.1 to 40 mg / kg, 0.1 to 30 mg / kg, 0.1 to 20 mg / kg, 0.1 to 10 mg / kg, 0.1 to 5 mg / kg, 0.1 to 4 mg / kg, 0.1 to 3 mg / kg, 0.1 to 2 mg / kg, 0.1 to 1 mg / kg, 1 to 50 mg / kg, 1 to 40 mg / kg, 1 to 30 mg / kg, 1 to 20 mg / kg, 1 to 10 mg / kg, 1 to 5 mg / kg, 1 to 4 mg / kg, 1 to 3 mg / kg, 1 to 2 mg / kg, or 1 to 1 mg / kg body weight. It is to be noted that dosage values may vary with the type and severity of the conditions to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0098] In various embodiments, the individual dosing range for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 0.3 mg / kg to 4.5 mg / kg administered subcutaneously under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 0.3 mg / kg administered subcutaneously under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 1 mg / kg administered subcutaneously under fasting conditions. In various embodiments, the individual dose for a therapeutically orprophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 3 mg / kg administered subcutaneously under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 4.5 mg / kg administered subcutaneously under fasting conditions.

[0099] In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 1 mg / kg administered subcutaneously weekly for 4 weeks under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 3 mg / kg administered subcutaneously weekly for 4 weeks under fasting conditions. In various embodiments, the individual dose for a therapeutically or prophylactically effective amount of a hybrid ActRIIB ligand trap of the disclosure is 4.5 mg / kg administered subcutaneously weekly for 4 weeks under fasting conditions.

[0100] In various embodiments, the total dose administered will achieve a plasma antibody concentration in the range of, e.g., about 1 to 1000 pg / ml, about 1 to 750 pg / ml, about 1 to 500 pg / ml, about 1 to 250 pg / ml, about 10 to 1000 pg / ml, about 10 to 750 pg / ml, about 10 to 500 pg / ml, about 10 to 250 pg / ml, about 20 to 1000 pg / ml, about 20 to 750 pg / ml, about 20 to 500 pg / ml, about 20 to 250 pg / ml, about 30 to 1000 pg / ml, about 30 to 750 pg / ml, about 30 to 500 pg / ml, about 30 to 250 pg / ml.

[0101] T oxicity and therapeutic index of the pharmaceutical compositions of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effective dose is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are generally preferred. The dosing frequency of the administration of the hybrid ActRIIB ligand trap pharmaceutical composition depends on the nature of the therapy and the particular disease being treated. The subject can be treated at regular intervals, such as weekly or monthly, until a desired therapeutic result is achieved. Exemplary dosing frequencies include but are not limited to: once weekly without break; once weekly, every other week; once every 2 weeks; once every 3 weeks; weakly without break for 2 weeks, then monthly; weakly without break for 3 weeks, then monthly; monthly; once every other month; once every three months; once every four months; once every five months; or once every six months, or yearly.

[0102] The following examples are offered to more fully illustrate the disclosure but are not construed as limiting the scope thereof.Example 1A Phase 1 a Single Ascending Dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of ALG-801 in Community Dwelling, Healthy Older Men

[0103] Prostate cancer is the most prevalent malignancy in men in the United States and the most common newly diagnosed cancer among men throughout the world (Torre LA et aL, Cancer Epidemiol Biomarkers Prev., 25(1 ):16-27, 2016). Prostate cancer accounts for 29% of all diagnosed cancers and 13% of all cancer deaths in the United States. Because of the important role of androgen receptor signaling in the biology of prostate cancer, androgen deprivation therapy (ADT) is used widely in men with locally-advanced and metastatic prostate cancer (Heidenreich A et aL, Eur UroL, 65(2):467-79, 2014; Roach M., Cancer, 120(11 ):1620-9, 2014). ADT improves overall survival and disease-free survival in men with locally advanced prostate cancer who are undergoing radiation therapy and in patients with lymph node positive cancer without distant metastases who have undergone radical prostatectomy and pelvic lymph node removal (Wong Y-N, et aL, Eur UroL, 56:609-16, 2009; Roach M., Cancer, 120(1 1):1620-9, 2014). ADT also is employed to treat men whose PSA levels rise after primary therapy (Wong Y-N, et aL, Eur UroL, 56:609-16, 2009; Heidenreich A et aL, Eur UroL, 65(2):467-79, 2014). More than half a million men with prostate cancer are receiving ADT in the United States.

[0104] Although ADT has improved the survival of men with locally-advanced and metastatic prostate cancer, its survival benefits are partly offset by its adverse effects. Men undergoing ADT experience increased risk of osteoporosis and fractures, loss of muscle mass, strength, and physical performance, frailty, and sexual dysfunction. Because prostate cancer is often a disease of older men, who have high baseline prevalence of functional limitations and frailty, the superimposition of androgen deprivation therapy augments age-related loss of skeletal muscle mass, muscle strength and physical function and increases the risk of disability and frailty. Moreover, interventions that increase skeletal muscle mass and contractile function have the potential to improve cardiometabolic outcomes (Basaria S, Bhasin S., N Engl J Med., 367(10):965-7, 2012; Woodhouse L et aL, J Frailty Aging., 5(1):62-70, 2016). However, there are no currently approved therapeutics that address this unmet medical need.

[0105] In several preclinical disease models, including experimentally-induced androgen deficiency, ALG-801 has been shown to promote muscle growth and enhancing bone formationand thereby reverse muscle and bone loss. Based on these preclinical data, we plan to determine its efficacy in improving bone health, muscle performance and physical function, and metabolic outcomes in men with prostate cancer who are receiving androgen deprivation therapy. The potential for a single therapeutic to provide beneficial effects on the muscle and bone loss as well as the metabolic complications associated with ADT would represent a significant advance for the care of these patients.

[0106] The proposed Phase 1 a trial will determine the safety, pharmacokinetics and pharmacodynamics of ALG-801 and is an essential first step towards the long-term goal of conducting a Phase 2 efficacy trial in men receiving androgen deprivation therapy. Healthy community dwelling men were selected as the proposed target population in this Phase 1 a study based on the favorable nonclinical safety profile of ALG-801 and the ability of this study population to provide reliable and robust safety, tolerability, pharmacokinetic and pharmacodynamic data essential for further development of ALG-801 .Phase 1 a Study

[0107] This was a single-center, randomized, double-blind, placebo-controlled, single ascending dose study to evaluate the safety, tolerability, PK and PD of ALG-801 in community dwelling, healthy men, 60 - 80 years of age, inclusive. Up to 3 successive cohorts of 8 subjects will be included in this trial.

[0108] ALG-801 and placebo will be manufactured and packaged by a certified CMO and distributed using a GCP-compliant clinical investigational product distribution procedure. ALG-801 is supplied as single-use glass vials formulated at 50 mg / mL in 20 mM sodium phosphate buffer, 9% sucrose, and 0.01% polysorbate 20, pH 7.0. ALG-801 is dispensed at 1 ,5mL in 2mL neutral borosilicate glass vials and is stored at -20±5° C. Three ascending dose levels (0.3, 1 .0 and 3.0 mg / kg) will be evaluated sequentially. Individual dose will be prepared by the Investigational Drug Services (IDS) and administered subcutaneously by a study staff member in the Clinical Research Center. Each subject will receive only a single dose of the study medication and can participate in only one dose cohort. A matching placebo will be supplied. The formulation of placebo is 20 mM sodium phosphate buffer, 9% sucrose, and 0.01% polysorbate 20, pH 7.0. Placebo will be presented in identical containers, dispensed at 1 .5 mL in 2mL neutral borosilicate glass vials, and stored at - 20±5° C at the IDS untildispensed. Subjects randomized to placebo will receive a single subcutaneous dose of an equal volume of the vehicle.

[0109] Based on the preclinical toxicological data, this study evaluated 3 doses: 0.3 mg / kg, 1 .0 mg / kg, and 3.0 mg / kg. Each subject in each dose cohort will receive a single subcutaneous (SC) dose of study drug. The first cohort will start with the lowest dose (0.3 mg / kg) and proceed to the next dose level if no dose limiting toxicity (DLT) is observed in the 0.3 mg / kg dose cohort. Each subject will be followed for up to 56 days after the administration of the investigational product.

[0110] The primary objective of the Phase 1 a was to evaluate the safety and tolerability of subcutaneously administered single ascending doses of ALG-801 (0.3, 1 .0 and 3.0 mg / kg) in community dwelling, healthy men, 60 - 80 years of age, inclusive. The secondary objectives of the Phase 1 a study were: 1 ) to evaluate the pharmacokinetics (PK) of subcutaneously administered single ascending doses of ALG-801 (0.3, 1 .0 and 3.0 mg / kg) in community dwelling, healthy men, 60 - 80 years of age, inclusive; and 2) to evaluate the immunogenicity of subcutaneously administered single ascending doses of ALG-801 (0.3, 1.0 and 3.0 mg / kg) in community dwelling men, 60 - 80 years of age, inclusive. An exploratory objective of the study is to evaluate the pharmacodynamics (PD) of subcutaneously administered single ascending doses of ALG-801 (0.3, 1 .0 and 3.0 mg / kg) in community dwelling men, 60 -80 years of age, inclusive.

[0111] Subjects were screened for conformity to inclusion and exclusion criteria and those meeting eligibility criteria on screening will be offered participation in the study. Eight subjects per cohort were randomized in a 3:1 ratio to receive a single dose of either ALG-801 or placebo. ALG-801 was administered to successive cohorts at each of three escalating dose levels (0.3, 1 .0 and 3.0 mg / kg) under fasting conditions. Subjects were followed for 56 days following dosing. Dose escalation was sequential and proceeded as long as no pre-defined DLT or stopping criteria was met.

[0112] PK assessments were performed pre-dose and at 1 , 4, 8, 12, 24, 48, 72, 120,168 and 336 hours after administration of investigational product, and on Day 29.

[0113] Immunogenicity assessments were performed pre-dose and on Days 15, 29, and 57 after administration of investigational product.Results

[0114] ALG-801 administered subcutaneously with dose escalation to 1 .0 and 3.0 mg / kg was well-tolerated and a MTD was not reached in this clinical trial. Peak ALG-801 serum concentrations were observed within 72.0 to 121 hours post-dose (median Tmax) for all doses, indicating a prolonged absorption following subcutaneous administration with a long terminal elimination. The mean ti / 2 ranged from 177 to 181 hours (~ 7 days). The pharmacokinetics of ALG-801 were dose-proportional or slightly greater than dose-proportional over a dose range of 0.3 to 3.0 mg / kg in community dwelling, healthy older men. Four subjects tested positive for anti-ALG-801 antibodies after study drug exposure over the course of the study. Additionally, two subjects tested positive for ADA prior to treatment with ALG-801 . Although ADAs are known to potentially affect the PK of a drug, there was no evidence of an impact of immunogenicity on ALG-801 PK in this study.

[0115] Pharmacodynamic assessment of free activin A showed a dose-dependent sequestration of activin A in the serum from subjects dosed with single doses of 0.3 to 3.0 mg / kg. Suppression of activin A was 100 % for up to 1 week following a subcutaneous dose of 1 mg / kg ALG-801 and was 100% for up to 2 weeks following administration of a subcutaneous dose of 3.0 mg / kg. A dose of 0.3 mg / kg gave 75% suppression of activin A, lasting for 120 hours.

[0116] As depicted in FIG. 1 , subcutaneous administration of ALG-801 resulted in a dose-dependent and long-lasting decrease in free activin A levels in clinical serum samples from community dwelling, healthy older men, thus demonstrating the ability of ALG-801 to sequester the target ligand activin A.

[0117] As depicted in FIG. 2, subcutaneous administration of ALG-801 had no effect on free BMP9 levels in clinical serum samples from community dwelling, healthy older men.This result demonstrates the ability of ALG-801 to spare BMP9.

[0118] As depicted in FIGS. 3A and 3B, a single SC administration of ALG-801 resulted in dose dependent decrease in bone resorption biomarker CTX and increase in bone formation biomarker BSAP in community dwelling, healthy older men.

[0119] As depicted in FIG. 4, a single subcutaneous administration of ALG-801 resulted in dose dependent decrease in BUN biomarker in community dwelling, healthy older men, suggesting anabolic and anti-catabolic effect on protein metabolism.Example 2A Phase lb Multiple Ascending Dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of ALG-801 in Postmenopausal Women

[0120] In preclinical disease models, ALG-801 has been shown to reverse muscle wasting, bone loss, pulmonary hypertension and cardiac failure. Based on these preclinical data, this study was designed to evaluate the safety and tolerability, as well as pharmacokinetics and immunogenicity, of ALG-801 in healthy postmenopausal women. The present study was also designed to explore the effects of ALG-801 on bone turnover, body composition, glucose metabolism, and insulin sensitivity in postmenopausal women. The rationale for conducting the present clinical study in postmenopausal women was based on ALG-801 ’s ability to inhibit activins (i.e., activins A and activin B), which stimulate FSH release (Vale W et aL, Nature, 321 :776-779, 1986; Bernard DJ, Tran S., Biol Reprod., Mar 28;88(3):78, 2013).

[0121] The subject group, postmenopausal women, is associated with highly elevated FSH. During menopause, the decline in estrogen triggers a marked increase in FSH in blood circulation. High FSH plays a critical role in bone turnover, muscle metabolism, adiposity, and insulin resistance. In postmenopausal women specifically, elevated FSH has been shown to accelerate aging, as high FSH leads to decreased bone density, reduced muscle mass, and increased fat mass, as well as increased risk of diabetes, metabolic syndrome, renal dysfunction, cardiovascular diseases, and Alzheimer’s disease (Jung ES et al., J Clin Med., 9(4):1161 , 2020; Zaidi M et al., Endocrinology, 159(10):3503-3514, 2018; Xiong J et al., Nature, 603, 470-476, 2022). In addition, increased activin levels have been connected with bone loss as well as insulin resistance in postmenopausal women (Wu H et al., Exp Diabetes Res., 2012:410579, 2012; Anastasilakis AD, et al., Osteoporos Int, 24(7):2127-32, 2013). Importantly, ALG-801 has been designed to potently neutralize activin A and activin B, thereby suppressing FSH release. ALG-801 elicits a series of changes via inhibiting activins and suppression of FSH including changes in biomarkers for bone turnover, muscle mass, fat mass, intramuscular fat infiltration and insulin resistance.Phase 1 b Study

[0122] This Phase 1 b trial was a single-center, randomized, double-blind, placebo- controlled, multiple ascending dose study to evaluate the safety, tolerability, PK and PD of ALG- 801 in postmenopausal women. Up to 3 successive cohorts of 10 or 11 participants (10participants in Cohort 1 and 11 participants for Cohorts 2 and 3, respectively) were enrolled in this study.

[0123] ALG-801 was supplied as an aqueous solution containing 50 mg / mL of the active drug (Vial size 2 mL, Fill volume 1 .5 mL / vial, 75 mg / vial). Three ascending dose levels (0.3, 1 .0 and 3.0 mg / kg) were evaluated sequentially. Individual doses were prepared by the Investigational Drug Services (IDS) and administered subcutaneously by a study staff member in the Clinical Research Center. Each subject received only a single dose of the study medication and can participate in only one dose cohort. A matching placebo was supplied. Subjects randomized to placebo received a single subcutaneous dose of an equal volume of the vehicle. A matching placebo was supplied. The formulation of placebo is 20 mM sodium phosphate buffer, 9% sucrose, and 0.01% polysorbate 20, pH 7.0. Placebo was presented in identical containers, dispensed at 1 .5 mL in 2mL neutral borosilicate glass vials, and stored at - 20±5° C at the IDS until dispensed. Subjects randomized to placebo received a single subcutaneous dose of an equal volume of the vehicle.

[0124] As complete suppression of free activin A is desired in postmenopausal women, a low dose of 1 mg / kg SC ALG-801 was the proposed starting dose in this Phase 1 b multiple ascending dose study, as this dose should be associated with 100% sequestration of free activin A for 1 week. To maintain inhibition, this dose was followed by 3 weekly doses of 1 mg / kg for a total of 4 doses over a period of 4 weeks. The mid dose for this study is 3 mg / kg dosed weekly for 4 weeks. An additional dose of 4.5 mg / kg given weekly for a total of 4 doses was proposed as the high dose, to ensure full suppression of activin A, and as no maximum tolerated dose has been identified. This dose has not been tested in the clinic, but it still below the NOAEL in the monkey repeat dose toxicity study. To ensure the safety and tolerability of this dose, the first dose of 4.5 mg / kg was not administered until subjects in the 3.0 mg / kg dose group have received at least 2 doses of ALG-801 , followed by at least a week of safety assessment following the second dose.

[0125] The primary objective of the Phase 1 b study was to evaluate the safety and tolerability of ALG-801 administered as 4 weekly subcutaneous doses at 3 ascending dose levels in postmenopausal women. The secondary objectives of the Phase 1 b study are: 1 ) to evaluate the pharmacokinetics (PK) of ALG-801 administered as 4 weekly subcutaneous doses at 3 ascending dose levels at 1.0 mg / kg, 3.0 mg / kg and 4.5 mg / kg, respectively, in postmenopausal women; and 2) to evaluate the immunogenicity of ALG-801 administered as 4 weekly subcutaneous doses at 3 ascending dose levels at 1.0 mg / kg, 3.0 mg / kg, and 4.5 mg / kg,evaluate the pharmacodynamics (PD) of ALG-801 administered as 4 weekly subcutaneous doses at 3 ascending dose levels at 1 .0 mg / kg, 3.0 mg / kg, and 4.5 mg / kg, respectively, in postmenopausal women using: a) Magnetic resonance imaging (MRI) at baseline (pre-dose) and on Days 29 and 43 of thigh muscle volume, thigh subcutaneous and intermuscular fat mass infiltration, visceral adipose tissue, abdominal subcutaneous adipose tissue, and liver fat; b) Bone Turnover markers on Days 1 , 15, 29, and 43 of Bone-Specific Alkaline Phosphatase (BSAP), Procollagen type I N-terminal propeptide (P1 NP) and C-telopeptide (CTx); c) Follicle- stimulating hormone (FSH) levels at baseline predose and on Days 15, 29, and 43; d) Metabolic Biomarkers on Days 1 , 15, 29, and 43 of Adiponectin and leptin, and Glycated hemoglobin (Hb A1 C), fasting glucose, insulin, and homeostatic model of insulin resistance (HOMA-IR); e) Biomarker on target coverage; f) Free activin A levels on Days 1 , 3, 5, 8, 15, 22, 29, 36, 43, and 50; and 2) to evaluate the potential relationship of PK parameters with immunogenicity and PD parameters.

[0126] A total of 32 subjects were enrolled in this Phase 1 b study (see Table 1 ),Table 1

[0127] Participants were screened for potential participation and those meeting eligibility criteria on screening was offered participation in the study. Ten or 1 1 subjects per cohort were randomized in an 8:2 or 8:3 ratio to receive 4 weekly subcutaneous doses (Days 1 , 8, 15 and 22) of either ALG-801 or placebo. ALG-801 was administered to successive cohorts at each of3 escalating dose levels under fasting conditions. Participants were followed for 28 days following the final dose according to the Schedule of Events. On Day 50, blood sampling was performed under fasting condition.

[0128] For each cohort, randomization occurred in 2 blocks as follows: In the first block, 2 subjects (sentinel subjects) were randomized 1 :1 to receive ALG-801 or placebo; in the second block, 8-9 subjects were randomized 7 ALG-801 : 1 -2 placebo. Based on Phasel a exposure data [mean ti / 2 from 177 to 181 hours (~ 7 days)], there was a minimum of 7 daysbetween dosing of the 2 sentinel subjects and the remainder of the cohort. If ALG-801 was well- tolerated in the sentinel subjects during the 7-day observation period, the remainder of the cohort (n = 8-9) was randomized.

[0129] PK assessments were performed pre-dose and at Hours 8, 24, and on Days3, 5, 8, 15, 22, 29, 36, 43, and 50 after administration of study drug.

[0130] Immunogenicity assessments were performed pre-dose (Day 1 ) and on Days22 and 50 after administration of study drug.

[0131] The sampling period is 50 days after administration of the first dose of ALG-801 . Including the screening period of up to 28 days, the total duration of the study may be up to 78 days from the day of signing consent.

[0132] Pharmacokinetic data was derived using all subjects who have sufficient plasma samples to allow for calculation of pharmacokinetic parameters. Pharmacokinetic parameters will include, but not be limited to, Tmax, Cmax, AUCot, AUC0~, T1 / 2, CL / F and Vz / F. PK parameters were summarized by dose group using descriptive statistics. Selected PK parameters were analyzed by comparative statistics. Dose proportionality of PK parameters was assessed by linear regression analysis.

[0133] Immunogenicity of ALG-801 was assessed using an ADA assay pre-dose and on Days 22 and 50 to evaluate generation of ADA titers and any change in ADA titers over time. Subjects with ADA titers on Day 50 may be requested to provide follow-up blood samples for analysis of the durability of the ADA response.

[0134] Pharmacodynamic data was assessed in all subjects according to theSchedule of Events. PD assessments was summarized using descriptive statistics for each dose group with respect to dose level and time point. PD effects will also be evaluated in relationship to systemic exposure.

[0135] As depicted in FIG. 5A and FIG. 5B, ALG-801 administration led to dosedependent and statistically significant increase in thigh muscle volume in postmenopausal women in volunteer subjects who received 4 weekly SC injections of ALG-801 at 1 .0mg / kg, 3.0mg / kg, and 4.5mg / kg, respectively, compared to placebo control at day 29 and day 43.

[0136] As depicted in FIG. 6, ALG-801 administration led to dose-dependent and statistically significant reduction in body fat in postmenopausal women in volunteer subjects who received 4 weekly SC injections of ALG-801 at 1 .0mg / kg, 3.0mg / kg, and 4.5mg / kg, respectively, compared to placebo control at day 29 and day 43.

[0137] As depicted in FIG. 7, ALG-801 administration significantly reduced leptin-to- adiponectin ratio (LAR), an acute index of insulin resistance in postmenopausal women in healthy postmenopausal volunteer subjects who received 4 weekly SC injections at 1 .Omg / kg, 3.0mg / kg, and 4.5mg / kg, respectively, compared to placebo control. This dose-dependent and statistically significant suppression of LAR seen upon ALG-801 administration demonstrates that the treatment is able to decrease insulin resistance and enhance insulin sensitivity. This insulin-sensitizing effect of ALG-801 forms a strong rationale-basis for treating a wide-range of chronic diseases including various cardiometabolic disorders, neuromuscular diseases and age related disorders.

[0138] The study results demonstrate, among other things, that ALG-801 was found to be safe and well-tolerated across all dosing cohorts, and that ALG-801 selectively inhibits Smad2 / 3 signaling, which is mediated by activin A, activin B, GDF8, and GDF11 , without interfering with BMP9-Smad 1 / 5 / 8 signaling. As a result, ALG-801 achieves an unparalleled efficacy and metabolic benefits. This collective data highlights the promising potential of ALG- 801 (and similar next-generation ligand traps) to address a wide range of unmet medical needs, such as muscle wasting, obesity, cardiometabolic disorders, neuromuscular diseases, chronic kidney disease (CKD), and osteosarcopenia.

[0139] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0140] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. It is also understood that the terminology used herein is for the purposes of describing particular embodiments.

[0141] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0142] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.Sequence Listings

[0143] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three letter code for amino acids, as defined in 37 C.F.R. 1 .822.SEQ ID NO: 1 is the amino acid sequence of a truncated wild-type human ActRIIB-ECD polypeptide.SEQ ID NO: 2 is the amino acid sequence of a truncated wild-type human ActRIIA-ECD polypeptide.SEQ ID NO: 3 is the amino acid sequence of a hybrid soluble ActRIIB-ECD polypeptide.SEQ ID NO: 4 is the amino acid sequence of a human immunoglobulin gamma-1 (lgG1 ) heavy chain constant regionSEQ ID NO: 5 is the amino acid sequence of an IgG 1 Fc DomainSEQ ID NO: 6 is the amino acid sequence of a human immunoglobulin gamma-2 chain heavy constant regionSEQ ID NO: 7 is the amino acid sequence of an lgG2 Fc DomainSEQ ID NO: 8 is the amino acid sequence of a human immunoglobulin gamma-4 chain heavy constant regionSEQ ID NO: 9 is the amino acid sequence of an lgG4 Fc DomainSEQ ID NO: 10 is the amino acid sequence of peptide linker.SEQ ID NO: 11 is the amino acid sequence of peptide linker.SEQUENCE LISTINGSTruncated wild-type ActRIIB-ECDETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 1)Truncated wild-type ActRIIA-ECDETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP(SEQ ID NO: 2)Hybrid hu-ActRIIB-ECDETRECIYYNANWELERTNQSGLERCEGDQDKRLHCYASWRNSSGTIELVKKGCWLDDINCYDRQECVATKENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 3)Human immunoglobulin gamma-1 heavy chain constant regionASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4) lgG1 Fc DomainVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)Human immunoglobulin gamma-2 heavy chain constant regionASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO: 6) lgG2 Fc DomainVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)Human immunoglobulin gamma-4 heavy chain constant regionASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK (SEQ ID NO: 8)lgG4 Fc DomainAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9)Peptide Linker sequenceGGGGS (SEQ ID NO: 10)Peptide Linker sequenceESKYGPPCPPCP (SEQ ID NO: 11 )

Claims

What is claimed is:1 . A method of treating myo statin -related or activin A-related disorders in a subject, comprising administering to said subject a therapeutically effective amount of an isolated protein comprising a hybrid soluble activin I IB receptor-extracellular domain (ActRIIB-ECD) polypeptide fused to a human Fc domain, wherein said myostatin-related or activin A-related disorder is selected from the group consisting of: a bone disorder, a muscle wasting disease, a cardiovascular disease, a metabolic disorder, a renal disease, an inflammatory / autoimmune disease, a fibrosis disease, and aging, and treating cancer cells; and wherein the individual dosing range for a therapeutically effective amount of the isolated protein is between about 0.3 mg / kg to 4.5 mg / kg administered subcutaneously under fasting conditions.

2. A method according to claim 1 , wherein the individual dose for a therapeutically effective amount of the isolated protein is selected from the group consisting of: 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg. and 4.5 mg / kg administered subcutaneously under fasting conditions.

3. A method according to any one of claims 1 -2, wherein the individual dose is administered subcutaneously weekly for 4 weeks under fasting conditions.

4. A method according to claim 1 , wherein the muscle wasting disease is selected from the group consisting of: muscular dystrophies (such as DMD, Becker MD, Limb-Girdle MD, Myotonic MD and FSHD), myositis, myopathies (including inherited myopathy and acquired myopathy), motoneuron diseases (such as Lou Gehrig's Disease or ALS), and neurodegenerative diseases (such as Parkinson’s disease, Huntington’s disease and Alzheimer’s disease).

5. A method according to claim 1 , wherein the bone disease is selected from the group consisting of: osteoporosis, osteomalacia, osteogenesis imperfecta, fibrodysplasia ossificans progressiva, corticosteroid-induced bone loss, bone fracture, and bone metastasis.

6. A method according to claim 1 , wherein the metabolic disorder is selected from the group consisting of: obesity, dyslipidemia, sarcopenic obesity, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis, alcoholic fatty liver disease, insulin resistance, diabetesand metabolic syndrome, as well as diabetic myopathy, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, and hemochromatosis.

7. A method according to claim 1 , wherein the fibrosis disease is selected from the group consisting of: interstitial lung disease, idiotypic pulmonary fibrosis, cystic fibrosis, liver fibrosis, cirrhosis, cardiac fibrosis, renal fibrosis, myelofibrosis, idiopathic retroperitoneal fibrosis, nephrogenic fibrosing dermopathy, inflammatory bowel disease, keloid, scleroderma and arthrofibrosis.

8. A method according to claim 1 , wherein the inflammatory / autoimmune disease is selected from the group consisting of: multiple sclerosis, systemic sclerosis, diabetes (type-1 ), glomerulonephritis, myasthenia gravis, psoriasis, systemic lupus erythematosus, polymyositis, Crohn's disease, ulcerative colitis, and primary biliary cirrhosis.

9. A method according to claim 1 , wherein the cardiovascular disease is selected from the group consisting of: heart failure, cardiac atrophy, hypertension, myocarditis, coronary artery disease, myocardial infarction, cardiac arrhythmias, heart valve disease, cardiomyopathy, pericardial disease, aorta disease and Marfan syndrome.

10. A method according to claim 1 , wherein the cancer cell is selected from the group consisting of: pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head-neck cancer, and rhabdomyosarcoma cells.

11. A method according to claim 1 , wherein the renal disease is selected from the group consisting of: Chronic Kidney Disease (CKD) including renal failure, interstitial fibrosis, and kidney dialysis as well as protein energy wasting (PEW) associated with CKD.

12. A method according to claim 1 , wherein the arthritis is selected from the group consisting of: rheumatoid arthritis and osteoarthritis.

13. A method according to claim 1 , wherein the pain is selected from the group consisting of: neuropathic pain, somatic pain, visceral pain, inflammatory pain, cancer pain, back pain, and joint pain.

14. A method according to claim 1 , wherein the aging condition is selected from the group consisting of: frailty of the elderly, age-related sarcopenia, and osteoarthritis.

15. A method according to any one of claims 1 -14, wherein the hybrid ActRIIB ligand trap proteins comprise hybrid soluble ActRIIB-ECD polypeptides having the amino acid sequence set forth in SEQ ID NO: 3, wherein the hybrid ActRIIB-ECD polypeptide is capable of binding myostatin and activin A, but demonstrates a decreased binding affinity for BMP9 relative to a wild-type ActRIIB-ECD polypeptide.

16. A method according to any one of claims 1 -15, wherein the human Fc domain is selected from the group consisting of the Fc domain is selected from the group consisting of a human Fc domain having the amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

17. A method according to any one of claims 1 -16, wherein a hybrid soluble ActRIIB-ECD polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 3 is attached to a human Fc domain having an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

18. A method according to any one of claims 1 -17, wherein a hybrid soluble ActRIIB-ECD polypeptide having an amino acid sequence of SEQ ID NO: 3 is attached to a human Fc domain having an amino acid sequence of SEQ ID NO: 9.

19. A method according to any one of claims 1 -18, wherein the Fc domain is attached to the hybrid soluble ActRIIB-ECD polypeptide by a linker and / or a hinge linker peptide, wherein the linker has a (GGGGS (SEQ ID NO: 10))nmotif, wherein n = 1 -6.

20. A method according to any one of claims 1 -19, wherein a linker having the amino acid sequence set forth in SEQ ID NO: 10 is used with a hinge linker having the amino acidsequence set forth in SEQ ID NO: 11 to link a human lgG4 Fc to a hybrid soluble ActRIIB-ECD polypeptide.21 . A method according to claim 20, wherein a hybrid soluble ActRIIB-ECD polypeptide having the amino acid sequence of SEQ ID NO: 3 is fused to a peptide linker having the amino acid sequence set forth in SEQ ID NO: 10, the peptide linker is fused to a hinge linker having the amino acid sequence set forth in SEQ ID NO: 11 , and the hinge linker is fused to an Fc domain having an amino acid sequence of SEQ ID NO: 9.

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