Methods of using activin receptor type ii chimeras

Abstract

The invention features methods of treating a subject who is undergoing treatment with or previously underwent treatment with an incretin-based therapy by administering a polypeptide including an activin receptor type II (ActRII) chimera. The extracellular ActRII chimera may be fused to an Fc domain monomer. The incretin-based therapy may be, e.g., a GLP-1 receptor agonist or a DPP-4 inhibitor.

Description

[0001] METHODS OF USING ACTIVIN RECEPTOR TYPE II CHIMERAS

[0002] SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 16, 2024, is named 51 184-055WO4_Sequence_Listing_12_16_24.xml and is 393,764 bytes in size.

[0004] Background of the Invention

[0005] Obesity is a complex and chronic disease defined as having a body mass index, or BMI, of greater than or equal to 30, and is caused by abnormal or excessive fat accumulation due to an imbalance in energy intake and consumption over a long period of time. There are many comorbidities associated with obesity, including type 2 diabetes, cardiovascular disease, hypertension, heart disease, sleep apnea and some cancers, as well as an increased risk of death. According to the World Health Organization, in 2016, more than 1 .9 billion adults worldwide were overweight, with 650 million considered to be obese. The prevalence of obesity has tripled since 1975 and is projected to affect one billion people globally by 2030. According to the Centers for Disease Control and Prevention, the estimated annual medical cost of obesity in the U.S. was nearly $173.0 billion in 2019 dollars.

[0006] Despite the prevalence of obesity and metabolic diseases, few therapeutic options are available. The first-line treatment for obesity is lifestyle change brought about through a combination of diet, exercise, and behavior therapy. While lifestyle modifications can produce weight loss, the magnitude required (approximately 10% to 15% of total body weight) to translate into clinical improvement for certain comorbid conditions, including obstructive sleep apnea and non-alcoholic steatotic hepatitis, is often difficult to achieve and sustain over time outside of a clinical trial setting. A third-party retrospective observational study of over 10,000 obese participants enrolled in a medically supervised weight management program demonstrated an average weight loss of 5.8% from baseline over a 5-year period.

[0007] Glucagon-like peptide-1 , or GLP-1 , is a metabolic hormone, or incretin, and GLP-1 receptor agonists represent an emerging class of therapies used to achieve weight loss. Third-party clinical data with incretin-targeted therapeutics have demonstrated substantial and sustained reductions in body weight. WEGOVY®, a GLP-1 receptor agonist, and ZEPOUND™, a dual GLP-1 / glucose-dependent insulinotropic polypeptide receptor agonist, are both approved in the United States for the treatment of obesity. In a double-blind Phase 3 clinical trial of WEGOVY®, adults with obesity who did not have diabetes were randomly assigned, in a 2:1 ratio, to 68 weeks of treatment with once-weekly subcutaneous WEGOVY® (at a dose of 2.4 mg) or placebo, plus lifestyle intervention. In this trial, the mean change in body weight from baseline to week 68 was -14.9% in the WEGOVY® group as compared with -2.4% with placebo. However, nearly 40% of the weight loss in a subpopulation assessed by dual energy X-ray absorptiometry (DEXA) was attributable to loss of lean mass, which could potentially lead to frailty. This also predisposes individuals to regain weight after discontinuation of therapy.

[0008] Accordingly, there exists a need for a treatment option that leads to weight loss without an associated loss of muscle. Summary of the Invention

[0009] The present invention features methods of treating type 2 diabetes (T2D), a body mass index (BMI) of 30 kg / m2or greater, or a BMI of 27 kg / m2or greater and a weight-related comorbid condition in a subject undergoing an incretin-based therapy, methods of reducing loss of lean mass in a subject undergoing treatment with an incretin-based therapy, and methods of increasing lean mass in a subject who is undergoing or previously underwent treatment with an incretin-based therapy by administering a polypeptide including an extracellular activin receptor type II (ActRI I) chimera. The invention also features methods of treating a subject having T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2or greater and a weight-related comorbid condition by administering an incretin-based therapy and a polypeptide including an extracellular ActRI I chimera (e.g., in combination or by first administering the incretin-based therapy and subsequently administering the polypeptide including an extracellular ActRI I chimera as a replacement therapy). The polypeptide can include an extracellular ActRI I chimera fused to the N- or C- terminus of an Fc domain monomer. A polypeptide including an extracellular ActRI I chimera fused to an Fc domain monomer may also form a dimer (e.g., a homodimer or heterodimer) through the interaction between two Fc domain monomers. The incretin-based therapy may be a GLP-1 receptor agonist (e.g., semaglutide, tirzepatide, exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, tirzepatide, albiglutide, or efpeglenatide) or a DPP-4 inhibitor (e.g., sitagliptin, saxagliptin, alogliptin, linagliptin, or vildagliptin). The methods described herein may increase lean mass, increase muscle mass (e.g., skeletal muscle mass), reduce fat gain, reduce fat mass (i.e., promote or increase fat loss), reduce white adipose tissue mass, increase fat metabolism, increase energy expenditure, reduce body fat, improve insulin resistance, improve cardiac function or cardiac health, improve insulin sensitivity, prevent or reduce the development of frailty, prevent or reduce the premature onset of sarcopenia, reduce the risk of sarcopenia, or reduce weight regain after discontinuation of the incretin-based therapy (e.g., discontinuation of incretin mimetic therapy, such as discontinuation of treatment with a GLP-1 receptor agonist).

[0010] Exemplary embodiments of the invention are described in the enumerated paragraphs below. E1 . A method of treating type 2 diabetes (T2D, also known as type 2 diabetes mellitus) , a body mass index (BMI) of 30 kg / m2or greater (or a BMI equivalent to 30 kg / m2or greater or at the 95thpercentile or greater for age and sex in a pediatric subject), or a BMI of 27 kg / m2or greater and a weight-related comorbid condition in a subject undergoing treatment with an incretin-based therapy, the method comprising administering to the subject an effective amount of a composition of Table 5.

[0011] E2. A method of reducing loss of lean mass in a subject undergoing treatment with an incretin-based therapy, the method comprising administering to the subject an effective amount of a composition of Table 5.

[0012] E3. A method of increasing lean mass in a subject who is undergoing or previously underwent treatment with an incretin-based therapy, the method comprising administering to the subject an effective amount of a composition of Table 5.

[0013] E4. The method of E2 or E3, wherein the subject is undergoing or previously underwent treatment with an incretin-based therapy for the treatment of T2D, a BMI of 30 kg / m2or greater (or a BMI equivalent to 30 kg / m2or greater or at the 95thpercentile or greater for age and sex in a pediatric subject), or a BMI of 27 kg / m2and a weight-related comorbid condition.

[0014] E5. A method of treating a subject having T2D, a BMI of 30 kg / m2or greater (or a BMI equivalent to 30 kg / m2or greater or at the 95thpercentile or greater for age and sex in a pediatric subject), or a BMI of 27 kg / m2and a weight-related comorbid condition, the method comprising administering to the subject effective amounts of an incretin-based therapy and a composition of Table 5.

[0015] E6. The method of E5, wherein the incretin-based therapy and the composition of Table 5 are administered together.

[0016] E7. The method of any one of E1 -E6, wherein the incretin-based therapy and the composition of Table 5 are administered with the same frequency.

[0017] E8. The method of E7, wherein the composition of Table 5 is administered once per week at a dose of 1 mg / kg or less.

[0018] E9. The method of E8, wherein the composition of Table 5 is administered once per week at a dose of 0.5 mg / kg or less.

[0019] E10. The method of E9, wherein the composition of Table 5 is administered once per week at a dose of 0.3 mg / kg or less.

[0020] E11 . The method of E5, wherein the incretin-based therapy is administered for a first period and the composition of Table 5 is subsequently administered for a second period.

[0021] E12. The method of E11 , wherein the composition of Table 5 is administered once every 28 days or less frequently (e.g., once every 28 days, once a month, once bimonthly, once every three months, once every four months, once every six months, or less frequently).

[0022] E13. The method of E12, wherein the composition of Table 5 is administered at a dose of 1 mg / kg or greater (e.g., 1 .0, 1 .25, 1 .5, 1 .75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5 mg / kg or greater).

[0023] E14. The method of any one of E1 -E13, wherein the subject has T2D or is undergoing or previously underwent treatment with an incretin-based therapy for T2D.

[0024] E15. The method of any one of E1 -E13, wherein the subject has a BMI of 30 kg / m2or greater (or a BMI equivalent to 30 kg / m2or greater or at the 95thpercentile or greater for age and sex in a pediatric subject) or is undergoing or previously underwent treatment with an incretin-based therapy for a BMI of 30 kg / m2or greater (or a BMI equivalent to 30 kg / m2or greater or at the 95thpercentile or greater for age and sex in a pediatric subject).

[0025] E16. The method of any one of E1 -E13, wherein the subject has a BMI of 27 kg / m2or greater and a weight-related comorbid condition (e.g. hypertension, type 2 diabetes mellitus, dyslipidemia, obstructive sleep apnea, or cardiovascular disease) or is undergoing or previously underwent treatment with an incretin-based therapy for a BMI of 27 kg / m2or greater and a weight-related comorbid condition.

[0026] E17. The method of any one of E1 -E16, wherein the composition of Table 5 is administered before the subject t exhibits a reduction in lean mass.

[0027] E18. The method of any one of E1 -E16, wherein the composition of Table 5 is administered after the subject exhibits a reduction in lean mass.

[0028] E19. The method of any one of E1 -E18, wherein the incretin-based therapy is an incretin mimetic. E20. The method of E19, wherein the incretin mimetic is a GLP-1 receptor agonist.

[0029] E21 . The method of E20, wherein the GLP-1 receptor agonist is exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, tirzepatide, albiglutide, or efpeglenatide.

[0030] E22. The method of any one of E1 -E18, wherein the incretin-based therapy is an incretin enhancer.

[0031] E23. The method of E22, wherein the incretin enhancer is a DPP-4 inhibitor.

[0032] E24. The method of E23, wherein the DPP-4 inhibitor is sitagliptin, saxagliptin, alogliptin, linagliptin, or vildagliptin.

[0033] E25. The method of any one of E1 -E24, wherein the composition of Table 5 is polypeptide comprising an extracellular ActRII chimera having a sequence of any one of SEQ ID NOs: 1 -21 , wherein Xi is D or R, X2is I, F, E, D, Y, S, N, Q, or T, X3is N or T, X4is A or E, X5is T or K, X6is E or K, X7is E or D, Xs is N or S, and Xg is Q, E, K, R, D, or N, optionally wherein the chimera is truncated from the N-terminus by deletion of 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, wherein the chimera retains the two amino acids before the first cysteine.

[0034] E26. The method of E25, wherein the chimera has the sequence of SEQ ID NO: 19.

[0035] E27. The method of E25, wherein the chimera has the sequence of SEQ ID NO: 12.

[0036] E28. The method of E25, wherein the chimera has the sequence of SEQ ID NO: 5.

[0037] E29. The method of any one of E25-E28, wherein Xi is D, X2is I, F, or E, Xs is N or T, X4is A or E, Xs is T or K, Xe is E or K, X? is E or

[0038] E30. The method of E29, wherein Xi is E or D, Xs is N or S, and Xg is

[0039] E31 . The method of E30, wherein Xi is D, X2is F, X3 is N, X4is E, X5 is K, Xs is K, X7is D, Xs is S, and

[0040] Xg is Q.

[0041] E32. The method of E25, wherein the chimera has the sequence of any one of SEQ ID NOs: 22-43.

[0042] E33. The method of E32, wherein the chimera has the sequence of SEQ ID NO: 41 .

[0043] E34. The method of E32, wherein the chimera has the sequence of SEQ ID NO: 25.

[0044] E35. The method of E32, wherein the chimera has the sequence of SEQ ID NO: 40.

[0045] E36. The method of any one of E25-E35, wherein the polypeptide further includes an Fc domain monomer fused to the C-terminus of the polypeptide (e.g., the C-terminus of the chimera) by way of a linker.

[0046] E37. The method of E36, wherein the polypeptide is in the form of a dimer (e.g., a homodimer).

[0047] E38. The method of E36, wherein the polypeptide has the sequence of any one of SEQ ID NOs: 107-

[0048] 110 and SEQ ID NOs: 184-263.

[0049] E39. The method of E39, wherein the polypeptide has the sequence of SEQ ID NO: 216.

[0050] E40. The method of E39, wherein the polypeptide has the sequence of SEQ ID NO: 110.

[0051] E41 . The method of E39, wherein the polypeptide has the sequence of SEQ ID NO: 215.

[0052] E42. The method of any one of E38-E41 , wherein the polypeptide is in the form of a homodimer.

[0053] E43. The method of any one of E1 -E42, wherein the method increases lean mass.

[0054] E44. The method of any one of E1 -E43, wherein the method increases muscle mass (e.g., skeletal muscle mass).

[0055] E45. The method of any one of E1 -E44, wherein the method reduces fat mass.

[0056] E46. The method of any one of E1 -E45, wherein the method reduces white adipose tissue mass. E47. The method of any one of E45 or E46, wherein administration of the composition of Table 5 leads to a greater loss of fat mass or white adipose tissue mass compared to treatment with an incretinbased therapy alone.

[0057] E48. The method of any one of E1 -E47, wherein the method improves glycemic control (e.g., in subjects with T2D).

[0058] E49. The method of any one of E1 -E48, wherein the method reduces the risk of major adverse cardiovascular events (e.g., in subjects with T2D and established cardiovascular disease or multiple cardiovascular risk factors).

[0059] E50. The method of any one of E1 -E49, wherein the method prevents or reduces the development of frailty.

[0060] E51 . The method of any one of E1 -E50, wherein the method prevents or reduces the premature onset of sarcopenia or reduces the likelihood of the premature onset of sarcopenia.

[0061] E52. The method of any one of E1 -E51 , wherein the method improves insulin sensitivity.

[0062] E53. The method of any one of E1 -E52, wherein the method improves cardiac function or cardiac health.

[0063] E54. The method of any one of E1 -E53, wherein the method increases fat metabolism.

[0064] E55. The method of any one of E1 -E54, wherein the method increases energy expenditure.

[0065] E56. The method of any one of E1 -E55, wherein the method improves (i.e., reduces) insulin resistance.

[0066] E57. The method of any one of E1 -E56, wherein the method reduces body fat (e.g., amount of body fat or body fat percentage, reduces the amount of subcutaneous, visceral, and / or hepatic fat, reduces adiposity, or reduces the weights of epididymal and perirenal fat pads)

[0067] E58. The method of any one of E1 -E57, wherein the method reduces body weight or body weight gain (e.g., reduce the percentage of body weight gain).

[0068] E59. The method of any one of E1 -E58, wherein the method reduces fat gain.

[0069] E60. The method of any one of E1 -E59, wherein the method reduces fasting insulin levels, reduces blood glucose levels (e.g., fasting glucose levels), increases glucose clearance, reduces LDL, reduces triglycerides, or improves serum lipid profile.

[0070] E61 . The method of any one of E1 -E60, wherein the method reduces the risk of sarcopenia.

[0071] E62. The method of any one of E1 -E61 , wherein the method reduces weight regain after discontinuation of the incretin-based therapy.

[0072] E63. The method of any one of E1 -E62, wherein the composition of Table 5 is administered in an amount sufficient to increase lean mass, reduce lean mass loss, increase muscle mass, reduce fat mass, reduce white adipose tissue mass, improve glycemic control, reduce the risk of major adverse cardiovascular events, prevent or reduce the development of frailty, prevent or reduce the premature onset of sarcopenia or reduce the likelihood of the premature onset of sarcopenia, improve insulin sensitivity, improve cardiac function or cardiac health, increase fat metabolism, increase energy expenditure, improve insulin resistance, reduces body fat, reduce body weight or body weight gain, reduce fat gain, reduce fasting insulin levels, reduce blood glucose levels, increase glucose clearance, reduce LDL, reduce triglycerides, improve serum lipid profile, reduce the risk of sarcopenia, or reduce weight regain after discontinuation of the incretin-based therapy. E64. The method of any one of E1 -E63, wherein the method does not cause a vascular complication in the subject.

[0073] E65. The method of E64, wherein the method does not increase vascular permeability or leakage.

[0074] E66. The method of any one of E1 -E65, wherein the subject is a human.

[0075] Definitions

[0076] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the invention. Terms such as "a", "an," and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

[0077] As used herein, the term "about” refers to a value that is within 10% above or below the value being described.

[0078] As used herein, any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.

[0079] As used herein, the terms “extracellular activin receptor type II (ActRII) chimera,” “extracellular ActRII chimera,” and “ActRII chimera” refer to a peptide including a soluble, extracellular portion of the single transmembrane receptor ActRIIB and a soluble, extracellular portion of the single transmembrane receptor ActRIIA. The ActRII chimeras described herein result from joining an N-terminal portion of extracellular ActRIIB to a C-terminal portion of extracellular ActRIIA such that the sequences are contiguous (e.g., the ActRIIA sequence continues where the ActRIIB sequence left off, starting with the next the amino acid located in the corresponding position of ActRIIA). The extracellular ActRII chimera may also include one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIB compared to a wild-type extracellular ActRIIB (e.g., bold portion of the sequence of SEQ ID NO: 46 shown below), and one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIA compared to a wild-type extracellular ActRIIA (e.g., bold portion of the sequence of SEQ ID NO: 47 shown below). The extracellular ActRII chimera may also have an N-terminal truncation of 1 -9 amino acids relative to the extracellular portion of ActRIIB or ActRIIA. The sequences of wild-type, human ActRIIB (SEQ ID NO: 46) and wild-type, human ActRIIA (SEQ ID NO: 47) are shown below, in which the signal peptide is italicized and the extracellular portion is bold.

[0080] Wild-type human ActRIIB (SEQ ID NO: 46):

[0081] MTAP A / .AZ. / .I4 / GS / .CAGSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYAS WRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEV TYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPP PSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMK HENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSY LHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVG TRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQ HPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSL IRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI

[0082] Wild-type, human ActRIIA precursor protein (SEQ ID NO: 47): MGAAAKMFA1 / FUSCSSGAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFAT WKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPT SNPVTPKPPYYNILLYSLVPLMLIAGMCAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPL QLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRG TSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHR DIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRID MYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKH AGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL

[0083] An extracellular ActRII chimera may have the sequence of any one of SEQ ID NOs: 1 -43. In particular embodiments, an extracellular ActRII chimera has the sequence of any one of SEQ ID NOs: 22- 43 (Table 2).

[0084] As used herein, the term “N-terminal truncation” refers to a deletion of 1 -9 amino acids (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) from the N-terminus of an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)). The N-terminal truncation can remove amino acids up to two amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE or QE) are retained in the N-terminally truncated ActRII chimeras).

[0085] As used herein, the term “linker” refers to a linkage between two elements, e.g., peptides or protein domains. A polypeptide described herein may include an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)), fused to an Fc domain monomer, which increases stability or improves pharmacokinetic properties of the polypeptide. The Fc domain monomer may be fused to the polypeptide by way of a linker. A linker can be a covalent bond or a spacer. The term “bond” refers to a chemical bond, e.g., an amide bond or a disulfide bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation. The term “spacer” refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1 -200 amino acid sequence) occurring between two elements, e.g., peptides or protein domains, to provide space and / or flexibility between the two elements. An amino acid spacer is part of the primary sequence of a polypeptide (e.g., fused to the spaced peptides via the polypeptide backbone). The formation of disulfide bonds, e.g., between two hinge regions that form an Fc domain, is not considered a linker.

[0086] As used herein, the term “Fc domain” refers to a dimer of two Fc domain monomers. An Fc domain has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity) to a human Fc domain that includes at least a CH2 domain and a CH3 domain. An Fc domain monomer includes second and third antibody constant domains (CH2 and CH3). In some embodiments, the Fc domain monomer also includes a hinge domain. An Fc domain does not include any portion of an immunoglobulin that is capable of acting as an antigen-recognition region, e.g., a variable domain or a complementarity determining region (CDR). In the wild-type Fc domain, the two Fc domain monomers dimerize by the interaction between the two CH3 antibody constant domains, as well as one or more disulfide bonds that form between the hinge domains of the two dimerizing Fc domain monomers. In some embodiments, an Fc domain may be mutated to lack effector functions, typical of a “dead Fc domain.” In certain embodiments, each of the Fc domain monomers in an Fc domain includes amino acid substitutions in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and an Fey receptor. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. An Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. Additionally, an Fc domain can be an IgG subtype (e.g., lgG1 , lgG2a, lgG2b, lgG3, or lgG4). The Fc domain can also be a non-naturally occurring Fc domain, e.g., a recombinant Fc domain.

[0087] As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell, e.g., a human red blood cell, platelet, neutrophil, or muscle cell).

[0088] As used herein, the term “fused” is used to describe the combination or attachment of two or more elements, components, or protein domains, e.g., peptides or polypeptides, by means including chemical conjugation, recombinant means, and chemical bonds, e.g., amide bonds. For example, two single peptides in tandem series can be fused to form one contiguous protein structure, e.g., a polypeptide, through chemical conjugation, a chemical bond, a peptide linker, or any other means of covalent linkage. In some embodiments of a polypeptide described herein, an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)), may be fused in tandem series to the N- or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NO: 48, SEQ ID NO: 100, or SEQ ID NO: 264) by way of a linker. For example, an extracellular ActRII chimera is fused to an Fc domain monomer by way of a peptide linker, in which the N- terminus of the peptide linker is fused to the C-terminus of the extracellular ActRII chimera through a chemical bond, e.g., a peptide bond, and the C-terminus of the peptide linker is fused to the N-terminus of the Fc domain monomer through a chemical bond, e.g., a peptide bond.

[0089] As used herein, the terms “increasing” and “decreasing” refer to modulating resulting in, respectively, greater or lesser amounts, of function, expression, or activity of a metric relative to a reference. For example, subsequent to administration of a polypeptide including an extracellular ActRII chimera in a method described herein, the amount of a marker of a metric (e.g., lean mass or fat mass) as described herein may be increased or decreased in a subject relative to the amount of the marker prior to administration. Generally, the metric is measured subsequent to administration at a time that the administration has had the recited effect, e.g., at least one week, one month, 3 months, or 6 months, after a treatment regimen has begun.

[0090] As used herein, the term “incretin-based therapy” refers to a drug that regulates post-meal glucagon and helps reduce post-meal blood sugars. One type of incretin-based therapy is an “incretin mimetic,” which works to mimic the incretin hormones that are naturally released by the body to stimulate the release of insulin in response to a meal. One class of incretin mimetics are GLP-1 receptor agonists, which activate the GLP-1 receptor, e.g., by mimicking the action of endogenous GLP-1 . GLP-1 receptor agonists are often designed to resist breakdown by the enzyme dipeptidyl peptidase 4 (DPP-4) so that they may remain in the bloodstream longer than naturally occurring GLP-1 . GLP-1 receptor agonists include exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, tirzepatide, albiglutide, and efpeglenatide. Another type of incretin-based therapy is an “incretin enhancer,” which increases endogenous incretin levels by inhibiting enzymes that degrade endogenous incretins, such as GLP-1 and GIP. Most incretin enhancers are DPP-4 inhibitors that slow the breakdown of GLP-1 . DPP-4 inhibitors include sitagliptin, saxagliptin, alogliptin, linagliptin, and vildagliptin.

[0091] The term “GLP-1 receptor agonist” refers to a compound that activates a GLP-1 receptor. Such exemplary compounds include exendins, exendin analogs, exendin agonists, GLP-1 or a variant, analog, or biologically active fragment thereof, GLP-1 (7-37), GLP-1 (7-37) analogs, GLP-1 (7-37) agonists, and the like. GLP-1 receptor agonists that have received FDA approval include exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, tirzepatide, and albiglutide.

[0092] As used herein, the term "DPP-4 inhibitor" refers to a compound capable of inhibiting dipeptidyl peptidase 4 (DPP-4). DPP-4, also known as CD26, is a serine protease that specifically cleaves dipeptides from the amino terminus of oligopeptides or proteins that contain an alanine or proline residue in position 2, thereby modifying or inhibiting their activity. Both GLP-1 and GIP are inactivated by DPP-4.

[0093] As used herein, the term “percentage of body weight gain” refers to the percentage of gained body weight compared to a prior body weight of a subject at a prior time. The percentage of body weight gain can be calculated as follows:

[0094] 100 X [(body weight at a later time - body weight at a prior time) I (body weight at a prior time)].

[0095] As used herein, the term “adiposity” refers to the fat stored in the adipose tissue of a subject. Administration of a polypeptide including extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)), a nucleic acid molecule encoding such a polypeptide, or vector containing such a nucleic acid molecule to a subject who is undergoing treatment with or previously underwent treatment with an incretin-based therapy can reduce the subject’s adiposity while maintaining or increasing lean mass.

[0096] As used herein, the term “epididymal and perirenal fat pads” refers to the tightly packed fat cells in the epididymis and around the kidney.

[0097] As used herein, the term “fasting insulin” refers to a subject’s level of insulin while the subject has not had any food intake for a length of time (i.e., 12-24 hours). Fasting insulin level is used in diagnosing metabolic diseases. Fasting insulin level is also used as an indication of whether a subject is at the risk of developing a metabolic disease. Normally, in a subject suffering from Type 1 diabetes, the subject’s fasting insulin level is low compared to that of a healthy subject. In a subject suffering from insulin resistance (i.e., Type 2 diabetes), the subject’s fasting insulin level is high compared to that of a healthy subject.

[0098] As used herein, the term “rate of glucose clearance” refers to the rate at which glucose is being cleared from the blood. The rate of glucose clearance can be measured in a glucose tolerance test (GTT). In a GTT, a subject is given a certain amount of glucose and blood samples are taken afterward to determine how quickly it is cleared from the blood. The rate of glucose clearance can be used as a parameter in diagnosing and / or determining the risk of developing metabolic diseases such as obesity, diabetes, and insulin resistance. As used herein, the term “serum lipid profile” refers to the measurement of the distribution of different types of lipids and lipoproteins in a subject’s serum. Such measurement can be accomplished by a panel of blood tests. The types of lipids and lipoproteins in a subject’s serum include, but are not limited to, cholesterol (e.g., high-density lipoprotein (HDL) and low-density lipoprotein (LDL)), triglyceride, and free fatty acid (FFA). The distribution of the different types of lipids and lipoproteins can be used as a parameter in diagnosing and / or determining the risk of developing metabolic diseases such as obesity, diabetes, and insulin resistance. High levels of cholesterol, especially low-density lipoprotein, are generally regarded as an indication or risk factor for developing certain metabolic diseases, or in some severe medical cases, cardiovascular diseases.

[0099] As used herein, the term “C-terminal extension” refers to the addition of one or more amino acids to the C-terminus of a an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)). The C-terminal extension can be one or more amino acids, such as 1 -6 amino acids (e.g., 1 , 2, 3, 4, 5, 6 or more amino acids). The C- terminal extension may include amino acids from the corresponding position of wild-type ActRIIA or ActRIIB. Exemplary C-terminal extensions are the amino acid sequence NP (a two amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 104) (a six amino acid C-terminal extension). Any amino acid sequence that does not disrupt the activity of the polypeptide can be used.

[0100] As used herein, the term “percent (%) identity” refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (which can alternatively be phrased as a given candidate sequence that has or includes a certain percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows:

[0101] 100 x (fraction of A / B) where A is the number of amino acid (or nucleic acid) residues scored as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where the length of the candidate sequence does not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence would not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0102] In particular embodiments, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits from 50% to 100% identity across the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purpose is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0103] As used herein, the term “serum half-life” refers to, in the context of administering a therapeutic protein to a subject, the time required for plasma concentration of the protein in the subject to be reduced by half. The protein can be redistributed or cleared from the bloodstream, or degraded, e.g., by proteolysis. Serum half-life comparisons can be made by comparing the serum half-life of Fc fusion proteins.

[0104] As used herein, the term “lean mass” refers to a component of body composition which includes, e.g., lean mass, body fat, and body fluid. Normally lean mass is calculated by subtracting the weights of body fat and body fluid from total body weight. Typically, a subject’s lean mass is between 60% and 90% of total body weight. In the present invention, administration of a polypeptide including an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43), a nucleic acid molecule encoding such a polypeptide, or vector containing such a nucleic acid molecule to a subject to a subject who is undergoing treatment with or previously underwent treatment with an incretinbased therapy maintains or increases the subject’s lean mass.

[0105] As used herein, the term “affinity” or “binding affinity” refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the strength of the sum total of non-covalent interactions between a molecule and its binding partner, such as an extracellular ActRII chimera and BMP9 or activin A. Unless indicated otherwise, binding affinity refers to intrinsic binding affinity, which reflects a 1 :1 interaction between members of a binding pair. The binding affinity between two molecules is commonly described by the dissociation constant (KD) or the affinity constant (KA). TWO molecules that have low binding affinity for each other generally bind slowly, tend to dissociate easily, and exhibit a large KD. TWO molecules that have high affinity for each other generally bind readily, tend to remain bound longer, and exhibit a small KD. The KD of two interacting molecules may be determined using methods and techniques well known in the art, e.g., surface plasmon resonance. KD is calculated as the ratio of k0« / k0n.

[0106] As used herein, the term “muscle mass” refers to the primary component of lean mass. Muscle mass can be measured experimentally by measuring muscle weight.

[0107] As used herein, the term “vascular complication” refers to a vascular disorder or any damage to the blood vessels, such as damage to the blood vessel walls. Damage to the blood vessel walls may cause an increase in vascular permeability or leakage. The term “vascular permeability or leakage” refers to the capacity of the blood vessel walls to allow the flow of small molecules, proteins, and cells in and out of blood vessels. An increase in vascular permeability or leakage may be caused by an increase in the gaps (e.g., an increase in the size and / or number of the gaps) between endothelial cells that line the blood vessel walls and / or thinning of the blood vessel walls.

[0108] As used herein, the term “polypeptide” describes a single polymer in which the monomers are amino acid residues which are covalently conjugated together through amide bonds. A polypeptide is intended to encompass any amino acid sequence, either naturally occurring, recombinant, or synthetically produced.

[0109] As used herein, the term “homodimer” refers to a molecular construct formed by two identical macromolecules, such as proteins or nucleic acids. The two identical monomers may form a homodimer by covalent bonds or non-covalent bonds. For example, an Fc domain may be a homodimer of two Fc domain monomers if the two Fc domain monomers contain the same sequence. In another example, a polypeptide described herein including an extracellular ActRII chimera fused to an Fc domain monomer may form a homodimer through the interaction of two Fc domain monomers, which form an Fc domain in the homodimer.

[0110] As used herein, the term “heterodimer” refers to a molecular construct formed by two different macromolecules, such as proteins or nucleic acids. The two monomers may form a heterodimer by covalent bonds or non-covalent bonds. For example, a polypeptide described herein including an extracellular ActRII chimera fused to an Fc domain monomer may form a heterodimer through the interaction of two Fc domain monomers, each fused to a different ActRII chimera, which form an Fc domain in the heterodimer.

[0111] As used herein, the term “host cell” refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express proteins from their corresponding nucleic acids. The nucleic acids are typically included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). A host cell may be a prokaryotic cell, e.g., a bacterial cell, or a eukaryotic cell, e.g., a mammalian cell (e.g., a CHO cell or a HEK293 cell).

[0112] As used herein, the terms “effective amount” and “therapeutically effective amount” of a composition, polypeptide, nucleic acid, or vector described herein refer to a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of treating a subject undergoing or who has undergone treatment with an incretin-based therapy (e.g., for the treatment of T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2or greater and a weight-related comorbid condition), it is an amount of the composition, polypeptide, nucleic acid, or vector sufficient to achieve a treatment response as compared to the response obtained without administration of the composition, polypeptide, nucleic acid, or vector. The amount of a given composition described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, a “therapeutically effective amount” of a composition, polypeptide, nucleic acid, or vector of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of a composition, polypeptide, nucleic acid, or vector of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen may be adjusted to provide the optimum therapeutic response. As used herein, the term “pharmaceutical composition” refers to a medicinal or pharmaceutical formulation that includes an active ingredient as well as excipients and diluents to enable the active ingredient suitable for the method of administration. The pharmaceutical composition of the present invention includes pharmaceutically acceptable components that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition may be in tablet or capsule form for oral administration or in aqueous form for intravenous or subcutaneous administration.

[0113] As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present invention, the pharmaceutically acceptable carrier or excipient must provide adequate pharmaceutical stability to the polypeptide including an extracellular ActRII chimera, the nucleic acid molecule(s) encoding the polypeptide, or a vector containing such nucleic acid molecule(s). The nature of the carrier or excipient differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.

[0114] As used herein, “treatment” and “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0115] As used herein, the terms “subject” and “patient” refer to a mammal, e.g., preferably a human. Mammals include, but are not limited to, humans and domestic and farm animals, such as monkeys (e.g., a cynomolgus monkey), mice, rats, dogs, cats, horses, sheep, goats, rabbits, and cows, etc.

[0116] Brief Description of the Drawings

[0117] FIGS. 1A-1 B are a series of graphs showing body weight measurements during twice weekly treatment of diet induced obese, 18-week-old, male C57BI / 6 mice with either Tris-buffered-saline (Vehicle) and Vehicle (n=10), Semaglutide (Serna) and Tris-buffered-saline (Vehicle) (n=10), Chimera 2 I65F E81 Q- mFc (a construct including SEQ ID NO: 25 fused to a mouse Fc domain monomer by a GGG linker in the form of a homodimer) and Vehicle (n=10), Chimera 1 / 2b-mFc (a construct including SEQ ID NO: 41 fused to a mouse Fc domain monomer by a GGG linker in the form of a homodimer) and Vehicle (n=10), or Serna in combination with Chimera 2 I65F E81 Q-mFc (n=10). Data are shown as absolute body weight in grams (FIG. 1 A) and as a percentage of body weight change from baseline measurements (FIG. 1 B). Data are shown as mean ± SEM.

[0118] FIG. 2 is a graph showing change in lean mass from baseline after twice weekly treatment for four weeks of diet induced obese, 18-week-old, male C57BI / 6 mice with either Vehicle and Vehicle (n=10), Chimera 2 I65F E81 Q-mFc and Vehicle (n=10), or Chimera 1 / 2b-mFc and Vehicle (n=10). On day 27 of the study, lean mass was determined through the use of nuclear magnetic resonance (NMR) body composition analysis (Bruker Minispec). Data are shown as a percent of lean mass change from baseline measurements. Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA with a Dunnett posttest, ****p<0.0001 .

[0119] FIGS. 3A-3D are a series of graphs showing changes in lean mass (FIGS. 3A and 3C) and fat mass (FIGS. 3B and 3D) after twice weekly treatment for four weeks of diet induced obese, 18-week-old, male C57BI / 6 mice with either Vehicle and Vehicle (n=10), Serna and Vehicle (n=10), Chimera 2 I65F E81 Q-mFc and Vehicle (n=10), or Serna in combination with Chimera 2 I65F E81 Q-mFc (n=10). On day 27 of the study, lean mass and fat mass were determined by NMR body composition analysis (Bruker Minispec). Data are shown as a percent of lean mass or fat mass change from baseline measurements (FIGS. 3A and 3B) and as a percent of lean mass or fat mass change from baseline measurements compared to vehicle (FIGS. 3C and 3D). Data are shown as mean + SEM. Statistics are shown using 1 - way ANOVA with a Dunnett posttest, **p<0.01 ; ****p<0.0001 .

[0120] FIGS. 4A-4B are a series of graphs showing tibialis anterior weight (FIG. 4A) and soleus weight measurements (FIG. 4B) after twice weekly treatment for four weeks of diet induced obese, 18-week-old, male C57BI / 6 mice with either Vehicle and Vehicle (n=10), Serna and Vehicle (n=10), Chimera 2 I65F E81 Q-mFc and Vehicle (n=10), or Serna in combination with Chimera 2 I65F E81 Q-mFc (n=10). At study termination (day 29), soleus muscles and tibialis anterior muscles were fine dissected and weighed. Muscle weight data are shown as a percentage of vehicle. Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA with a Dunnett posttest, ns= not significant, ***p<0.001 ; ****p<0.0001 .

[0121] FIG. 5 is a graph showing inguinal white adipose tissue weight measurements after twice weekly treatment for four weeks of diet induced obese, 18-week-old, male C57BI / 6 mice with either Vehicle and Vehicle (n=10), Serna and Vehicle (n=10), Chimera 2 I65F E81 Q-mFc and Vehicle (n=10), or Serna in combination with Chimera 2 I65F E81 Q-mFc (n=10). At study termination (day 29), inguinal white adipose tissue (IWAT) depots were fine dissected and weighed. Data are shown as absolute weight in grams. Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA with a Dunnett posttest, *p<0.05.

[0122] FIGS. 6A-6C are a series of graphs showing changes in body weight (FIG. 6A) and lean mass and fat mass (FIGS. 6B and 6C) after twice weekly treatment for two weeks of diet induced obese, 18- week-old, male C57BI / 6 mice with either Vehicle and Vehicle (n=10), Serna and Vehicle (n=10), or Serna in combination with Chimera 1 / 2b-mFc (n=10). Data are shown as a percentage of body weight change from baseline measurements (FIG. 6A), as a percent of lean mass or fat mass change from baseline measurements (FIG. 6B), and as a ratio of lean to fat mass (FIG. 6C). Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA and Dunnett's multiple comparisons test, *p<0.05, **p<0.01 , “**p<0.0001. FIGS. 7A-7B are a series of graphs showing changes in lean mass (FIG. 7A) and functional strength as measured by evoked force in the gastrocnemius (FIG. 7B) after twice weekly treatment for two weeks of 14-week-old male C57BL / 6 mice with Tris-buffered-saline (vehicle, n=10), taldefgrobep alfa (n=10), bimagrumab (n=10), landogrozumab (n=10), or Chimera 1 / 2b-mFc (n=10) at 10 mg / kg. Data are shown as a percent of lean mass change from baseline (FIG. 7A) and as a maximum force (mN) (FIG. 7B). Data are shown as mean ± SEM. Statistics are shown using ordinary one-way ANOVA with a Tukey’s post-hoc test, *p< 0.05 *" p<0.001 , and ****p< 0.0001 .

[0123] FIGS. 8A-8F are a series of graphs showing lean mass (FIG. 8A), fat mass (FIG. 8B), ratio of fat mass / lean mass (FIG. 8C), tibialis anterior weight (FIG. 8D), gastrocnemius weight (FIG. 8E), and soleus weight (FIG. 8F) after twice weekly treatment for four weeks of diet induced obese 18-week-old male C57BL / 6 mice with Tris-buffered-saline (vehicle, n=10) or Chimera 1 / 2b-mFc (n=10) at 10 mg / kg. Muscles were collected bilaterally and graphed as a sum where applicable. Data are shown as absolute weight in grams (FIGS. 8A, 8B, and 8D-8F) and as a ratio of fat mass / lean mass (FIG. 8C). Data are shown as mean ± SEM. Statistics are shown using an unpaired t-test, *p<0.05, ***p<0.001 , ****p<0.0001 .

[0124] FIGS. 9A-9C are a series of graphs showing body weight measurements (FIG. 9A), changes in lean mass (FIG. 9B), and changes in fat mass (FIG. 9C) during treatment of diet induced obese (DIO), 6- month-old, male C57BL / 6 mice for 14 days with either Tris-buffered-saline (DIO Vehicle) (n=10), 0.120 mg / kg Semaglutide (Serna) and Vehicle (Sema / Veh; n=10), 0.120 mg / kg Serna in combination with 1 mg / kg Chimera 1 / 2b-mFc (Serna / 1 mg / kg Chimera 1 / 2b-mFc; n=10), or 0.120 mg / kg Serna in combination with 3 mg / kg Chimera 1 / 2b-mFc (Serna / 3 mg / kg Chimera 1 / 2b-mFc; n=15). Age-matched chow-fed C57 / BL6 mice were used as a control group (Chow vehicle; n=10). Data are shown as absolute body weight in grams (FIG. 9A), percent of lean mass change from baseline (FIG. 9B), or percent of fat mass change from baseline (FIG. 9C). Data are shown as mean ± SEM. Statistical analysis was done using a 2-way ANOVA and Tukey’s multiple comparisons test, *p<0.05, **p<0.01 , ***p<0.001 , *“*p<0.0001.

[0125] FIGS. 10A-10D are a series of graphs showing fat mass / lean mass ratio (FIG. 10A), interscapular brown adipose tissue (iBAT) weight (FIG. 10B), epididymal white adipose tissue (eWAT) weight (FIG. 10C), and tibialis anterior weight (FIG. 10D) measurements after treatment of diet induced obese (DIO), 6-month-old, male C57BL / 6 mice for 14 days with either Tris-buffered-saline (DIO Vehicle) (n=10), 0.120 mg / kg Serna and Vehicle (Sema / Veh; n=10), 0.120 mg / kg Serna in combination with 1 mg / kg Chimera 1 / 2b-mFc (Serna 1 1 mg / kg Chimera 1 / 2b-mFc; n=10), or 0.120 mg / kg Serna in combination with 3 mg / kg Chimera 1 / 2b-mFc (Serna / 3 mg / kg Chimera 1 / 2b-mFc; n=15). Age-matched chow-fed C57 / BL6 mice were used as a control group (Chow vehicle; n=10). Data are shown as fat mass / lean mass ratio (FIG. 10A), or absolute weight in grams (FIGS. 10B-D). Data are shown as mean ± SEM. Statistics are shown using a 2-way ANOVA and Tukey’s multiple comparisons for the fat-to-lean ratio. For lean and fat mass, a one-way ANOVA was run with Tukey’s multiple comparisons test, *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 , ns=not significant.

[0126] Detailed Description of the Invention

[0127] The invention features methods of treating a subject who is currently undergoing or previously underwent treatment with an incretin-based therapy (e.g., a subject being treated for T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2or greater and a weight-related comorbid condition) by administering a polypeptide including an extracellular activin receptor type II (ActRII) chimera. The polypeptide including an extracellular ActRII chimera may be fused to an Fc domain monomer, and a polypeptide including an extracellular ActRII chimera fused to an Fc domain monomer may also form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers. The incretin-based therapy may be an incretin mimetic, such as a GLP-1 receptor agonist, or an incretin enhancer, such as a DPP-4 inhibitor. The methods may include co-administration of the incretin-based therapy and the polypeptide including an extracellular ActRII chimera or the polypeptide including an extracellular ActRII chimera can be administered after treatment with the incretin-based therapy has ceased. The methods described herein can increase lean mass or prevent or reduce a loss of lean mass (e.g., a loss of lean mass associated with incretin-based therapy, such as incretin mimetic therapy), increase muscle mass, reduce fat mass, reduce white adipose tissue, increase fat metabolism, increase energy expenditure, reduce body fat, improve insulin resistance, improve cardiac function or cardiac health, and / or improve insulin sensitivity. Accordingly, these methods may further reduce fat mass (e.g., compared to incretin-based therapy alone) and ameliorate muscle loss. Increasing lean mass or preventing or reducing lean mass loss (e.g., muscle loss associated with incretin mimetic therapy) may prevent or reduce frailty in subjects undergoing or who previously underwent incretin-based therapy and may reduce the risk of sarcopenia or reduce or prevent the premature onset of sarcopenia. The methods described herein may also reduce weight regain after discontinuation of the incretin-based therapy.

[0128] Activin type II receptors

[0129] Activin type II receptors are single transmembrane domain receptors that modulate signals for ligands in the transforming growth factor p (TGF-p) superfamily. Ligands in the TGF-p superfamily are involved in a host of physiological processes, such as muscle growth, vascular growth, cell differentiation, homeostasis, hematopoiesis, and osteogenesis. Examples of ligands in the TGF-p superfamily include, e.g., activin (e.g., activin A and activin B), inhibin, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin, and GDF1 1 ), and bone morphogenetic proteins (BMPs) (e.g., BMP9).

[0130] There exist two types of activin type II receptors: ActRIIA and ActRIIB. Studies have shown that BMP9 binds ActRIIB with about 300-fold higher binding affinity than ActRIIA (see, e.g., Townson et al., J. Biol. Chem. 287:27313, 2012) and ActRIIA-Fc is known to have a longer half-life compared to ActRIIB-Fc. Described herein are extracellular ActRII chimeras that are constructed by combining portions of extracellular ActRIIA and ActRIIB with the goal of generating proteins that bind to ActRII ligands (e.g., activin A, activin B, myostatin, and GDF11 ) and retain the function of wild-type extracellular ActRII proteins (e.g., the ability to increase muscle mass and / or lean mass). In some embodiments, the ActRII chimeras exhibit reduced BMP9 binding relative to wild-type extracellular ActRIIB, which can prevent or reduce disruption of endogenous BMP9 signaling. In some embodiments, the chimeras have properties of both ActRIIA (e.g., low binding affinity to BMP9 and / or longer serum half-life as an Fc fusion protein) and ActRIIB (e.g., the ability to increase muscle mass).

[0131] The present invention is based, in part, on the discovery that administration of a polypeptide including an extracellular ActRII chimera described herein (an ActRII chimera-Fc polypeptide) to obese mice alone or in combination with semaglutide resulted in an increase in lean mass, an increase in muscle weight, a decrease in fat mass, and a decrease in inguinal white adipose tissue. Administration of the ActRII chimera-Fc polypeptide in combination with semaglutide reversed the loss of lean mass observed with semaglutide alone and further reduced fat mass (resulting in fat loss) and inguinal white adipose tissue compared to administration of semaglutide or the ActRII chimera-Fc alone. Accordingly, these data suggest that administration of a polypeptide containing an extracellular ActRII chimera to subjects who are currently undergoing or previously underwent treatment with an incretin-based therapy (e.g., treatment with semaglutide or another incretin mimetic) could prevent or reduce lean mass (muscle) loss (e.g., muscle loss associated with the incretin-based therapy), increase lean mass, and / or further reduce fat mass (i.e. , increase fat loss), which could reduce or prevent the development of frailty or the premature onset of sarcopenia.

[0132] Extracellular activin receptor type II chimeras

[0133] The extracellular ActRII chimeras described herein contain sequence from both the extracellular portion of ActRIIB and the extracellular portion of ActRIIA. The ActRII chimeras may exhibit similar or improved binding to activins (e.g., activin A and / or activin B) and / or myostatin compared to wild-type extracellular ActRIIA and / or ActRIIB, allowing them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous activin receptor signaling.

[0134] The wild-type amino acid sequences of the extracellular portions of human ActRIIA and ActRIIB are shown below.

[0135] Human ActRIIA, extracellular portion (SEQ ID NO: 44):

[0136] GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGC WLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS

[0137] Human ActRIIB, extracellular portion (SEQ ID NO: 45):

[0138] GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWL DDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT

[0139] The ActRII chimeras described herein result from joining an N-terminal portion of extracellular ActRIIB (SEQ ID NO: 45 shown above) to a C-terminal portion of extracellular ActRIIA (SEQ ID NO: 44 shown above) such that the sequences are contiguous (e.g., the ActRIIA sequence continues where the ActRIIB sequence left off, starting with the next the amino acid located in the corresponding position of ActRIIA). In some embodiments, the N-terminus of the ActRII chimera includes the six amino acids found at the N-terminus of extracellular ActRIIA joined to the fifth amino acid of extracellular ActRIIB. In some embodiments, the N-terminus of the ActRII chimera begins with the first amino acid located at the N- terminus of extracellular ActRIIB. Accordingly, in some embodiments, the N-terminal portion of ActRIIB begins with the amino acid in the fifth position of SEQ ID NO: 45 (A), while in other embodiments (e.g., in embodiments in which the six amino acids found at the N-terminus of extracellular ActRIIA are not included in the chimera), the N-terminal portion of ActRIIB begins with the amino acid in the first position of SEQ ID NO: 45 (G). In some embodiments, the N-terminus of the ActRII chimera includes the first ten amino acids found at the N-terminus of extracellular ActRIIA joined to the ninth amino acid of extracellular ActRIIB, in which case the N-terminal portion of ActRIIB begins with the amino acid in the ninth position of SEQ ID NO: 45 (E). The extracellular ActRII chimera may also include one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIB compared to wildtype extracellular ActRIIB (e.g., SEQ ID NO: 45 shown above), and / or one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIA compared to wildtype extracellular ActRIIA (e.g., SEQ ID NO: 44 shown above). Amino acid substitutions at 9 different positions may be introduced into an extracellular ActRII chimera (Table 1 ). An extracellular ActRII chimera may have one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions relative to the sequence of a wild-type sequence (e.g., relative to the sequence of wild-type extracellular ActRIIB (SEQ ID NO: 45) if the portion of the chimera corresponds to a region of wild-type extracellular ActRIIB, or relative to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 44) if the portion of the chimera corresponds to a region of wild-type extracellular ActRIIA). The positions at which amino acid substitutions may be made, as well as the amino acids that may be substituted at these positions, are listed in Table 1 .

[0140] Amino acid substitutions can alter the activity and / or binding affinity of the extracellular ActRII chimeras. In some embodiments, the extracellular ActRII chimeras bind to activin A, activin B, myostatin, and / or GDF1 1 with sufficient affinity to compete with endogenous activin receptors for binding to one or more of these ligands. In some embodiments, the extracellular ActRII chimeras have reduced, weak, or no substantial binding to BMP9 (e.g., compared to wild-type ActRIIB). BMP9 binding may be reduced in extracellular ActRII chimeras containing the amino acid sequence TEEN (SEQ ID NO: 105) or TKEN (SEQ ID NO: 106) at positions X3, X4, X5, and Xe. In some embodiments, a polypeptide including an extracellular ActRII chimera (e.g., any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) with the sequence TEEN (SEQ ID NO: 105) at positions X3, X4, X5, and Xe can have a substitution of the amino acid K for the amino acid E at position X4. In some embodiments, a polypeptide including an extracellular ActRII chimera (e.g., any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) with the sequence TKEN (SEQ ID NO: 106) at positions X3, X4, X5, and Xs can have a substitution of the amino acid E for the amino acid K at position X4. The sequences TEEN (SEQ ID NO: 105) and TKEN (SEQ ID NO: 106) can be used interchangeably in the extracellular ActRII chimeras (e.g., the chimeras in Tables 1 and 2, e.g., SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)). The extracellular ActRII chimeras may further include a C- terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension can add one or more additional amino acids at the C-terminus (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids) to any of the ActRII chimeras shown in Tables 1 and 2 (e.g., SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)). The C-terminal extension may correspond to sequence from the same position in wild-type ActRIIA or ActRIIB. For example, C-terminal extensions that can be included in the extracellular ActRII chimeras are the amino acid sequence NP and the amino acid sequence NPVTPK (SEQ ID NO: 104), which correspond to sequence found in the same position in wild-type ActRIIA. Table 1. Amino acid substitutions in an extracellular ActRII chimera having a sequence of any one of SEQ ID NOs: 1-21

[0141] In some embodiments, in the extracellular ActRII chimeras of SEQ ID NOs: 1 -21 (shown in Table

[0142] 1 ) Xi is D, X2 is I, F, or E, X3 is N or T, X4 is A or E, X5 is T or K, Xe is E or K, X7 is E or D, Xs is N or S, and X9 is E or Q. In some embodiments, in the extracellular ActRII chimeras of SEQ ID NOs: 1 -21 Xi is D, X2 is I or F, X3 is N, X4 is A or E, X5 is T or K, Xe is E or K, X7 is E or D, Xs is N or S, and Xg is E or Q. In some embodiments, in the extracellular ActRII chimeras of SEQ ID NOs: 1 -21 Xi is D, X2 is F, X3 is N, X4 is E, X5 is K, Xe is K, X7 is D, Xs is S, and Xg is Q.

[0143] In some embodiments, a polypeptide described herein includes an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 22-43 (Table 2). Table 2. Extracellular ActRII chimeras having the sequences of SEQ ID NOs: 22-43

[0144] In some embodiments, the extracellular ActRII chimeras described herein have an N-terminal truncation of 1 -9 amino acids (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). The N-terminal truncation can involve the removal of 1 -9 amino acids from the N-terminus of any of the chimeras shown in Tables 1 and 2 (e.g., SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)). The N-terminal truncation can remove amino acids up to two amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE or QE) are retained in the N-terminally truncated ActRII chimeras). Exemplary ActRII chimeras having N-terminal truncations are provided in Table 3, below. Table 3. Extracellular ActRII chimeras having N-terminal truncations

[0145] In some embodiments, a polypeptide including an extracellular ActRII chimera may further include an Fc domain monomer, which may be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRII chimera by way of a linker or other covalent bonds. A polypeptide including an extracellular ActRII chimera fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer. Exemplary polypeptides containing an ActRII chimera, an Fc domain monomer, and a linker are provided in Table 4, below. In some embodiments, the terminal lysine is absent from the Fc domain monomer amino acid sequence. The C-terminal lysine of the Fc domain monomer of the polypeptides provided in Table 4 (i.e., the C-terminal lysine in each polypeptide sequence) may or may not be present, without affecting the structure or stability of the polypeptide. The disclosure specifically contemplates SEQ ID NOs: 107-110 and 184-263 that do not include the C-terminal lysine at the end of the polypeptide sequence. The polypeptides of SEQ ID NOs: 107-110 and 184-263 may be expressed including a C-terminal lysine, which then may be proteolytically cleaved upon expression of the polypeptide (e.g., the polypeptides of SEQ ID NOs: 107-110 and 184-263 are expressed using nucleic acid constructs encoding the polypeptide including a C-terminal lysine residue). The polypeptides of SEQ ID NOs: 107-110 and 184-263 may also be expressed without including the C-terminal lysine residue.

[0146] Table 4. Polypeptides containing an extracellular ActRII chimera fused to an Fc domain monomer by way of a linker

[0147] Furthermore, in some embodiments, a polypeptide described herein (e.g., an ActRII chimera-Fc fusion protein) has a serum half-life of at least 7 days in humans. The polypeptide may bind to activin A with a KD of 10 pM or higher. In some embodiments, the polypeptide binds to activin A, activin B, and / or myostatin and exhibits reduced (e.g., weak) binding to BMP9 (e.g., compared to wild-type extracellular ActRIIB). In some embodiments, the polypeptide that has reduced or weak binding to BMP9 has the sequence TEEN (SEQ ID NO: 105) or TKEN (SEQ ID NO: 106) at positions X3, X4, X5, and Xe. In some embodiments, the polypeptide that has reduced or weak binding to BMP9 has the sequence KKDS (SEQ ID NO: 265) or TKDS (SEQ ID NO: 266) at positions X3, X4, X5, and Xe. In some embodiments, the polypeptide does not substantially bind to human BMP9.

[0148] In some embodiments, the polypeptide may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 30 pM). In some embodiments, the polypeptide may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 5 pM) The polypeptide may also bind to growth and differentiation factor 1 1 (GDF-11 ) with a KD of approximately 5 pM or higher (e.g., a KD of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher). Incretin-based therapies

[0149] A polypeptide including an extracellular ActRII chimera described herein can be administered to a subject who is currently undergoing treatment with an incretin-based therapy (e.g., a subject having T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2or greater and a weight-related comorbid condition) or to a subject who was previously treated with an incretin-based therapy, incretins are gut-derived hormones, members of the glucagon superfamily, that are released in response to nutrient ingestion, mainly glucose and fat. They stimulate pancreatic p-cells to secrete insulin m a glucose-dependent manner and play an important roie in the local gastrointestinal and whole-body physiology. There are two main incretin hormones in humans: glucose-dependent insulinotropic peptide (GIP, also known as gastric inhibitory peptide) and glucagon-like peptide-1 (GLP-1 ). Incretin-based therapies include incretin mimetics (e.g., glucagon-like peptide-1 (GLP-1 ) receptor agonists) and incretin enhancers.

[0150] Incretin mimetics

[0151] In some embodiments, a polypeptide including an extracellular ActRII chimera described herein is administered to a subject who is currently undergoing treatment with an incretin mimetic (e.g., a subject having T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2or greater and a weight-related comorbid condition) or to a subject who was previously treated with an incretin mimetic. Most incretin mimetics are GLP-1 receptor agonists that mimic endogenous GLP-1 . They are often designed to resist breakdown by the enzyme dipeptidyl peptidase 4 (DPP-4) so that they may remain in the bloodstream longer than naturally occurring GLP-1 . GLP-1 receptor agonists include exenatide, lixisenatide, liraglutide, semaglutide, du laglutide, tirzepatide, albig lutide, and efpeglenatide.

[0152] Exenatide is a GLP-1 receptor agonist FDA approved for the treatment of patients with T2D (e.g., to improve glycemic control as an adjunct to diet and exercise in adults and pediatric patients 10 years or older). It can be administered by subcutaneous injection at an initial dose of 5 micrograms (mcg) twice daily, with an increase to 10 mcg twice daily after one month based on clinical response (under brand name BYETTA™) or at 2 milligrams (mg) once every seven days (weekly) for the extended-release form (under brand name BYDUREON BCise®).

[0153] Lixisenatide is a GLP-1 receptor agonist FDA approved for treatment of patients with T2D (e.g., to improve glycemic control as an adjunct to diet and exercise in adults). It can be administered by subcutaneous injection at an initial dose of 10 mcg once daily for 14 days, with a dosage increase on day 15 to 20 mcg once daily (under brand name ADLYXIN®).

[0154] Liraglutide is a GLP-1 receptor agonist FDA approved for the treatment of patients with T2D (under brand name VICTOZA®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults and pediatric patients 10 years or older or to reduce the risk of major adverse cardiovascular events in adults with T2D and established cardiovascular disease) and for chronic weight management in adult patients with a BMI of 30 kg / m2or greater (obese), or 27 kg / m2or greater (overweight) in the presence of at least one weight-related comorbid condition (e.g. hypertension, type 2 diabetes mellitus, or dyslipidemia), and in pediatric patients aged 12 years and older with body weight above 60 kg and an initial BMI corresponding to 30 kg / m2for adults (obese) by international cut-offs (under brand name SAXENDA®, e.g., as an adjunct to a reduced-calorie diet and increased physical activity). For treating T2D, liraglutide can be administered by subcutaneous injection at an initial dose of 0.6 mg once daily for one week, followed by an increase to 1 .2 mg daily. If additional glycemic control is required, the dose can be increased to 1 .8 mg daily after one week of treatment with the 1 .2 mg daily dose. For treating adult patients with a BMI of 30 kg / m2or greater, or 27 kg / m2or greater in the presence of at least one weight- related comorbid condition, or pediatric patients aged 12 years and older with body weight above 60 kg and an initial BMI corresponding to 30 kg / m2for adults by international cut-offs, liraglutide can be administered by subcutaneous injection at an initial dose of 0.6 mg per day for one week, with weekly dose increases until a dose of 3 mg daily is reached (recommended dosage regimen is 0.6 mg for week 1 , 1 .2 mg for week 2, 1 .8 mg for week 3, 2.4 mg for week 4 and maintenance at 3 mg for week 5 and onward). Pediatric patients who do not tolerate 3 mg daily may have their dose reduced to 2.4 mg daily.

[0155] Semaglutide is a GLP-1 receptor agonist FDA approved for the treatment of patients with T2D (under brand names OZEMPIC®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults or to reduce the risk of major adverse cardiovascular events in adults with T2D and established cardiovascular disease, and RYBELSUS®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults) and for chronic weight management in adult patients with an initial BMI of 30 kg / m2or greater (obese), or 27 kg / m2or greater (overweight) in the presence of at least one weight-related comorbid condition (e.g. hypertension, type 2 diabetes mellitus, or dyslipidemia), and in pediatric patients aged 12 years and older with an initial BMI at the 95,hpercentile or greater for age and sex (obesity) (under brand name WEGOVY®, e.g., as an adjunct to a reduced-calorie diet and increased physical activity). For the treatment of patients with T2D, semaglutide can be administered by subcutaneous injection at an initial dose of 0.25 mg once weekly that is increased to 0.5 mg once weekly after four weeks. If additional glycemic control is needed, the dosage can be increased to 1 mg once weekly after at least four weeks on the 0.5 mg dose, and if additional glycemic control is needed, the dosage can be increased to 2 mg once weekly after at least four weeks on the 1 mg dosage. Alternatively, semaglutide can be administered orally as a tablet at an initial dose of 3 mg once daily for 30 days. After 30 days on the 3 mg dosage, the dosage is increased to 7 mg once daily. If additional glycemic control is needed, the dosage may be increased to 14 mg once daily after at least 30 days on the 7 mg dosage. For the treatment of adult patients with an initial BMI of 30 kg / m2or greater, or 27 kg / m2or greater in the presence of at least one weight-related comorbid condition, and pediatric patients aged 12 years and older with an initial BMI at the 95thpercentile or greater for age and sex, semaglutide can be administered by subcutaneous injection at an initial dose of 0.25 mg once weekly for four weeks, with dose increases in four week intervals until a dose of 2.4 mg is reached (recommended dosage regimen is 0.25 mg for weeks 1 -4, 0.5 mg for weeks 5-8, 1 mg for weeks 9-12, 1 .7 mg for weeks 13-16, and maintenance at 1 .7 mg or 2.4 mg for week 17 and onward). The maintenance dose is 2.4 mg or 1 .7 mg once weekly.

[0156] Dulaglutide is a GLP-1 receptor agonist FDA approved for the treatment of patients with T2D (under brand name TRULICITY®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults and pediatric patients 10 years or older or to reduce the risk of major adverse cardiovascular events in adults with T2D and established cardiovascular disease or multiple cardiovascular risk factors). Dulaglutide can be administered by subcutaneous injection at an initial dose of 0.75 mg once weekly, which can be increased to 1 .5 mg once weekly for additional glycemic control (for adult and pediatric patients), and further increased in 1 .5 mg increments after at least four weeks on the current dosage if additional glycemic control is needed, up to a maximum recommended dosage of 4.5 mg once weekly (for adult patients).

[0157] Tirzepatide is GIP receptor and GLP-1 receptor agonist approved for the treatment of patients with T2D (under brand name MOUNJARO®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults) and for chronic weight management in adult patients with an initial BMI of 30 kg / m2or greater (obese) or 27 kg / m2or greater (overweight) in the presence of at least one weight-related comorbid condition (e.g. hypertension, type 2 diabetes mellitus, dyslipidemia, obstructive sleep apnea, or cardiovascular disease) (under brand name ZEPBOUND™, e.g., as an adjunct to a reduced-calorie diet and increased physical activity). For treating patients with T2D, tirzepatide can be administered by subcutaneous injection at a recommended starting dosage of 2.5 mg once weekly, with an increase to 5 mg once weekly after four weeks. If additional glycemic control is needed, the dosage can be increased in 2.5 mg increments after at least four weeks on the current dose, up to a maximum dosage of 15 mg once weekly. For treating patients with an initial BMI of 30 kg / m2or greater or 27 kg / m2or greater in the presence of at least one weight-related comorbid condition, tirzepatide can be administered by subcutaneous injection at a recommended starting dosage of 2.5 mg once weekly, with an increase to 5 mg once weekly after four weeks. The dosage can be increased in 2.5 mg increments after at least four weeks on the current dose, and the recommended maintenance dosages are 5 mg, 10 mg, or 15 mg once weekly, with a maximum dosage of 15 mg once weekly.

[0158] Albig I utide is a GLP-1 receptor agonist FDA approved for the treatment of patients with T2D (under brand name TANZEUM®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults). Albiglutide can be administered by subcutaneous injection at an initial dose of 30 mg once weekly. Dose can be increased to 50 mg once weekly in patients requiring additional glycemic control.

[0159] Efpeglenatide is a GLP-1 receptor agonist administered to adult patients with T2D in a phase 3, double-blind, placebo-controlled, multicenter trial. Efpeglenatide was administered by subcutaneous injection once weekly at 2, 4, or 6 mg and was found to improve glycemic control and body weight.

[0160] Incretin enhancers

[0161] In some embodiments, a polypeptide including an extracellular ActRII chimera described herein is administered to a subject who is currently undergoing treatment with an incretin enhancer (e.g., a subject having T2D) or to a subject who was previously treated with an incretin enhancer. Most incretin enhancers are DPP-4 inhibitors that slow the breakdown of GLP-1 . DPP-4 inhibitors include sitagliptin, saxagliptin, alogliptin, linagliptin, and vildagliptin.

[0162] Sitagliptin is a DPP-4 inhibitor FDA approved for the treatment of patients with T2D (under brand name JANUVIA®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults). Sitagliptin can be administered orally as a tablet at a recommended dose of 100 mg once daily, which can be adjusted to 50 mg once daily for subjects with an eGFR greater than or equal to 30 mL / min / 1 .73 m2to less than 45 mL / min / 1 .73 m2or 25 mg once daily for subjects with an eGFR less than 30 mL / min / 1 .73 m2(including patients with end stage renal disease (ESRD) on dialysis).

[0163] Saxagliptin is a DPP-4 inhibitor FDA approved for the treatment of patients with T2D (under brand name ONGLYZA®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults). Saxagliptin can be administered orally as a tablet at a recommended dose of 2.5 mg or 5 mg once daily. A dose of 2.5 mg once daily is recommended for patients with eGFR less than 45 mUmin / 1 .73 m2(with moderate or severe renal impairment or ESRD) and for patients also taking strong cytochrome P450 3A4 / 5 (CYP3A4 / 5) inhibitors (e.g., ketoconazole).

[0164] Alogliptin is a DPP-4 inhibitor FDA approved for the treatment of patients with T2D (under brand name NESINA®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults). Alogliptin can be administered orally as a tablet at a recommended dose of 25 mg once daily. A dose of 12.5 mg once daily is recommended for patients with moderate renal impairment (creatinine clearance >30 to <60 mL / min) and a dose of 6.25 mg once daily is recommended for patients with severe renal impairment or ESRD (creatinine clearance <30 mL / min).

[0165] Linagliptin is a DPP-4 inhibitor FDA approved for the treatment of patients with T2D (under brand name TRADJENTA®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults). Linagliptin can be administered orally as a tablet at a recommended dose of 5 mg once daily.

[0166] Vildagliptin is a DPP-4 inhibitor for the treatment of patients with T2D (under brand name GALVUS®, e.g., to improve glycemic control as an adjunct to diet and exercise in adults). Vildagliptin can be administered orally as a tablet at a recommended dose of 50 mg twice daily (for a total daily dose of 100 mg).

[0167] Fc domains

[0168] In some embodiments, a polypeptide described herein may include an extracellular ActRII chimera fused to an Fc domain monomer of an immunoglobulin or a fragment of an Fc domain to increase the serum half-life of the polypeptide. A polypeptide including an extracellular ActRII chimera fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which form an Fc domain in the dimer. As conventionally known in the art, an Fc domain is the protein structure that is found at the C-terminus of an immunoglobulin. An Fc domain includes two Fc domain monomers that are dimerized by the interaction between the CH3 antibody constant domains. A wild-type Fc domain forms the minimum structure that binds to an Fc receptor, e.g., FcyRI, FcyRlla, FcyRllb, FcyRllla, FcyRlllb, FcyRIV. In some embodiments, an Fc domain may be mutated to lack effector functions, typical of a “dead” Fc domain. For example, an Fc domain may include specific amino acid substitutions that are known to minimize the interaction between the Fc domain and an Fey receptor. In some embodiments, an Fc domain is from an lgG1 antibody and includes amino acid substitutions L234A, L235A, and G237A. In some embodiments, an Fc domain is from an lgG1 antibody and includes amino acid substitutions D265A, K322A, and N434A. The aforementioned amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 )). The Kabat numbering of amino acid residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard" Kabat numbered sequence. Furthermore, in some embodiments, an Fc domain does not induce any immune system- related response. For example, the Fc domain in a dimer of a polypeptide including an extracellular ActRII chimera fused to an Fc domain monomer may be modified to reduce the interaction or binding between the Fc domain and an Fey receptor. The sequence of an Fc domain monomer that may be fused to an extracellular ActRII chimera is shown below (SEQ ID NO: 48): THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0169] The sequence of a wild-type Fc domain monomer that may be fused to an extracellular ActRII chimera is shown below in SEQ ID NO: 100:

[0170] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0171] In some embodiments, the Fc domain monomer fused to an extracellular ActRII chimera lacks a terminal lysine. An exemplary sequence for a wild-type Fc domain monomer lacking a terminal lysine is provided below (SEQ ID NO: 264):

[0172] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0173] In some embodiments, an Fc domain is from an lgG1 antibody and includes amino acid substitutions L12A, L13A, and G15A, relative to the sequence of SEQ ID NO: 48. In some embodiments, an Fc domain is from an lgG1 antibody and includes amino acid substitutions D43A, K100A, and N212A, relative to the sequence of SEQ ID NO: 48. In some embodiments, the terminal lysine is absent from the Fc domain monomer having the sequence of SEQ ID NO: 48 or SEQ ID NO: 100. In some embodiments, an extracellular ActRII chimera described herein (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) may be fused to the N- or C- terminus of an Fc domain monomer (e.g., SEQ ID NO: 48, SEQ ID NO: 100, or SEQ ID NO: 264) through conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRII chimera and the Fc domain monomer. The Fc domain monomer can be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRII chimera. The Fc domain monomer can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. Additionally, the Fc domain monomer can be an IgG subtype (e.g., lgG1 , lgG2a, lgG2b, lgG3, or lgG4). In some embodiments, the Fc domain monomer is an IgG 1 Fc domain monomer (e.g., a human IgG 1 Fc domain monomer).

[0174] In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. In some embodiments, the Fc domain contains a hinge domain. The Fc domain can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. Additionally, the Fc domain can be an IgG subtype (e.g., IgG 1 , lgG2a, lgG2b, lgG3, or lgG4). The Fc domain can also be a non- naturally occurring Fc domain, e.g., a recombinant Fc domain.

[0175] Methods of engineering Fc domains that have reduced dimerization are known in the art. In some embodiments, one or more amino acids with large sidechains (e.g., tyrosine or tryptophan) may be introduced to the CH3-CH3 dimer interface to hinder dimer formation due to steric clash. In other embodiments, one or more amino acids with small sidechains (e.g., alanine, valine, or threonine) may be introduced to the CH3-CH3 dimer interface to remove favorable interactions. Methods of introducing amino acids with large or small side-chains in the CH3 domain are described in, e.g., Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Publication No. 2006 / 0074225, U.S. Patent Nos. 8,216,805 and 5,731 ,168, Ridgway et al. {Protein Eng. 9:617-612, 1996), Atwell et al. {J Mol Biol. 270:26-35, 1997), and Merchant et al. {Nat Biotechnol. 16:677-681 , 1998), all of which are incorporated herein by reference in their entireties.

[0176] In yet other embodiments, one or more amino acid residues in the CH3 domain that make up the CH3-CH3 interface between two Fc domains are replaced with positively charged amino acid residues (e.g., lysine, arginine, or histidine) or negatively charged amino acid residues (e.g., aspartic acid or glutamic acid) such that the interaction becomes electrostatically unfavorable depending on the specific charged amino acids introduced. Methods of introducing charged amino acids in the CH3 domain to disfavor or prevent dimer formation are described in, e.g., Ying et al. {J Biol Chem. 287:19399-19408, 2012), U.S. Patent Publication Nos. 2006 / 0074225, 2012 / 0244578, and 2014 / 0024111 , all of which are incorporated herein by reference in their entireties.

[0177] In some embodiments of the invention, an Fc domain includes one or more of the following amino acid substitutions: T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351 T, L351 H, L351 N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I, relative to the sequence of human IgG 1 . In some embodiments, the terminal lysine is absent from the Fc domain amino acid sequence. In one particular embodiment, an Fc domain includes the amino acid substitution T366W, relative to the sequence of human IgG 1 .

[0178] Linkers

[0179] A polypeptide described herein may include an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having a sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) fused to an Fc domain monomer by way of a linker. In some embodiments, the Fc domain monomer increases stability of the polypeptide. In the present invention, a linker between an Fc domain monomer (e.g., the sequence of SEQ ID NO: 48, SEQ ID NO: 100, or SEQ ID NO: 264) and an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) can be an amino acid spacer including 1 -200 amino acids. Suitable peptide spacers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 49), GGGS (SEQ ID NO: 50), GGGG (SEQ ID NO: 51 ), GGGGA (SEQ ID NO: 52), GGGGS (SEQ ID NO: 53), GGGGG (SEQ ID NO: 54), GGAG (SEQ ID NO: 55), GGSG (SEQ ID NO: 56), AGGG (SEQ ID NO: 57), or SGGG (SEQ ID NO: 58). In some embodiments, a spacer can contain 2 to 12 amino acids including motifs of GA or GS, e.g., GA, GS, GAGA (SEQ ID NO: 59), GSGS (SEQ ID NO: 60), GAGAGA (SEQ ID NO: 61 ), GSGSGS (SEQ ID NO: 62), GAGAGAGA (SEQ ID NO: 63), GSGSGSGS (SEQ ID NO: 64), GAGAGAGAGA (SEQ ID NO: 65), GSGSGSGSGS (SEQ ID NO: 66), GAGAGAGAGAGA (SEQ ID NO: 67), and GSGSGSGSGSGS (SEQ ID NO: 68). In some embodiments, a spacer can contain 3 to 12 amino acids including motifs of GGA or GGS, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 69), GGSGGS (SEQ ID NO: 70), GGAGGAGGA (SEQ ID NO: 71 ), GGSGGSGGS (SEQ ID NO: 72), GGAGGAGGAGGA (SEQ ID NO: 73), and GGSGGSGGSGGS (SEQ ID NO: 74). In yet some embodiments, a spacer can contain 4 to 12 amino acids including motifs of GGAG (SEQ ID NO: 55), GGSG (SEQ ID NO: 56), e.g., GGAG (SEQ ID NO: 55), GGSG (SEQ ID NO: 56), GGAGGGAG (SEQ ID NO: 75), GGSGGGSG (SEQ ID NO: 76), GGAGGGAGGGAG (SEQ ID NO: 77), and GGSGGGSGGGSG (SEQ ID NO: 78). In some embodiments, a spacer can contain motifs of GGGGA (SEQ ID NO: 52) or GGGGS (SEQ ID NO: 53), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 79) and GGGGSGGGGSGGGGS (SEQ ID NO: 80). In some embodiments of the invention, an amino acid spacer between an Fc domain monomer and an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) may be GGG, GGGA (SEQ ID NO: 49), GGGG (SEQ ID NO: 51 ), GGGAG (SEQ ID NO: 81 ), GGGAGG (SEQ ID NO: 82), or GGGAGGG (SEQ ID NO: 83).

[0180] In some embodiments, a spacer can also contain amino acids other than glycine, alanine, and serine, e.g., AAAL (SEQ ID NO: 84), AAAK (SEQ ID NO: 85), AAAR (SEQ ID NO: 86), EGKSSGSGSESKST (SEQ ID NO: 87), GSAGSAAGSGEF (SEQ ID NO: 88), AEAAAKEAAAKA (SEQ ID NO: 89), KESGSVSSEQLAQFRSLD (SEQ ID NO: 90), GENLYFQSGG (SEQ ID NO: 91 ), SACYCELS (SEQ ID NO: 92), RSIAT (SEQ ID NO: 93), RPACKIPNDLKQKVMNH (SEQ ID NO: 94), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 95), AAANSSIDLISVPVDSR (SEQ ID NO: 96), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 97). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of EAAAK (SEQ ID NO: 98). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of prolinerich sequences such as (XP)n(SEQ ID NO: 267), in which X may be any amino acid (e.g., A, K, or E) and n is from 1 -5, and PAPAP (SEQ ID NO: 99).

[0181] The length of the peptide spacer and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0182] In some embodiments, the linker between an Fc domain monomer (e.g., the sequence of SEQ ID NO: 48, SEQ ID NO: 100, or SEQ ID NO: 264) and an extracellular ActRII chimera described herein (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) is an amino acid spacer having the sequence GGG. For example, a polypeptide for use in the methods described herein can contain an extracellular ActRII chimera (e.g., any one of SEQ ID NOs: 22- 43) fused to an Fc domain monomer (e.g, SEQ ID NO: 100) by a GGG linker. An exemplary polypeptide containing an ActRII chimera of SEQ ID NO: 41 , a GGG linker, and an Fc domain monomer (SEQ ID NO: 100) is provided below (SEQ ID NO: 216): GAILGRSETQECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIEIVKQG CWLDDFNCYDRTDCVEKKDSPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0183] An exemplary polypeptide containing an ActRII chimera of SEQ ID NO: 25, a GGG linker, and an Fc domain monomer (SEQ ID NO: 100) is provided below (SEQ ID NO: 110):

[0184] GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIEIVKQGC WLDDFNCYDRTDCVEKKDSPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0185] An exemplary polypeptide containing an ActRII chimera of SEQ ID NO: 40, a GGG linker, and an Fc domain monomer (SEQ ID NO: 100) is provided below (SEQ ID NO: 215):

[0186] GAILGRAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIEIVKQG CWLDDFNCYDRTDCVEKKDSPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0187] The C-terminal Lys339 of the polypeptide of SEQ ID NO: 216 or SEQ ID NO: 215 (the C-terminal Lys in the Fc region of SEQ ID NO: 216 or SEQ ID NO: 215) and the C-terminal Lys337 of the polypeptide of SEQ ID NO: 110 (the C-terminal Lys in the Fc region of SEQ ID NO: 110) may or may not be present, without affecting the structure or stability of the polypeptide. The disclosure specifically contemplates SEQ ID NOs: 216 and 215 that do not include the C-terminal Lys corresponding to Lys339 and SEQ ID NO: 110 that does not include the C-terminal Lys corresponding to Lys337. The polypeptides of SEQ ID NO: 216 and SEQ ID NO: 215 may be expressed including a C-terminal Lys339 which then may be proteolytically cleaved upon expression of the polypeptide, and the polypeptide of SEQ ID NO: 110 may be expressed including a C-terminal Lys337 which then may be proteolytically cleaved upon expression of the polypeptide (e.g., the polypeptides of SEQ ID NOs: 216, 215, and 110 are expressed using nucleic acid constructs encoding the polypeptide including a C-terminal lysine residue). The polypeptides of SEQ ID NOs: 216 and 215 and SEQ ID NO: 110 may also be expressed without including the C-terminal Lys339 and the C-terminal Lys337, respectively. Vectors, host cells, and protein production

[0188] The polypeptides described herein can be produced from a host cell. A host cell refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. The nucleic acids may be included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, or the like). The choice of nucleic acid vectors depends in part on the host cells to be used. Generally, preferred host cells are of either eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0189] Nucleic acid vector construction and host cells

[0190] A nucleic acid sequence encoding the amino acid sequence of a polypeptide described herein may be prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, ligation, and overlap extension PCR. A nucleic acid molecule encoding a polypeptide described herein may be obtained using standard techniques, e.g., gene synthesis. Alternatively, a nucleic acid molecule encoding a wild-type extracellular ActRIIA or ActRIIB may be mutated to include specific amino acid substitutions using standard techniques in the art, e.g., QuikChange™ mutagenesis. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.

[0191] A nucleic acid sequence encoding a polypeptide described herein may be inserted into a vector capable of replicating and expressing the nucleic acid molecule in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purpose of the invention. Each vector may include various components that may be adjusted and optimized for compatibility with the particular host cell. For example, the vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, the nucleic acid sequence encoding protein of interest, and a transcription termination sequence.

[0192] In some embodiments, mammalian cells may be used as host cells for the invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coli cells may also be used as host cells for the invention. Examples of E. co / / strains include, but are not limited to, E. coli 294 (ATCC®31 ,446), E. coli A 1776 (ATCC®31 ,537, E. coli BL21 (DE3) (ATCC® BAA- 1025), and E. coli RV308 (ATCC®31 ,608). Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of protein products (e.g., glycosylation). Appropriate cell lines or host systems may be chosen to ensure the correct modification and processing of the polypeptide expressed. The above-described expression vectors may be introduced into appropriate host cells using conventional techniques in the art, e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vectors are introduced into host cells for protein production, host cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Methods for expression of therapeutic proteins are known in the art, see, for example, Paulina Baibas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012.

[0193] Protein production, recovery, and purification

[0194] Host cells used to produce the polypeptides described herein may be grown in media known in the art and suitable for culturing of the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as a selection agent, e.g., ampicillin. Host cells are cultured at suitable temperatures, such as from about 20 °C to about 39 °C, e.g., from 25 °C to about 37 °C, preferably 37 °C, and CO2 levels, such as 5 to 10%. The pH of the medium is generally from about 6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter.

[0195] In some embodiments, depending on the expression vector and the host cells used, the expressed protein may be secreted from the host cells (e.g., mammalian host cells) into the cell culture media. Protein recovery may involve filtering the cell culture media to remove cell debris. The proteins may be further purified. A polypeptide described herein may be purified by any method known in the art of protein purification, for example, by chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. For example, the protein can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column (e.g., POROS Protein A chromatography) with chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultra filtration, salting-out and dialysis procedures.

[0196] In other embodiments, host cells may be disrupted, e.g., by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are disrupted, cell debris may be removed by centrifugation or filtration. In some instances, a polypeptide can be conjugated to marker sequences, such as a peptide to facilitate purification. An example of a marker amino acid sequence is a hexa-histidine peptide (His- tag), which binds to nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).

[0197] Alternatively, the polypeptides described herein can be produced by the cells of a subject (e.g., a human), e.g., in the context of gene therapy, by administrating a vector (such as a viral vector (e.g., a retroviral vector, adenoviral vector, poxviral vector (e.g., vaccinia viral vector, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vector, and alphaviral vector)) containing a nucleic acid molecule encoding the polypeptide. The vector, once inside a cell of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.) will promote expression of the polypeptide, which is then secreted from the cell. If treatment of a disease or disorder is the desired outcome, no further action may be required. If collection of the protein is desired, blood may be collected from the subject and the protein purified from the blood by methods known in the art.

[0198] Pharmaceutical compositions and preparations

[0199] The invention features pharmaceutical compositions that include the polypeptides described herein (e.g., a polypeptide including an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)). In some embodiments, a pharmaceutical composition of the invention includes a polypeptide including an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) fused to an Fc domain monomer, or a dimer thereof, as the therapeutic protein. In some embodiments, a pharmaceutical composition of the invention including a polypeptide described herein may be used in combination with other agents (e.g., therapeutic biologies and / or small molecules) or compositions in a therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition may include one or more pharmaceutically acceptable carriers or excipients, which can be formulated by methods known to those skilled in the art. In some embodiments, a pharmaceutical composition of the invention includes a nucleic acid molecule (DNA or RNA, e.g., mRNA) encoding a polypeptide described herein, or a vector containing such a nucleic acid molecule.

[0200] Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine, arginine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol. Pharmaceutical compositions of the invention can be administered parenterally in the form of an injectable formulation. Pharmaceutical compositions for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), a-Modified Eagles Medium (a-MEM), F-12 medium). Formulation methods are known in the art, see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd ed.) Taylor & Francis Group, CRC Press (2015).

[0201] The pharmaceutical compositions may be prepared in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule. The pharmaceutical compositions of the invention may also be prepared in other drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Such techniques are described in Remington: The Science and Practice of Pharmacy 22ndedition (2012). The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0202] The pharmaceutical compositions may also be prepared as a sustained-release formulation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptides described herein. Examples of sustained release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and y ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™, and poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations enable release of molecules over a few months, e.g., one to six months, while other formulations release pharmaceutical compositions of the invention for shorter time periods, e.g., days to weeks.

[0203] The pharmaceutical composition may be formed in a unit dose form as needed. The amount of active component, e.g., a polypeptide described herein, included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided (e.g., a dose within the range of 0.01 - 100 mg / kg of body weight).

[0204] The pharmaceutical composition for gene therapy can be in an acceptable diluent, or can include a slow-release matrix in which the gene delivery vehicle is imbedded. If hydrodynamic injection is used as the delivery method, the pharmaceutical composition containing a nucleic acid molecule encoding a polypeptide described herein or a vector (e.g., a viral vector) containing the nucleic acid molecule is delivered rapidly in a large fluid volume intravenously. Vectors that may be used as in vivo gene delivery vehicle include, but are not limited to, retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vaccinia viral vectors, such as Modified Vaccinia Ankara), adeno-associated viral vectors, and alphaviral vectors.

[0205] Routes, dosage, and administration

[0206] Pharmaceutical compositions that include the polypeptides described herein as the therapeutic proteins may be formulated for, e.g., intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intra-arterial administration, intrathecal administration, or intraperitoneal administration. The pharmaceutical composition may also be formulated for, or administered via, oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, various effective pharmaceutical carriers are known in the art. See, e.g., ASHP Handbook on Injectable Drugs, Toissel, 18th ed. (2014).

[0207] In some embodiments, a pharmaceutical composition that includes a nucleic acid molecule encoding a polypeptide described herein or a vector containing such nucleic acid molecule may be administered by way of gene delivery. Methods of gene delivery are well-known to one of skill in the art. Vectors that may be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vaccinia viral vectors, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vectors, and alphaviral vectors. In some embodiments, mRNA molecules encoding polypeptides described herein may be administered directly to a subject.

[0208] In some embodiments of the present invention, nucleic acid molecules encoding a polypeptide described herein or vectors containing such nucleic acid molecules may be administered using a hydrodynamic injection platform. In the hydrodynamic injection method, a nucleic acid molecule encoding a polypeptide described herein is put under the control of a strong promoter in an engineered plasmid (e.g., a viral plasmid). The plasmid is often delivered rapidly in a large fluid volume intravenously. Hydrodynamic injection uses controlled hydrodynamic pressure in veins to enhance cell permeability such that the elevated pressure from the rapid injection of the large fluid volume results in fluid and plasmid extravasation from the vein. The expression of the nucleic acid molecule is driven primarily by the liver. In mice, hydrodynamic injection is often performed by injection of the plasmid into the tail vein. In certain embodiments, mRNA molecules encoding a polypeptide described herein may be administered using hydrodynamic injection.

[0209] The dosage of the pharmaceutical compositions of the invention depends on factors including the route of administration, the disease to be treated, and physical characteristics, e.g., age, weight, general health, of the subject. A pharmaceutical composition may include a dosage of a polypeptide described herein ranging from 0.01 to 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 1 .25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg) and, in a more specific embodiment, about 0.1 to about 30 mg / kg and, in a more specific embodiment, about 0.3 to about 30 mg / kg. The dosage may be adapted by the physician in accordance with conventional factors such as the extent of the disease and different parameters of the subject.

[0210] The pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The pharmaceutical compositions are administered in a variety of dosage forms, e.g., intravenous dosage forms, subcutaneous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug release capsules). Generally, therapeutic proteins are dosed at 0.1 -100 mg / kg, e.g., 0.5-50 mg / kg. Pharmaceutical compositions that include a polypeptide described herein may be administered to a subject in need thereof, for example, one or more times (e.g., 1 -10 times or more) daily, weekly, biweekly, every four weeks, monthly, bimonthly, quarterly, biannually, annually, or as medically necessary. In some embodiments, pharmaceutical compositions that include a polypeptide described herein may be administered to a subject in need thereof weekly, biweekly, every four weeks, monthly, bimonthly, or quarterly. Dosages may be provided in either a single or multiple dosage regimens. The timing between administrations may increase as the medical condition improves or decrease as the health of the patient declines.

[0211] Methods of treatment

[0212] The extracellular ActRII chimeras described herein have improved properties (e.g., improved ligand binding properties) compared to wild-type extracellular ActRIIA and ActRIIB. The ActRII chimeras generated by combining extracellular portions of ActRIIA and ActRIIB may possess beneficial properties of both ActRIIB (e.g., an ability to increase muscle mass and strong binding affinity to activins A and B) and ActRIIA (e.g., reduced binding affinity to BMP9 and / or longer serum half-life as an Fc fusion protein (e.g., compared to ActRIIB-Fc)). As the ActRII chimeras contain extracellular portions of ActRIIA and ActRIIB, they will be soluble and able to compete with endogenous activin receptors by binding to and sequestering ligands (e.g., activins A and B, myostatin, GDF1 1 ) without activating intracellular signaling pathways. Based on the discovery that administration of a polypeptide including an extracellular ActRII chimera described herein (an ActRII chimera-Fc polypeptide) to obese mice alone or in combination with semaglutide resulted in an increase in lean mass, an increase in muscle weight, a decrease in fat mass, and a decrease in inguinal white adipose tissue and administration of the ActRII chimera-Fc polypeptide in combination with semaglutide reversed the loss of lean mass observed with semaglutide alone and further reduced fat mass (resulting in fat loss) and inguinal white adipose tissue compared to administration of semaglutide or the ActRII chimera-Fc alone, polypeptides containing an ActRII chimera described herein can be used to treat subjects who are currently undergoing or previously underwent treatment with an incretin-based therapy (e.g., treatment with semaglutide or another incretin mimetic) to prevent or reduce lean mass (muscle) loss (e.g., muscle loss associated with the incretin-based therapy), increase lean mass, and / or further reduce fat mass (i.e., increase fat loss).

[0213] A polypeptide including an ActRII chimera described herein (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43), e.g., an effective amount of an ActRII chimera) can be used to treat a subject who is undergoing treatment with an incretinbased therapy or a subject who has previously been treated with an incretin-based therapy (i.e., the polypeptide including an ActRII chimera can be administered in combination with the incretin-based therapy or may be administered after treatment with the incretin-based therapy has ceased, e.g., as a replacement therapy). In some embodiments, the subject has T2D or is undergoing or was previously treated with an incretin-based therapy for T2D. In some embodiments, the subject has a BMI of 30 kg / m2or greater (e.g., a BMI of 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, or greater) or is undergoing or was previously treated with an incretin-based therapy for having a BMI of 30 kg / m2or greater. In some embodiments, the subject has a BMI of 27 kg / m2or greater (e.g., a BMI of 27 or greater and less than 30, such as a BMI of 27, 28, or 29) and a weight-related comorbid condition (e.g. hypertension, type 2 diabetes mellitus, dyslipidemia, obstructive sleep apnea, or cardiovascular disease) or is undergoing or was previously treated with an incretin-based therapy for having a BMI of 27 kg / m2or greater and a weight-related comorbid condition. In some embodiments, treatment with the incretin-based therapy and the polypeptide including an extracellular ActRII chimera is started concurrently (e.g., the subject begins treatment with both agents at approximately the same time, e.g., begins treatment with both agents during the same day, week, or month). In some embodiments, the subject has been receiving treatment with the incretin-based therapy for at least four weeks (e.g., 4 weeks or longer, such as 4, 5, 6, 7 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or more weeks, or 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more months) prior to co-administration of a polypeptide including an ActRII chimera described herein. In some embodiments, the subject has been receiving treatment with the incretin-based therapy for at least eight weeks and has been on a stable dose of the incretin-based therapy for at least 4 weeks (e.g., 4 weeks or longer, such as 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or more weeks, or 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more months) prior to co-administration of a polypeptide including an ActRII chimera described herein. In some embodiments, the subject has finished treatment with the incretin-based therapy for at least one week before starting treatment with a polypeptide including an ActRII chimera described herein (e.g., the subject finished taking the incretin-based therapy 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or more weeks, or 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more months before beginning treatment with a polypeptide including an ActRII chimera described herein). The method can further include evaluating body weight, lean mass, muscle mass, fat mass, and / or body fat after administration of a polypeptide including an extracellular ActRII chimera described herein (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 1 , 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 months or more after the start of treatment with a polypeptide including an ActRII chimera described herein). In some embodiments, the incretin-based therapy is an incretin mimetic (e.g., exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, tirzepatide, albiglutide, or efpeglenatide). In some embodiments, the incretin-based therapy is an incretin enhancer (e.g., sitagliptin, saxagliptin, alogliptin, linagliptin, or vildagliptin).

[0214] Administration of a polypeptide including an ActRII chimera described herein can maintain or increase lean mass (e.g., muscle mass, such as skeletal muscle mass) in a subject undergoing treatment with or previously treated with an incretin-based therapy or can prevent or reduce lean mass loss (e.g., muscle loss, such as skeletal muscle loss) in a subject undergoing treatment with an incretin-based therapy (e.g., lean mass loss (such as muscle loss) associated with incretin mimetic therapy). The polypeptide including an ActRII chimera may increase lean mass or reduce lean mass loss as compared to baseline measurements prior to treatment initiation with the polypeptide including an ActRII chimera or as compared to treatment with an incretin-based therapy alone. In some embodiments, administration of a polypeptide including an ActRII chimera described herein reduces fat mass (e.g., reduces body fat or body fat percentage, i.e., increases fat loss) or reduces white adipose tissue (e.g., inguinal white adipose tissue) mass. The polypeptide including an ActRII chimera may reduce fat mass or white adipose tissue mass as compared to baseline measurements prior to treatment initiation with the polypeptide including an ActRII chimera or as compared to treatment with an incretin-based therapy alone. In some embodiments, co-administration of the polypeptide including an ActRII chimera and the incretin-based therapy (e.g., an incretin mimetic) leads to a greater reduction in fat mass (i.e., greater fat loss) or white adipose tissue mass compared to administration of either agent alone. In some embodiments, coadministration of the polypeptide including an ActRII chimera and the incretin-based therapy (e.g., an incretin mimetic) leads to a reduction in body weight while preserving or increasing lean mass and reducing fat mass. In some embodiments, co-administration of the polypeptide including an ActRII chimera and the incretin-based therapy (e.g., an incretin mimetic) leads to a maintenance of body weight or an increase in body weight while reducing fat mass.

[0215] In some embodiments, the methods described herein (e.g., co-administration of an incretin-based therapy and a polypeptide including an ActRII chimera or administration of a polypeptide including an ActRII chimera to a subject who previously underwent treatment with an incretin-based therapy) improve glycemic control (e.g., in subjects with T2D), reduce the risk of major adverse cardiovascular events (e.g., in subjects with T2D and established cardiovascular disease or multiple cardiovascular risk factors), prevent or reduce the development of frailty, prevent or reduce the premature onset of sarcopenia, reduce the likelihood of the premature onset of sarcopenia, improve insulin sensitivity, improve cardiac function or cardiac health, increase fat metabolism, increase energy expenditure, improve (i.e., reduce) insulin resistance, increase lean mass, increase skeletal muscle, reduce fat gain, reduce body fat (e.g., amount of body fat or body fat percentage, reduce the amount of subcutaneous, visceral, and / or hepatic fat, reduce adiposity, or reduce the weights of epididymal and perirenal fat pads), reduce body weight or body weight gain (e.g., reduce the percentage of body weight gain), reduce fasting insulin levels, reduce blood glucose levels (e.g., fasting glucose levels), increase glucose clearance, reduce LDL, reduce triglycerides, improve serum lipid profile, reduce the risk of sarcopenia, or reduce weight regain after discontinuation of the incretin-based therapy (e.g., after discontinuation of an incretin mimetic therapy, such as treatment with a GLP-1 receptor agonist). The methods described herein may improve glycemic control, reduce the risk of major adverse cardiovascular events, prevent or reduce the development of frailty, prevent or reduce the premature onset of sarcopenia, reduce the likelihood of the premature onset of sarcopenia, improve insulin sensitivity, improve cardiac function or cardiac health, increase fat metabolism, increase energy expenditure, improve insulin resistance, increase lean mass, increase skeletal muscle, reduce body fat, reduce body weight or body weight gain, reduce fasting insulin levels, reduce blood glucose levels, increase glucose clearance, reduce LDL, reduce triglycerides, improve serum lipid profile, reduce the risk of sarcopenia, or reduce weight regain after discontinuation of the incretin-based therapy compared to measurements obtained prior to treatment (e.g., treatment with the combination of the incretin-based therapy and the polypeptide including an ActRII chimera or treatment with the polypeptide including an ActRII chimera for subjects who have ceased treatment with the incretin-based therapy) or compared to measurements obtained from subjects treated with an incretinbased therapy or a polypeptide including an extracellular ActRII chimera alone. The method can further include evaluating glycemic control, major adverse cardiovascular events, frailty, risk or onset of sarcopenia, insulin sensitivity, cardiac function or cardiac health, fat metabolism, energy expenditure, insulin resistance, lean mass, skeletal muscle, body fat, body weight or body weight gain, fasting insulin levels, blood glucose levels, glucose clearance, LDL, triglycerides, serum lipid profile, or weight regain after administration of a polypeptide including an extracellular ActRII chimera described herein (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 1 , 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 months or more after the start of treatment with a polypeptide including an ActRII chimera described herein).

[0216] In some embodiments, the methods described herein do not cause any vascular complications in the subject, such as increased vascular permeability or leakage.

[0217] In any of the methods described herein, a polypeptide including an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) that further includes a C-terminal extension of one to six amino acids (e.g., 1 , 2, 3, 4, 5, 6 or more amino acids from extracellular ActRIIA or ActRIIB) may be used as the therapeutic protein. In any of the methods described herein, a dimer (e.g., homodimer or heterodimer) formed by the interaction of two Fc domain monomers that are each fused to a polypeptide including an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) may be used as the therapeutic protein. In any of the methods described herein, a polypeptide including an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43)) fused to an Fc domain monomer may be used as the therapeutic protein. Nucleic acids encoding the polypeptides described herein, or vectors containing said nucleic acids can also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptide, nucleic acid, or vector can be administered as part of a pharmaceutical composition.

[0218] Compositions that can be administered to a subject according to the methods described herein are provided in Table 5, below.

[0219] Combination therapy

[0220] A polypeptide including an extracellular ActRII chimera described herein may be administered to the subject in combination with an incretin-based therapy. The incretin-based therapy may be, e.g., an incretin mimetic, such as semaglutide (e.g., OZEMPIC®, RYBELSUS®, or WEGOVY®), dulag lutide (e.g., TRULICITY®), tirzepatide (e.g., MOUNJARO® or ZEPBOUND™), albiglutide (e.g., TANZEUM®), exenatide (e.g., BYETTA™ or BYDUREON BCise®), lixisenatide (e.g., ADLYXIN®), liraglutide (e.g., VICTOZA® or SAXENDA®), or efpeglenatide, or an incretin enhancer, such as sitagliptin (e.g., JANUVIA®), saxagliptin (e.g., ONGLYZA®), alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®), or vildagliptin (e.g., GALVUS®). The incretin-based therapy may be administered at the same time (e.g., administration of all agents occurs within 15 minutes, 10 minutes, 5 minutes, 2 minutes or less) as the polypeptide including an extracellular ActRI I chimera. The agents can also be administered simultaneously via co-formulation. The polypeptide including an extracellular ActRII chimera and the incretin-based therapy can also be administered sequentially, such that the action of the two overlaps and their combined effect is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the polypeptide including an extracellular ActRII chimera and the incretin-based therapy can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each of the polypeptide including an extracellular ActRII chimera and the incretin-based therapy treatment can be performed by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, local routes, and direct absorption through mucous membrane tissues. The polypeptide including an extracellular ActRII chimera and the incretin-based therapy can be administered by the same route or by different routes. For example, a polypeptide including an extracellular ActRII chimera may be administered by subcutaneous injection while the incretin-based therapy can be administered orally (for sitagliptin, saxagliptin, alogliptin, linagliptin, vildagliptin, or semaglutide (e.g., RYBELSUS®)) or by subcutaneous injection (for semaglutide (e.g., OZEMPIC® or WEGOVY®), dulaglutide, tirzepatide, albiglutide, exenatide, lixisenatide, liraglutide, or efpeglenatide). The polypeptide including an extracellular ActRII chimera may be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours or up to 1 -7, 1 -14, 1 -21 or 1 -30 days before or after the incretinbased therapy. In some embodiments, the polypeptide including an extracellular ActRII chimera and the incretin-based therapy are administered at different frequencies. For example, the polypeptide including an extracellular ActRII chimera can be administered once a week, once every two weeks, once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months and the incretin-based therapy can be administered once or twice daily (e.g., for exenatide (e.g., BYETTA™), lixisenatide (e.g., ADLYXIN®), semaglutide (e.g., RYBELSUS®), sitagliptin (e.g., JANUVIA®), saxagliptin (e.g., ONGLYZA®), alogliptin (e.g., NESINA®), linagliptin (e.g., TRADJENTA®), or vildagliptin (e.g., GALVUS®). In some embodiments, the polypeptide including an extracellular ActRII chimera and the incretin-based therapy are administered at the same or at similar frequencies. For example, both the polypeptide including an extracellular ActRII chimera and the incretin-based therapy can be administered once a week, once every two weeks, once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months (e.g., when the incretin-based therapy is exenatide (e.g., BYDUREON BCise®), liraglutide (e.g., VICTOZA® or SAXENDA®), semaglutide (e.g., OZEMPIC® or WEGOVY®), dulaglutide (e.g., TRULICITY®), tirzepatide (e.g., MOUNJARO® or ZEPBOUND™), albiglutide (e.g., TANZEUM®), or efpeglenatide). In a particular embodiment of the combination therapy, the polypeptide including an extracellular ActRII chimera and the incretin-based therapy are both administered by subcutaneous injection once a week (e.g., when the incretin-based therapy is exenatide (e.g., BYDUREON BCise®), liraglutide (e.g., VICTOZA® or SAXENDA®), semaglutide (e.g., OZEMPIC® or WEGOVY®), dulaglutide (e.g., TRULICITY®), tirzepatide (e.g., MOUNJARO® or ZEPBOUND™), albiglutide (e.g., TANZEUM®), or efpeglenatide). In this embodiment, the polypeptide including an extracellular ActRII chimera is administered at a dose of 1 mg / kg or less (e.g., 1 .0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg / kg or less).

[0221] In an alternative embodiment, the polypeptide including an extracellular ActRII chimera is administered after the subject has ceased treatment with the incretin-based therapy (e.g., the polypeptide including an extracellular ActRII chimera is administered as a replacement therapy). In this embodiment, the polypeptide including an extracellular ActRII chimera is administered by subcutaneous injection once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months at a dose of 1 mg / kg or more (e.g., 1 .0, 1 .25, 1 .5, 1 .75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5 mg / kg or more).

[0222] In some embodiments, the incretin-based therapy is administered as indicated on the label. For example, exenatide can be administered by subcutaneous injection at an initial dose of 5 micrograms (mcg) twice daily, with an increase to 10 mcg twice daily after one month based on clinical response or at 2 milligrams (mg) once every seven days (weekly) for the extended-release form. Lixisenatide can be administered by subcutaneous injection at an initial dose of 10 mcg once daily for 14 days, with a dosage increase on day 15 to 20 mcg once daily. Liraglutide can be administered by subcutaneous injection at an initial dose of 0.6 mg once daily for one week, followed by an increase to 1 .2 mg daily, with an additional increase to 1 .8 mg daily after one week of treatment with the 1 .2 mg daily dose if additional glycemic control is required (e.g., for T2D) or at an initial dose of 0.6 mg per day for one week, with weekly dose increases until a dose of 3 mg daily is reached (e.g., for chronic weight management, recommended dosage regimen is 0.6 mg for week 1 , 1 .2 mg for week 2, 1 .8 mg for week 3, 2.4 mg for week 4 and maintenance at 3 mg for week 5 and onward). Semaglutide can be administered by subcutaneous injection at an initial dose of 0.25 mg once weekly that is increased to 0.5 mg once weekly after four weeks. If additional glycemic control is needed, the dosage can be increased to 1 mg once weekly after at least four weeks on the 0.5 mg dose, and if additional glycemic control is needed, the dosage can be increased to 2 mg once weekly after at least four weeks on the 1 mg dosage (e.g., for T2D). Alternatively, semaglutide can be administered by subcutaneous injection at an initial dose of 0.25 mg once weekly for four weeks, with dose increases in four week intervals until a dose of 2.4 mg is reached (e.g., for chronic weight management, recommended dosage regimen is 0.25 mg for weeks 1 -4, 0.5 mg for weeks 5-8, 1 mg for weeks 9-12, 1 .7 mg for weeks 13-16, and maintenance at 1 .7 mg or 2.4 mg for week 17 and onward). The maintenance dose is 2.4 mg or 1 .7 mg once weekly. When administered orally as a tablet, semaglutide can be administered at an initial dose of 3 mg once daily for 30 days. After 30 days on the 3 mg dosage, the dosage is increased to 7 mg once daily. If additional glycemic control is needed, the dosage may be increased to 14 mg once daily after at least 30 days on the 7 mg dosage (e.g., for T2D). Dulaglutide can be administered by subcutaneous injection at an initial dose of 0.75 mg once weekly, which can be increased to 1 .5 mg once weekly for additional glycemic control (for adult and pediatric patients), and further increased in 1 .5 mg increments after at least four weeks on the current dosage if additional glycemic control is needed, up to a maximum recommended dosage of 4.5 mg once weekly (for adult patients). Tirzepatide can be administered by subcutaneous injection at a recommended starting dosage of 2.5 mg once weekly, with an increase to 5 mg once weekly after four weeks. If additional glycemic control is needed, the dosage can be increased in 2.5 mg increments after at least four weeks on the current dose, up to a maximum dosage of 15 mg once weekly (e.g., for T2D). For chronic weight management, the dosage can be increased in 2.5 mg increments after at least four weeks on the current dose, and the recommended maintenance dosages are 5 mg, 10 mg, or 15 mg once weekly, with a maximum dosage of 15 mg once weekly. Albiglutide can be administered by subcutaneous injection at an initial dose of 30 mg once weekly, which can be increased to 50 mg once weekly in patients requiring additional glycemic control. Efpeglenatide can be administered by subcutaneous injection once weekly at 2, 4, or 6 mg. Sitagliptin can be administered orally as a tablet at a recommended dose of 100 mg once daily, which can be adjusted to 50 mg once daily for subjects with an eGFR greater than or equal to 30 mL / min / 1 .73 m2to less than 45 mL / min / 1 .73 m2or 25 mg once daily for subjects with an eGFR less than 30 mL / min / 1 .73 m2(including patients with end stage renal disease (ESRD) on dialysis). Saxagliptin can be administered orally as a tablet at a recommended dose of 2.5 mg or 5 mg once daily. A dose of 2.5 mg once daily is recommended for patients with eGFR less than 45 mL / min / 1 .73 m2(with moderate or severe renal impairment or ESRD) and for patients also taking strong cytochrome P450 3A4 / 5 (CYP3A4 / 5) inhibitors (e.g., ketoconazole). Alogliptin can be administered orally as a tablet at a recommended dose of 25 mg once daily. A dose of 12.5 mg once daily is recommended for patients with moderate renal impairment (creatinine clearance >30 to <60 mL / min) and a dose of 6.25 mg once daily is recommended for patients with severe renal impairment or ESRD (creatinine clearance <30 mL / min). Linagliptin can be administered orally as a tablet at 5 mg once daily. Vildagliptin can be administered orally as a tablet at 50 mg twice daily (for a total of 100 mg per day). The polypeptide including an extracellular ActRII chimera can be administered by subcutaneous injection at a dose of from about 0.01 to about 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg) and, in a more specific embodiment, about 0.1 to about 30 mg / kg and, in a more specific embodiment, about 0.3 to about 30 mg / kg, once a week, once every two weeks, once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months, or once a year.

[0223] In some embodiments, combination therapy with a polypeptide including an ActRII chimera and an incretin-based therapy maintains or increases lean mass (e.g., muscle mass, such as skeletal muscle mass) or prevents or reduces lean mass loss (e.g., lean mass loss, such as skeletal muscle loss, associated with incretin mimetic therapy). This may prevent or reduce the development of frailty in subjects treated with an incretin-based therapy (e.g., an incretin mimetic) and / or prevent or reduce the premature onset of sarcopenia or reduce the risk of sarcopenia in subjects treated with an incretin-based therapy (e.g., an incretin mimetic). In some embodiments, combination therapy reduces fat mass (e.g., reduces body fat or body fat percentage, i.e. , induces or increases fat loss) or reduces white adipose tissue (e.g., inguinal white adipose tissue) mass. In some embodiments, combination therapy reduces the risk of major adverse cardiovascular events (e.g., in subjects with T2D and established cardiovascular disease or multiple cardiovascular risk factors) or improves cardiac function or cardiac health. In some embodiments, combination therapy increases fat metabolism, increases energy expenditure, improves (i.e., reduces) insulin resistance, or improves insulin sensitivity. In some embodiments, combination therapy improves glycemic control (e.g., in subjects with T2D). In some embodiments, combination therapy reduces weight regain after discontinuation of the incretin-based therapy (e.g., discontinuation of an incretin mimetic therapy, such as treatment with a GLP-1 receptor agonist).

[0224] Kits

[0225] A polypeptide including an ActRII chimera and an incretin-based therapy described herein can be provided in a kit for use in treating a subject with T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2or greater and a weight-related comorbid condition (e.g. hypertension, type 2 diabetes mellitus, dyslipidemia, obstructive sleep apnea, or cardiovascular disease). Each agent may be provided in unit dosage form, optionally in a pharmaceutically acceptable excipient (e.g., saline), in an amount sufficient to treat the subject. The kit can further include a package insert that instructs a user of the kit, such as a physician, to perform the methods described herein. The kit may optionally include a syringe or other device for administering the polypeptide including an ActRII chimera or incretin-based therapy.

[0226] Table 5

[0227] Examples

[0228] The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0229] Example 1 -Effect of an extracellular ActRII chimera alone or in combination with Serna in a mouse model of obesity Diet induced obese, 18-week-old, male C57BI / 6 mice were dosed twice weekly with either Tris- buffered-saline (Vehicle) and Vehicle (n=10), Semaglutide (Serna) and Vehicle (n=10), Chimera 2 I65F E81 Q-mFc (a construct including SEQ ID NO: 25 fused to a mouse Fc domain monomer by a GGG linker in the form of a homodimer) and Vehicle (n=10), Chimera 1 / 2b-mFc (a construct including SEQ ID NO: 41 fused to a mouse Fc domain monomer by a GGG linker in the form of a homodimer) and Vehicle (n=10), or Serna in combination with Chimera 2 I65F E81 Q-mFc (n=10). Serna was administered by subcutaneous injection at a dose of 0.041 mg / kg, Chimera 2 I65F E81 Q-mFc was administered by intraperitoneal injection at a dose of 10 mg / kg, and Chimera 1 / 2b-mFc was administered by intraperitoneal injection at a dose of 10 mg / kg.

[0230] All test articles were administered twice weekly and body mass measured concurrent to this. Body mass results are shown in FIGS. 1 A-1 B. Data are shown as absolute body weight in grams (FIG.

[0231] 1 A) and as a percentage of body weight change from baseline measurements (FIG. 1 B). Data are shown as mean + SEM.

[0232] On day 27 of the study, lean mass and fat mass were determined through the use of nuclear magnetic resonance (NMR) body composition analysis (Bruker Minispec). Lean mass results for Chimera

[0233] 2 I65F E81 Q-mFc and Chimera 1 / 2b-mFc treatment are shown in FIG. 2. These data indicate that administration of either Chimera 2 I65F E81 Q-mFc or Chimera 1 / 2b-mFc (10 mg / kg, twice weekly for four weeks) in obese mice on a high calorie diet resulted in equivalent increases in lean mass. Data are shown as a percent of lean mass change from baseline measurements. Data are shown as mean + SEM. Statistics are shown using 1 -way ANOVA with a Dunnett posttest, **** p<0.0001 .

[0234] Lean mass and fat mass results for Chimera 2 I65F E81 Q-mFc alone and in combination with Serna are shown in FIGS. 3A-3D. Data are shown as a percent of lean mass or fat mass change from baseline measurements (FIGS. 3A and 3B) or as a percent of lean mass or fat mass change from baseline measurements compared to vehicle (FIGS. 3C and 3D). These data show that obese mice treated with Serna lost lean mass and that administration Chimera 2 I65F E81 Q-mFc did not only preserve, but also increased lean mass in Sema-treated obese mice, ameliorating the loss of lean mass observed in Sema-treated obese mice. The observed increase in lean mass with combination therapy (0.041 mg / kg Serna and 10 mg / kg Chimera 2 I65F E81 Q-mFc) was equivalent to Chimera 2 I65F E81 Q- mFc treatment alone. In addition, these data show that obese mice on a high calorie diet continued to gain weight and fat mass, and that treatment with Serna (0.041 mg / kg twice weekly) resulted in reduced fat gain compared to untreated obese mice, treatment with Chimera 2 I65F E81 Q-mFc (10 mg / kg twice weekly) demonstrated reduced fat gain equivalent to Serna, and treatment with 10 mg / kg Chimera 2 I65F E81 Q-mFc and 0.041 mg / kg Serna not only reduced the fat gain, but also resulted in a loss of fat mass compared to baseline. Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA with a Dunnett posttest, **p<0.01 ; ****p<0.0001 .

[0235] At study termination (day 29), soleus muscles, tibialis anterior muscles, and inguinal white adipose tissue (IWAT) depots were fine dissected and weighed. Soleus muscle and tibialis anterior muscle results for Chimera 2 I65F E81 Q-mFc alone and in combination with Serna are shown in FIGS. 4A-4B. Data for muscle weights are shown as a percent of vehicle. Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA with a Dunnett posttest, ns=not significant, ***p<0.001 ; ****p<0.0001 . Inguinal white adipose tissue results for Chimera 2 I65F E81 Q-mFc alone and in combination with Serna are shown in FIG. 5. Data are shown as absolute weight in grams. Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA with a Dunnett posttest, *p<0.05.

[0236] In summary, treatment with Chimera 1 / 2b-mFc and Chimera 2 I65F E81 Q-mFc showed similar increases in skeletal muscle. As a single treatment, both Chimera 1 / 2b-mFc and Chimera 2 I65F E81 Q- mFc increased lean (muscle) mass and reduced fat mass. Combination treatment with Chimera 2 I65F E81 Q-mFc and Sema led to a reversal of lean mass loss mediated by Serna and enhanced loss of fat mass through increased energy expenditure from increased lean mass.

[0237] Example 2 -Effect of an extracellular ActRII chimera in combination with Sema in a mouse model of obesity

[0238] To discern the effects of ActRII chimera adjunctive treatment on lean mass loss, diet induced obese, 18-week-old, male C57BI / 6 mice were dosed twice weekly with either Tris-buffered-saline (Vehicle) and Vehicle (n=10), Semaglutide (Sema) and Vehicle (Sema / Veh; n=10), or Sema in combination with Chimera 1 / 2b-mFc (a construct including SEQ ID NO: 41 fused to a mouse Fc domain monomer by a GGG linker in the form of a homodimer) (Sema / Chimera 1 / 2b-mFc; n=10). Sema was administered by subcutaneous injection at a dose of 0.082 mg / kg and Chimera 1 / 2b-mFc was administered by intraperitoneal injection at a dose of 10 mg / kg.

[0239] All test articles were administered twice weekly for 14 days. Body mass was measured twice a week while lean mass and fat mass were assessed via NMR at baseline and study termination after a total of four doses.

[0240] Body mass results are shown in FIG. 6A. Data are shown as a percentage of body weight change from baseline measurements. Data are shown as mean ± SEM.

[0241] Lean mass and fat mass results are shown in FIGS. 6B-6C. Data in FIG. 6B are shown as a percent of lean mass or fat mass change from baseline measurements. Data in FIG. 6C are shown as ratios of lean to fat mass. Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA and Dunnett's multiple comparisons test, *p<0.05, **p<0.01 , ****p<0.0001 .

[0242] Mice dosed with Sema showed a decrease in body mass (FIG. 6A) due to the loss of both lean and fat mass (FIG. 6B). In contrast, mice treated with Sema in combination with Chimera 1 / 2b-mFc showed an increase in lean mass and a significantly greater reduction in fat mass compared to Vehicle or Sema and Vehicle (FIG. 6B), resulting in a more favorable ratio of lean mass to fat mass from a metabolic standpoint (FIG. 6C).

[0243] In summary, Chimera 1 / 2b-mFc mitigated loss of lean mass and augmented loss of fat mass in obese mice treated with semaglutide. Combination treatment of diet induced obese mice with Sema in combination with Chimera 1 / 2b-mFc resulted in a significantly greater loss of fat mass and greater gain of lean mass compared to Sema monotherapy. These data support a combination treatment with Chimera 1 / 2b-mFc in combination with a GLP-1 agonist to augment fat loss and ameliorate GLP-1 agonist- associated loss of lean mass.

[0244] Example 3 - Effect of an extracellular ActRII chimera on lean mass and muscle strength in C57BL / 6 mice compared to other myostatin and activin inhibitors

[0245] To compare in vivo activity of Chimera 1 / 2b-mFc with other investigational therapeutic modalities that inhibit activins and / or myostatin, 14-week-old male C57BL / 6 mice were dosed with Tris-buffered- saline (vehicle, n=10), taldefgrobep alfa (an anti-myostatin adnectin, n=10), bimagrumab (monoclonal antibody (mAb) directed to ActRIIA and ActRIIB, n=10), landogrozumab (mAb directed to myostatin, n=10), or Chimera 1 / 2b-mFc (n=10) at 10 mg / kg twice weekly for 2 weeks by intraperitoneal injection. Mice were weighed twice weekly and quantitative nuclear magnetic resonance imaging (NMR) was used to measure lean mass at baseline and study termination. In order to measure muscle strength, an evoked force apparatus (AURORA SCIENTIFIC®) was used to measure maximum force potential of the gastrocnemius.

[0246] Lean mass results are shown in FIG. 7A. Data are shown as a percent of lean mass change from baseline. Data are shown as mean ± SEM. Statistics are shown using ordinary one-way ANOVA with a Tukey’s post-hoc test, *** p<0.001 , and ****p< 0.0001 .

[0247] Gastrocnemius results are shown in FIG. 7B. Data are shown as a maximum force (mN). Data are shown as mean ± SEM. Statistics are shown using ordinary one-way ANOVA with a Tukey’s post-hoc test, *p< 0.05.

[0248] Chimera 1 / 2b-mFc significantly increased lean mass (FIG. 7A) and muscle function (FIG. 7B) in C57BL / 6 mice compared to other myostatin and activin inhibitors.

[0249] In summary, in lean mice, Chimera 1 / 2b-mFc significantly increased lean mass and muscle strength to a greater extent than other therapeutics targeting myostatin, activin, or signaling through activin type II receptors.

[0250] Example 4 - Effect of an extracellular ActRII chimera in a mouse model of obesity

[0251] Eighteen-week-old male diet induced obese (DIO), C57BL / 6 mice were dosed with Tris-buffered- saline (vehicle, n=10) or Chimera 1 / 2b-mFc (n=10) at 10 mg / kg twice weekly for 4 weeks by intraperitoneal injection.

[0252] Quantitative nuclear magnetic resonance imaging (NMR) was used to measure lean mass and fat mass at baseline and at study termination. Skeletal muscle (tibialis anterior, gastrocnemius, and soleus muscle) was collected upon study termination and weighed. Muscles were collected bilaterally and graphed as a sum where applicable.

[0253] Lean mass results are shown in FIG. 8A. Fat mass results are shown in FIG. 8B. The ratio of fat mass to lean mass is shown in FIG. 8C. Tibialis anterior mass results are shown in FIG. 8D. Gastrocnemius mass results are shown in FIG. 8E. Soleus mass results are shown in FIG. 8F. Data are shown as absolute weight in grams (FIGS. 8A, 8B, and 8D-8F) and as a ratio of fat mass / lean mass (FIG. 8C). Data are shown as mean + SEM. Statistics are shown using an unpaired t-test, *p<0.05, ***p<0.001 , ****p<0.0001.

[0254] Compared to vehicle, Chimera 1 / 2b-mFc treatment resulted in a significant increase in lean mass (FIG. 8A), and no significant change in fat mass (FIG. 8B), resulting in a metabolically favorable decrease in fat to lean mass ratio (FIG. 8C). The observed increase in lean mass was a result of significant increases in individual skeletal muscle groups, as shown in FIGS. 8D-8F.

[0255] In summary, Chimera 1 / 2b-mFc increased lean muscle mass and improved fat to lean ratio in diet induced obese mice.

[0256] Example 5 - Effect of an extracellular ActRII chimera in combination with Serna in a mouse model of obesity

[0257] To evaluate the effect of Chimera 1 / 2b-mFc + semaglutide adjunct therapy in mitigating lean muscle mass loss due to treatment with GLP-1 receptor agonists in diet-induced obese (DIO) mice, 6- month-old, male C57BL / 6 mice were dosed for 14 days with either Tris-buffered-saline (DIO Vehicle; / j n=10), 0.120 mg / kg Semaglutide (Sema) and Vehicle (Sema / Veh; n=10), 0.120 mg / kg Sema in combination with 1 mg / kg Chimera 1 / 2b-mFc (Sema / 1 mg / kg Chimera 1 / 2b-mFc; n=10), or 0.120 mg / kg Sema in combination with 3 mg / kg Chimera 1 / 2b-mFc (Sema I 3 mg / kg Chimera 1 / 2b-mFc; n=15). Sema was administered daily by subcutaneous injection and Chimera 1 / 2b-mFc was administered twice weekly by intraperitoneal injection. Age-matched chow-fed C57 / BL6 mice were used as a control group (Chow vehicle; n=10).

[0258] Mice were weighed daily and quantitative nuclear magnetic resonance imaging (NMR) was used to measure lean mass and fat mass. Test articles were administered starting at study day 7. NMR was done at study day 0, 7, 14, and 21 . Upon study termination fat depots and muscles were collected and weighed.

[0259] Body weight results are shown in FIG. 9A, lean mass results are shown in FIG. 9B, and fat mass results are shown in FIG. 9C. Data are shown as absolute body weight in grams (FIG. 9A) , percent of lean mass change from baseline (FIG. 9B), or percent of fat mass change from baseline (FIG. 9C). Data are shown as mean ± SEM. Statistics are shown using a 2-way ANOVA and Tukey’s multiple comparisons test, *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 .

[0260] Treatment of diet induced obese mice with Sema / Veh resulted in a decrease in body mass (FIG. 9A) due to the loss of both fat and lean mass (FIGS. 9B-C). In contrast, the combination of Sema with Chimera 1 / 2b-mFc showed an increase in lean mass and a significantly greater reduction in fat mass compared to DIO vehicle or Sema / Veh (FIGS. 9B-C).

[0261] Fat mass / lean mass ratio results are shown in FIG. 10A, interscapular brown adipose tissue (iBAT) weight results are shown in FIG. 10B, epididymal white adipose tissue (eWAT) weight results are shown in FIG. 10C, and tibialis anterior weight results are shown in FIG. 10D. Data are shown as fat mass / lean mass ratio (FIG. 10A), or absolute weight in grams (FIGS. 10B-D). Data are shown as mean ± SEM. Statistics are shown using a 2-way ANOVA and Tukey’s multiple comparisons for the fat-to-lean ratio. For lean mass and fat mass, a one-way ANOVA was run with Tukey’s multiple comparisons test, *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 , ns=not significant.

[0262] The combination of Sema with Chimera 1 / 2b-mFc in diet induced obese mice resulted in, from a metabolic standpoint, a more favorable ratio of fat mass to lean mass (FIG. 10A), when compared to baseline. The 3 mg / kg dosed group, when compared to DIO vehicle, resulted in a significant decrease in iBAT (FIG. 10B) suggesting a reduction of white adipose tissue content of this fat pad and a significant decrease in the eWAT (FIG. 10C), which is indicative of a reduction in lipid content. Both doses of the combination of Sema with Chimera 1 / 2b-mFc showed significant increases in the tibialis anterior as compared to Sema / Veh (FIG. 10D).

[0263] In summary, Chimera 1 / 2b-mFc increased lean mass and enhanced fat mass loss in DIO mice treated with semaglutide. Semaglutide + Chimera 1 / 2b-mFc combination therapy of DIO mice resulted in a significantly greater loss of fat mass and preservation of lean mass compared to Sema monotherapy. These data support the use of Chimera 1 / 2b-mFc in combination with a GLP-1 agonist in patients with obesity to augment fat loss and ameliorate GLP-1 agonist-associated loss of lean mass. Example 6 - Treatment of a subject undergoing incretin-based therapy by administration of an extracellular ActRII chimera

[0264] According to the methods disclosed herein, a physician of skill in the art can treat a subject, such as a human patient, receiving an incretin-based therapy (e.g., an incretin mimetic, such as semaglutide or tirzepatide) for T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2and a weight-related comorbid condition so as to increase lean mass (e.g., skeletal muscle mass), reduce lean mass loss (e.g., skeletal muscle loss associated with the incretin-based therapy), reduce fat mass, reduce white adipose tissue mass, increase fat metabolism, increase energy expenditure, improve insulin resistance, improve insulin sensitivity, improve cardiac function or cardiac health, reduce or prevent the development of frailty, reduce or prevent the premature onset of sarcopenia, reduce the risk of sarcopenia, or reduce weight regain after discontinuation of the incretin-based therapy.

[0265] To treat the subject, a physician of skill in the art can administer to the subject a composition containing a polypeptide including an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43), such as an extracellular ActRII chimera fused to an Fc domain monomer by way of a linker, which may be in the form of a homodimer). The composition containing the extracellular ActRII chimera may be administered to the subject, for example, by parenteral injection (e.g., subcutaneous injection) in combination with the incretin-based therapy, which may be administered orally (for sitagliptin, saxagliptin, alogliptin, linagliptin, vildagliptin, or semaglutide (e.g., RYBELSUS®)) or by subcutaneous injection (for semaglutide (e.g., OZEMPIC® or WEGOVY®), dulaglutide, tirzepatide, albiglutide, exenatide, lixisenatide, liraglutide, or efpeglenatide). The polypeptide including an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43), such as an extracellular ActRII chimera fused to an Fc domain monomer, which may be in the form of a homodimer) is administered in a therapeutically effective amount, such as from 0.01 to 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRII chimera is administered bimonthly, once a month, once every four weeks, once every two weeks, or at least once a week or more (e.g., 1 , 2, 3, 4, 5, 6, or 7 times a week or more). In some embodiments, the ActRII chimera is administered at the same frequency as the incretinbased therapy (e.g., both agents are administered once a week). The extracellular ActRII chimera is administered in an amount sufficient to increase lean mass, reduce lean mass loss, reduce fat mass, reduce white adipose tissue mass, increase fat metabolism, increase energy expenditure, improve insulin resistance, improve insulin sensitivity, improve cardiac function or cardiac health, reduce or prevent the development of frailty, reduce or prevent the premature onset of sarcopenia, reduce the risk of sarcopenia, or reduce weight regain after discontinuation of the incretin-based therapy.

[0266] Following administration of the composition to a patient, a practitioner of skill in the art can monitor the patient’s improvement in response to the therapy by a variety of methods. For example, a physician can monitor the patient’s lean mass, fat mass, insulin sensitivity, or cardiac function, using standard clinical tests. A finding that the patient exhibits increased lean mass, reduced fat mass, improved insulin sensitivity, or improved cardiac function following administration of the composition compared to test results prior to administration of the composition indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.

[0267] Example 7 - Treatment of a subject who previously underwent treatment with an incretin-based therapy by administration of an extracellular ActRII chimera

[0268] According to the methods disclosed herein, a physician of skill in the art can treat a subject, such as a human patient, who previously underwent treatment with an incretin-based therapy (e.g., for T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2and a weight-related comorbid condition, such as treatment with an incretin mimetic, e.g., semaglutide or tirzepatide) so as to so as to increase lean mass (e.g., skeletal muscle mass), reduce or prevent the development of frailty, or reduce or prevent the premature onset of sarcopenia, reduce the risk of sarcopenia, or reduce weight regain after discontinuation of the incretin-based therapy. The method of treatment can include diagnosing or identifying a subject as a candidate for treatment by measuring lean mass (e.g., to evaluate whether treatment with the incretin-based therapy led to lean mass loss). To treat the subject, a physician of skill in the art can administer to the subject a composition containing a polypeptide including an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 - 43 (e.g., SEQ ID NOs: 22-43), such as an extracellular ActRII chimera fused to an Fc domain monomer by way of a linker, which may be in the form of a homodimer). The composition containing the extracellular ActRII chimera may be administered to the subject, for example, by parenteral injection (e.g., subcutaneous injection) to increase lean mass. The polypeptide including an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 1 -43 (e.g., SEQ ID NOs: 22-43), such as an extracellular ActRII chimera fused to an Fc domain monomer, which may be in the form of a homodimer) is administered in a therapeutically effective amount, such as from 0.01 to 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRII chimera is administered bimonthly, once a month, once every four weeks, once every two weeks, or at least once a week or more (e.g., 1 , 2, 3, 4, 5, 6, or 7 times a week or more). The extracellular ActRII chimera is administered in an amount sufficient to increase lean mass (e.g., skeletal muscle mass), reduce or prevent the development of frailty, or reduce or prevent the premature onset of sarcopenia, reduce the risk of sarcopenia, or reduce weight regain after discontinuation of the incretin-based therapy.

[0269] Following administration of the composition to a patient, a practitioner of skill in the art can monitor the patient’s improvement in response to the therapy by a variety of methods. For example, a physician can monitor the subject’s lean mass using dual-energy X-ray absorptiometry (DEXA). A finding that the patient’s lean mass is increased following administration of the composition compared to test results prior to administration of the composition indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.

[0270] Other Embodiments

[0271] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0272] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0273] Other embodiments are within the following claims.

Claims

CLAIMS1 . A method of treating type 2 diabetes (T2D), a body mass index (BMI) of 30 kg / m2or greater, or a BMI of 27 kg / m2or greater and a weight-related comorbid condition in a patient undergoing treatment with an incretin-based therapy, the method comprising administering an effective amount of an extracellular activin receptor type II (ActRII) chimera of Table 5.

2. A method of reducing loss of lean mass in a patient undergoing treatment with an incretin-based therapy, the method comprising administering an effective amount of an extracellular ActRII chimera of Table 5.

3. A method of increasing lean mass in a patient who is undergoing or previously underwent treatment with an incretin-based therapy, the method comprising administering an effective amount of an extracellular ActRII chimera of Table 5.

4. The method of claim 2 or 3, wherein the patient is undergoing or previously underwent treatment with an incretin-based therapy for the treatment of T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2and a weight-related comorbid condition.

5. A method of treating a patient having T2D, a BMI of 30 kg / m2or greater, or a BMI of 27 kg / m2and a weight-related comorbid condition, the method comprising administering effective amounts of an incretinbased therapy and an extracellular ActRII chimera of Table 5.

6. The method of claim 5, wherein the incretin-based therapy and the extracellular ActRII chimera are administered together.

7. The method of any one of claims 1 -6, wherein the incretin-based therapy and the extracellular ActRII chimera are administered with the same frequency.

8. The method of claim 7, wherein the extracellular ActRII chimera is administered once per week at a dose of 1 mg / kg or less.

9. The method of claim 5, wherein the incretin-based therapy is administered for a first period and the extracellular ActRII chimera is subsequently administered for a second period.

10. The method of claim 9, wherein the extracellular ActRII chimera is administered once every 28 days or less frequently.11 . The method of any one of claims 1 -10, wherein the patient has T2D or is undergoing or previously underwent treatment with an incretin-based therapy for T2D.

12. The method of any one of claims 1 -10, wherein the patient has a BMI of 30 kg / m2or greater or is undergoing or previously underwent treatment with an incretin-based therapy for a BMI of 30 kg / m2or greater.

13. The method of any one of claims 1 -10, wherein the patient has a BMI of 27 kg / m2or greater and a weight-related comorbid condition or is undergoing or previously underwent treatment with an incretinbased therapy for a BMI of 27 kg / m2or greater and a weight-related comorbid condition.

14. The method of any one of claims 1 -13, wherein the extracellular ActRII chimera is administered before the patient exhibits a reduction in lean mass.

15. The method of any one of claims 1 -13, wherein the extracellular ActRII chimera is administered after the patient exhibits a reduction in lean mass.

16. The method of any one of claims 1 -15, wherein the incretin-based therapy is an incretin mimetic.

17. The method of claim 16, wherein the incretin mimetic is a GLP-1 receptor agonist.

18. The method of claim 17, wherein the GLP-1 receptor agonist is exenatide, lixisenatide, liraglutide, semaglutide, du laglutide, tirzepatide, albig lutide, or efpeglenatide.

19. The method of any one of claims 1 -15, wherein the incretin-based therapy is an incretin enhancer.

20. The method of claim 19, wherein the incretin enhancer is a DPP-4 inhibitor.21 . The method of claim 20, wherein the DPP-4 inhibitor is sitagliptin, saxagliptin, alogliptin, linagliptin, or vildagliptin.

22. The method of any one of claims 1 -21 , wherein the method increases lean mass.

23. The method of any one of claims 1 -22, wherein the method increases muscle mass.

24. The method of any one of claims 1 -23, wherein the method reduces fat mass.

25. The method of any one of claims 1 -24, wherein the method reduces white adipose tissue mass.

26. The method of any one of claims 24 or 25, wherein administration of the extracellular ActRII chimera leads to a greater loss of fat mass or white adipose tissue mass compared to treatment with the incretinbased therapy alone.

27. The method of any one of claims 1 -26, wherein the method improves insulin sensitivity.

28. The method of any one of claims 1 -27, wherein the method improves insulin resistance.

29. The method of any one of claims 1 -28, wherein the method improves cardiac function or cardiac health.

30. The method of any one of claims 1 -29, wherein the method reduces the risk of major adverse cardiovascular events.31 . The method of any one of claims 1 -30, wherein the method prevents or reduces the development of frailty.

32. The method of any one of claims 1 -31 , wherein the method prevents or reduces the premature onset of sarcopenia.

33. The method of any one of claims 1 -32, wherein the method reduces the risk of sarcopenia.

34. The method of any one of claims 1 -33, wherein the method reduces weight regain after discontinuation of the incretin-based therapy.

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