Combination therapy to treat overweight, obesity, and related health conditions

Abstract

Provided herein are methods of treating metabolic disorders, obesity and / or overweight and / or obesity-related symptoms, and type II diabetes using the anti-myostatin Adnectins described herein (e.g., BHV-2000) in combination with a GLP-1 receptor agonist (e.g., semaglutide).

Description

[0001] COMBINATION THERAPY TO TREAT OVERWEIGHT, OBESITY, AND RELATED

[0002] HEALTH CONDITIONS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to, and the benefit of, U.S. Provisional Application Nos. 63 / 633,814 filed on April 14, 2024, 63 / 652,347 filed on May 28, 2024, 63 / 669,416 filed on July 10, 2024, 63 / 715,616 filed on November 3, 2024, 63 / 715,617 filed on November 3, 2024, and 63 / 760,516 filed on February 19, 2025. The entire contents of the aforementioned applications are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 7, 2025, is named BHJ-004PC_SequenceListing.xml and is 181 kilobytes in size.

[0007] FIELD OF THE INVENTION

[0008] The present invention relates to methods of treating overweight, obesity, and metabolic disorders, such as type II diabetes, using a combination of a polypeptide that binds myostatin (e.g., an anti-myostatin Adnectin) and GLP-1 receptor agonists (e.g., semaglutide).

[0009] BACKGROUND

[0010] Obesity-related morbidity and mortality is driven by the presence of excess abnormal adipose tissue. Approved anti-obesity medications not only lead to meaningful reductions in body fat but also undesirable loss of lean muscle mass with unknown long-term consequences. Incretin-based obesity treatments (e.g., GLP-1 receptor agonists) demonstrate significant weight reduction and metabolic benefits; however, along with tolerability issues, patients exhibit a significant loss of lean muscle. This loss of lean muscle is documented, for example, in patients treated with approved GLP-1 receptor agonists, such as semaglutide (see, e.g., Wilding JPH et al., J Endocrine Soc. 2021;5(Supplement_l):A16-17; McCrimmon RJ et al., Diabetologia 2020;63:473-85). Accordingly, there remains a need for improved methods of treating metabolic disorders, such as diabetes, and obesity / overweight that address the sarcopenic effects of approved anti-obesity drugs, in particular, GLP-1 receptor agonists.

[0011] SUMMARY

[0012] Provided herein are methods for decreasing body weight and preventing significant loss of lean muscle mass, methods of decreasing body weight and increasing lean muscle mass, methods of treating a metabolic disease, disorder, or condition, methods of increasing the ratio of lean muscle mass to fat, methods of treating, preventing, or reducing overweight or obesity and related comorbidities, methods of treating or preventing type II diabetes, and methods of improving glycemic control in a subject (e.g., a human patient) by administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., BHV-2000) in combination with an effective amount of a GLP-1 receptor agonist (e.g., semaglutide).

[0013] Also provided are methods of increasing muscle mass in a human patient undergoing treatment with a GLP-1 receptor agonist (e.g., semaglutide) and methods of preventing the reduction of muscle mass in a human patient undergoing treatment with a GLP-1 receptor agonist (e.g., semaglutide), the method comprising administering to the patient an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., BHV-2000) in addition to the GLP-1 receptor agonist.

[0014] In some embodiments, the polypeptide comprises a10Fn3 domain comprising BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively. In some embodiments, the polypeptide comprises a10Fn3 domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the polypeptide is taldefgrobep alpha (BHV-2000).

[0015] In some embodiments, the polypeptide is administered subcutaneously. In some embodiments, the polypeptide is administered as a formulation comprising: (i) at least 10 mg / mL of the polypeptide; (ii) a disaccharide at a concentration of at least 5%; (iii) a histidine buffer at a concentration of between about 20 to about 60 mM; and (iv) a pharmaceutically acceptable aqueous carrier, wherein the formulation has a pH range of about 6.5 to about 7.8.

[0016] In some embodiments, the polypeptide is administered once a week, once every two weeks, or once a month.

[0017] In some embodiments, the GLP-1 receptor agonist (e.g., semaglutide) is administered subcutaneously or orally. In some embodiments, the GLP-1 receptor agonist is administered subcutaneously once weekly, for example, at a dose of between about 0.25 mg to about 2 mg. In some embodiments, the GLP-1 receptor agonist is administered orally once daily, for example, at a dose of between about 3 mg / day and about 15 mg / day.

[0018] In some embodiments, the first dose of the polypeptide and the first dose of the GLP-1 receptor agonist are administered simultaneously. In some embodiments, the polypeptide, or formulation comprising the polypeptide, is administered prior to administration of the GLP-1 receptor agonist. In some embodiments, the polypeptide, or formulation comprising the polypeptide, is administered after administration of the GLP-1 receptor agonist.

[0019] In some embodiments, the GLP-1 receptor agonist is semaglutide or tirzepatide.

[0020] Further provided are kits that include a pharmaceutical formulation containing a polypeptide which comprises a fibronectin type III tenth (10Fn3) domain which binds to myostatin, such as BHV-2000, and a GLP-1 receptor agonist, in therapeutically effective amounts adapted for use in methods of decreasing body weight and preventing significant loss of lean muscle mass, methods of decreasing body weight and increasing lean muscle mass, methods of treating a metabolic disease, disorder, or condition, methods of increasing the ratio of lean muscle mass to fat, methods of treating, preventing, or reducing overweight or obesity and related comorbidities, and methods of treating or preventing type II diabetes, as described herein.

[0021] In some embodiments, the kits comprise a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively (e.g., BHV-2000), a GLP-1 receptor agonist (e.g., semaglutide), and instructions for using the polypeptide and GLP-1 receptor agonist in the methods described herein. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the polypeptide is taldefgrobep alpha (BHV-2000). In some embodiments, the polypeptide is for administration as a unit dosage form comprising about 1.0 mL or less of a formulation comprising (i) about 10-75 mg / mL of the polypeptide; (ii) about 5-25% trehalose dihydrate; (iii) about 20-30 mM histidine; (iv) about 0.02-0.06 mM DTPA; (v) about 0.01-0.05% polysorbate 80; and (vi) a pharmaceutically acceptable aqueous carrier, wherein the pH of the formulation is about 6.8 to 7.3.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1A is a graph showing mean concentrations of BHV-2000 in mice administered the indicated doses (45 mg, 90 mg, and 180 mg) over the course of 0, 24, 48, 72, 96, 120, 144, and 168 hours. FIG. IB is a graph showing the effects of activin A in vitro activity (as determined by SMAD2 / 3 phosphorylation) across a range of concentrations when activin A administered alone or in the presence of BHV-2000 / myostatin complex, or in the presence of BHV- 2000 / GDF-l 1 complex. FIG. 2A is a schematic of the in vitro experiments described in Example 2.

[0024] FIGs. 2B-2D are graph showing the effects of Control, BHV-2000, activin II receptor (ActRII) ligand cocktail (activin A, myostatin, and GDF11), BHV-2000 + ActRII ligand cocktail, myostatin, and BHV-2000 + myostatin on lipid storage as assessed by BODIPY staining (FIG. 2B), intracellular granularity as assessed by side scatter (SSC) (FIG. 2C), SMAD2 / 3 signaling as assessed by a luciferase reporter assay (FIG. 2D), and mitochondrial mass as assessed by MitoTracker Green staining (FIG. 2E). BODIPY: 4,4-difluoro-4-bora- 3a,4a-diaza-s-indacene; NS: not significant; gMFI: geographic mean fluorescence intensity; SSC-A: side scatter area. **P<0.01, ****P<0.0001.

[0025] FIG. 2F is a heat map reflecting the levels of intracellular metabolites in the indicated treatment groups. FIGs. 2G and 2H are graphs showing the levels (relative abundance) of intracellular phosphocreatine and proline, respectively, in cells treated with Control, BHV-2000, activin II receptor (ActRII) ligand cocktail (activin A, myostatin, and GDF11), BHV-2000 + ActRII ligand cocktail, myostatin, and BHV-2000 + myostatin.

[0026] FIG. 3 is a schematic of in vivo experiments using a mouse diet-induced obesity (DIO) model.

[0027] FIGs. 4A and 4B are graphs showing the effects of Vehicle, BHV-2000, Semaglutide (20 pg / kg), Semaglutide (40 pg / kg), BHV-2000 + Semaglutide (20 pg / kg), and BHV-2000 + Semaglutide (40 pg / kg) on body weight (FIG. 4A) and % change in body weight relative to baseline (pre-treatment) (FIG. 4B) of DIO mice. BHV-2000 was administered twice weekly subcutaneously and semaglutide was administered once daily orally

[0028] FIGs. 5A-5C are graphs showing the effects of Vehicle, BHV-2000, Semaglutide (20 pg / kg), Semaglutide (40 pg / kg), BHV-2000 + Semaglutide (20 pg / kg), and BHV-2000 + Semaglutide (40 pg / kg) on percent changes in fat mass relative to baseline in DIO mice. FIG. 5C is a bar graph showing percent changes in fat mass relative to baseline at 8 weeks. BHV- 2000 was administered twice weekly subcutaneously and semaglutide was administered once daily orally. n=15 for vehicle; n=16 for all other groups. Error bars represent standard error of the mean. Significance evaluated using Tukey’s multiple comparisons test. **P<0.01, ***P<0.001, ****P<0.0001.

[0029] FIGs. 6A and 6B are graphs showing the effects of Vehicle, BHV-2000, Semaglutide (20 pg / kg), Semaglutide (40 pg / kg), BHV-2000 + Semaglutide (20 pg / kg), and BHV-2000 + Semaglutide (40 pg / kg) on percent changes in lean muscle mass relative to baseline in DIO mice. FIG. 6B is a bar graph showing percent changes in lean muscle mass relative to baseline at 8 weeks. BHV-2000 was administered twice weekly subcutaneously and semaglutide was administered once daily orally. n=15 for vehicle; n=16 for all other groups. Error bars represent standard error of the mean. Significance evaluated using Tukey’s multiple comparisons test. *P<0.05, **P<0.01, ****P<0.0001.

[0030] FIG. 7 shows a series of graphs the effects of Vehicle, BHV-2000, Semaglutide (20 pg / kg), Semaglutide (40 pg / kg), BHV-2000 + Semaglutide (20 pg / kg), and BHV-2000 + Semaglutide (40 pg / kg) on levels of ALP, ALT, cholesterol, LDLD cholesterol, glucose, HDL cholesterol, triglycerides, and NEFA. BHV-2000 was administered twice weekly subcutaneously and semaglutide was administered once daily orally. *P<0.05, **P<0.01, ***P<0.001. FIG. 8 shows a series of graphs the effects of Vehicle, BHV-2000, Semaglutide (20 pg / kg), Semaglutide (40 pg / kg), BHV-2000 + Semaglutide (20 pg / kg), and BHV-2000 + Semaglutide (40 pg / kg) on levels of insulin, leptin, and corticosterone. BHV-2000 was administered twice weekly subcutaneously and semaglutide was administered once daily orally.

[0031] FIGs. 9A-9C are graphs showing the effects of Vehicle, BHV-2000, Semaglutide (20 pg / kg), Semaglutide (40 pg / kg), BHV-2000 + Semaglutide (20 pg / kg), and BHV-2000 + Semaglutide (40 pg / kg) on glucose levels at 8 weeks after a 4 hour fast (FIG. 9A) and in a insulin sensitivity test, whereby a fixed dose of insulin (1.5 U / kg) is given and then impact on glucose is measured at fixed intervals (15, 30, 60, 90, and 120 minutes after insulin injection) in DIO mice (FIGs. 9B and 9C). BHV-2000 was administered twice weekly subcutaneously and semaglutide was administered once daily orally. Error bars represent standard error of the mean. Significance evaluated using Tukey’s multiple comparisons test. *P<0.05, ***P<0.001, ****p<0.0001. AUC: area under the curve.

[0032] DETAILED DESCRIPTION

[0033] I. Definitions

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the skilled artisan. Although any methods and compositions similar or equivalent to those described herein can be used in practice or testing of the present invention, the preferred methods and compositions are described herein.

[0035] The singular form “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0036] The term “about”, particularly in reference to a given quantity or number, is meant to encompass deviations within plus or minus ten percent (± 10%), (e.g., ± 5%).

[0037] “Full-length myostatin” as used herein refers to the full-length polypeptide sequence described in McPherron et al. (1997), supra, as well as related full-length polypeptides including allelic variants and interspecies homologs. The term “myostatin” or “mature myostatin” refers to fragments of the biologically active mature myostatin, as well as related polypeptides including allelic variants, splice variants, and fusion peptides and polypeptides. The mature C-terminal protein has been reported to have 100% sequence identity among many species including human, mouse, chicken, porcine, turkey, and rat (Lee et al., PNAS 2001;98:9306). The sequence for human prepromyostatin is:

[0038] MQKLQLCVYIYLFMLIVAGPVDLNENSEQKENVEKEGLCNACTWRQNTK SSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSS DGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKW I<AQLWIYLRPVETPTTVFVQILRLII<PMI<DGTRYTGIRSLI<LDMNPGTGIW QSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLE VKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRY KANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYF

[0039] NGKEQIIYGKIPAMWDRCGCS (SEQ ID NO: 1).

[0040] The sequence for human pro-myostatin is:

[0041] NENSEQI<ENVEI<EGLCNACTWRQNTI<SSRIEAII<IQILSI<LRLETAPNISI<D VIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDF LMQVDGI<PI<CCFFI<FSSI<IQYNI<VVI<AQLWIYLRPVETPTTVFVQILRLII< PMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIK ALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTE SRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLV HQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMWDRCGCS (SEQ ID NO: 2).

[0042] The sequence for mature myostatin (conserved in human, murine, rat, chicken, turkey, dog, horse, and pig) is:

[0043] DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFV FLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPA MWDRCGCS (SEQ ID NO: 3).

[0044] As used herein, a "fibronectin based scaffold" or "FBS" protein or moiety refers to proteins or moieties that are based on a fibronectin type III ("Fn3") repeat. Fibronectin has 18 Fn3 repeats, and while the sequence homology between the repeats is low, they all share a high similarity in tertiary structure. For reviews see Bork et al., Proc. Natl. Acad. Set. USA, 89(19): 8990-8994 (1992); Bork et al., J. Mol. Biol., 242(4):309-320 (1994); Campbell et al., Structure, 2(5):333-337 (1994); Harpez et al., J. Mol. Biol., 238(4):528-539 (1994)). An Fn3 domain is small, monomeric, soluble, and stable. It lacks disulfide bonds and, therefore, is stable under reducing conditions. Fn3 domains comprise, in order from N-terminus to C-terminus, a beta or beta-like strand, A; a loop, AB; a beta or beta-like strand, B; a loop, BC; a beta or betalike strand, C; a loop, CD; a beta or beta-like strand, D; a loop, DE; a beta or beta-like strand, E; a loop, EF; a beta or beta-like strand, F; a loop, FG; and a beta or beta-like strand, G. The seven antiparallel P-strands are arranged as two beta sheets that form a stable core, while creating two "faces" composed of the loops that connect the beta or beta-like strands. Loops AB, CD, and EF are located at one face ("the south pole") and loops BC, DE, and FG are located on the opposing face ("the north pole").

[0045] Adnectins are a class of therapeutic FBS proteins with high-affinity and specific targetbinding properties that are derived from the tenth human fibronectin type III domain (10Fn3):

[0046] VSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVOEFTV PGSKSTAHSGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 4) (BC, DE, and FG loops are underlined).

[0047] Accordingly, as used herein, a "10Fn3 domain" or "10Fn3 moiety" or "10Fn3 molecule" refers to wild-type10Fn3 and biologically active variants thereof, e.g., biologically active variants that specifically bind to a target, such as a target protein.

[0048] A "region" of a10Fn3 domain (or moiety or molecule) as used herein refers to either a loop (AB, BC, CD, DE, EF and FG), a P-strand (A, B, C, D, E, F and G), the N-terminus (corresponding to amino acid residues 1-7 of SEQ ID NO: 4), or the C-terminus (corresponding to amino acid residues 93-94 of SEQ ID NO: 4).

[0049] A "scaffold region" refers to any non-loop region of a human10Fn3 domain. The scaffold region includes the A, B, C, D, E, F and G P-strands as well as the N-terminal region (amino acids corresponding to residues 1-7 of SEQ ID NO: 4) and the C-terminal region (amino acids corresponding to residues 93-94 of SEQ ID NO: 4).

[0050] The term "anti-myostatin Adnectin" refers to a protein molecule that binds to and antagonizes myostatin and that comprises at least a one10Fn3 domain derived from the human wild-type10Fn3 domain (SEQ ID NO: 4). The anti-myostatin Adnectin can further comprise additional protein domains (e.g., an Fc domain), and can also refer to multimer forms of the polypeptide, such as dimers, tetramers and hexamers. In some embodiments, the anti-myostatin Adnectin can be an “anti-myostatin Fc engineered Adnectin,” which refers to an anti-myostatin Adnectin which is linked to an engineered Fc region which comprises an amino acid sequence that differs from a wild-type Fc region by at least one amino acid (e.g., an amino acid modification, such as an amino acid substitution). In some embodiments, the engineered Fc region is a variant IgGl Fc region or a variant IgG4 Fc region.

[0051] "Polypeptide" as used herein refers to any sequence of two or more amino acids, regardless of length, post-translation modification, or function. "Polypeptide," "peptide," and "protein" are used interchangeably herein. Polypeptides can include natural amino acids and nonnatural amino acids such as those described in U.S. Pat. No. 6,559,126, incorporated herein by reference. Polypeptides can also be modified in any of a variety of standard chemical ways (e.g., an amino acid can be modified with a protecting group; the carboxy-terminal amino acid can be made into a terminal amide group; the amino-terminal residue can be modified with groups to, e.g., enhance lipophilicity; or the polypeptide can be chemically glycosylated or otherwise modified to increase stability or in vivo half-life). Polypeptide modifications can include the attachment of another structure such as a cyclic compound or other molecule to the polypeptide and can also include polypeptides that contain one or more amino acids in an altered configuration (i.e., R or S; or, L or D). The peptides of the invention are proteins derived from the tenth type III domain of fibronectin that have been modified to bind to myostatin and are referred to herein as, "anti-myostatin Adnectin" or "myostatin Adnectin."

[0052] A "polypeptide chain", as used herein, refers to a polypeptide wherein each of the domains thereof is joined to other domain(s) by peptide bond(s), as opposed to non-covalent interactions or disulfide bonds.

[0053] An "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing condition using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

[0054] "Percent (%) amino acid sequence identity" herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence 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, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR™) software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For example, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

[0055] As used herein, "conservative substitution" denotes the replacement of an amino acid residue by another, without altering the overall conformation and function of the peptide, including, but not limited to, replacement of an ammo acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, shape, hydrophobic, aromatic, and the like). Exemplary conservative substitutions include those fulfilling the criteria defined for an accepted point mutation in Dayhoff el al., Atlas of Protein Sequence and Structure, 5:345-352 (1978 and Supp.) Examples of conservative substitutions include substitutions within the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine. By "substituted"' or "modified" the present invention includes those ammo acids that have been altered or modified from naturally occurring amino acids. As such i t should be understood that in the context of the present invention a conservative substitution is recognized in the art as a substitution of one amino acid for another ammo acid that has similar properties.

[0056] The terms “specifically binds,” “specific binding,” “selective binding,” and “selectively binds,” as used interchangeably herein refers to an Adnectin that exhibits affinity for a myostatin, but does not significantly bind (e.g., less than about 10% binding) to a different polypeptide as measured by a technique available in the art such as, but not limited to, Scatchard analysis and / or competitive binding assays (e.g., competition ELISA, BIACORE assay). The term is also applicable where e.g., a binding domain of an Adnectin of the invention is specific for myostatin.

[0057] The term "preferentially binds" as used herein refers to the situation in which an Adnectin described herein binds myostatin at least about 20% greater than it binds a different polypeptide as measured by a technique available in the art such as, but not limited to, Scatchard analysis and / or competitive binding assays (e.g., competition ELISA, BIACORE assay).

[0058] The term “KD,” as used herein, is intended to refer to the dissociation equilibrium constant of a particular Adnectin-protein (e.g., myostatin) interaction or the affinity of an Adnectin for a protein (e.g., myostatin), as measured using a surface plasmon resonance assay or a cell binding assay. A “desired KD,” as used herein, refers to a KD of an Adnectin that is sufficient for the purposes contemplated. For example, a desired KD may refer to the KD of an Adnectin required to elicit a functional effect in an in vitro assay, e.g., a cell-based luciferase assay.

[0059] The term “kass”, as used herein, is intended to refer to the association rate constant for the association of an Adnectin into the Adnectin / protein complex.

[0060] The term “kdiSS”, as used herein, is intended to refer to the dissociation rate constant for the dissociation of an Adnectin from the Adnectin / protein complex.

[0061] The term “IC50”, as used herein, refers to the concentration of an Adnectin that inhibits a response, either in an in vitro or an in vivo assay, to a level that is 50% of the maximal inhibitory response, i.e., halfway between the maximal inhibitory response and the untreated response.

[0062] The term "myostatin activity" as used herein refers to one or more of growth-regulatory or morphogenetic activities associated with the binding of active myostatin protein to ActRIIb and the subsequent recruitment of Alk4 or Alk5. For example, active myostatin is a negative regulator of skeletal muscle mass. Active myostatin can also modulate the production of musclespecific enzymes (e.g., creatine kinase), stimulate myoblast proliferation, and modulate preadipocyte differentiation to adipocytes. Myostatin activity can be determined using art- recognized methods, such as those described herein.

[0063] The phrases “inhibit myostatin activity” or “antagonize myostatin activity” or “antagonize myostatin” are used interchangeably to refer to the ability of the anti-myostatin Adnectins of the present invention to neutralize or antagonize an activity of myostatin in vivo or in vitro. The terms "inhibit" or "neutralize" as used herein with respect to an activity of an Adnectin of the invention means the ability to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, eliminate, stop, reduce or reverse e.g., progression or severity of that which is being inhibited including, but not limited to, a biological activity or property, a disease or a condition. The inhibition or neutralization is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher.

[0064] For example, an anti-myostatin Adnectin may reduce circulating levels of biologically active myostatin normally found in a vertebrate subject, or a reduction of circulating levels of biologically active myostatin in subjects with disorders that result in elevated circulating levels of myostatin. A reduction of myostatin activity may be determined using in vitro assays, e.g., binding assays, as described herein.

[0065] The term “Glucagon-like peptide- 1 (GLP-1) receptor agonist,” also known as GLP-1 analogs, GLP-1 RAs, GLP-1 agonists, incretin mimetics, and nutrient-stimulated hormone (NuSH) therapies are a class of anorectic drugs that reduce blood sugar and energy intake by activating the GLP-1 receptor, triggering insulin release and blocking glucagon secretion. They mimic the actions of the endogenous incretin hormone GLP-1. GLP-1 receptor agonists are recommended as an add-on therapy to lifestyle intervention (calorie restriction and exercise) in people with a BMI over 30 or a BMI over 27 with at least one weight-related comorbidity. Various GLP-1 receptor agonists are FDA approved, including, for example, semaglutide, exenatide, liraglutide, dulaglutide, lixisenatide, and tirzepatide.

[0066] The term "PK" is an acronym for "pharmacokinetic" and encompasses properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject. A "PK modulation protein" or "PK moiety" as used herein refers to any protein, peptide, or moiety that affects the pharmacokinetic properties of a biologically active molecule when fused to or administered together with the biologically active molecule. Examples of a PK modulation protein or PK moiety include PEG, human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005 / 0287153 and 2007 / 0003549, PCT Publication Nos. WO 2009 / 083804 and WO 2009 / 133208), human serum albumin, Fc or Fc fragments and variants thereof, and sugars (e.g., sialic acid).

[0067] The notations "mpk", "mg / kg", or "mg per kg" refer to milligrams per kilogram. All notations are used interchangeably throughout the present disclosure.

[0068] The terms "individual," "subject," and "patient," used interchangeably herein, refer to an animal, preferably a mammalian (including a non-primate and a primate) or avian species, including, but not limited to, murines, simians, humans, mammalian farm animals (e.g., bovine, porcine, ovine), mammalian sport animals (e.g., equine), and mammalian pets (e.g., canine and feline); preferably the term refers to humans. The term also refers to avian species, including, but not limited to, chickens and turkeys. In a certain embodiment, the subject, preferably a mammal, preferably a human, is further characterized with a disease or disorder or condition that would benefit from a decreased level or decreased bioactivity of myostatin. In another embodiment the subject, preferably a mammal, preferably a human, is further characterized as being at risk of developing a disorder, disease or condition that would benefit from a decreased level of myostatin or a decreased bioactivity of myostatin. In some embodiments, the subject is an obese patient, patient having obesity-related comorbidities, and / or a patient having type II diabetes.

[0069] The term "effective amount" refers to an amount of an agent sufficient to achieve the stated purpose or otherwise achieve the effects for which it is administered. In the context of a combination therapy of a polypeptide with a highly differentiated mode of action described herein (e.g., an anti-myostatin Adnectin, such as BHV-2000) and a GLP-1 receptor agonist (e.g., semaglutide), an effective amount of the polypeptide is an amount effective to prevent, halt, or reverse the decrease in lean muscle mass which results from GLP-1 receptor agonist treatment, or an amount effective to increase lean muscle mass relative to baseline (i.e., before administration of the polypeptide) in a patient undergoing the combination therapy or a patient who is already undergoing GLP-1 receptor agonist therapy. An effective mount of a GLP-1 receptor agonist is an amount efficient to reduce total body weight or fat mass in the patient, and can be, for example, the recommended dose on the label of an FDA-approved GLP-1 receptor agonist (e.g., semaglutide or tirzepatide). The term "therapeutically effective amount" refers to at least the minimal dose, but less than a toxic dose, of an agent which is necessary to impart a therapeutic benefit to a subject. For example, a therapeutically effective amount of an anti-myostatin Adnectin of the invention is an amount which in mammals, preferably humans, results in one or more of the following: an increase in muscle volume and / or muscle strength, an increase in muscle mass, a decrease in body fat, an increase in insulin sensitivity, or the treatment of conditions wherein the presence of myostatin causes or contributes to an undesirable pathological effect or a decrease in myostatin levels results in a beneficial therapeutic effect.

[0070] The term “frail” or “frailty” as used herein refers to a condition that can be characterized by two or more symptoms from weakness, weight loss, slowed mobility, fatigue, low activity levels, poor endurance, and impaired behavioral response to sensory cues. One hallmark of frailty is “sarcopenia,” or the age-related loss of muscle mass.

[0071] The term “cachexia” as used herein refers to the condition of accelerated muscle wasting and loss of lean body mass that can result from various diseases.

[0072] The term “obesity” is defined as a condition of abnormal or excessive fat accumulation in adipose tissue. The amount of excess fat in absolute terms, and its regional distribution between different fat depots both play an important role in determining the health impact of obesity. Obesity can be categorized into central / android obesity and peripheral / gynoid obesity, android obesity being more typical for men while gynoid obesity being more characteristic for women. Obesity is based on Body Mass Index (BMI) for both youth and adults, but the definitions are not directly comparable. Among adults, there is a set cut point based on health risk, while among children the definition is statistical and is based on a comparison to a reference population. BMI is calculated as weight in kilograms divided by height in meters squared, rounded to one decimal place. Obesity in adults is defined as a BMI of greater than or equal to 30 kg / m2. Obesity in youth is defined as a BMI of greater than or equal to the age- and sex-specific 95th percentile of the 2000 CDC growth charts.

[0073] The terms "overweight” or “overweight condition" are based on a BMI > 25 - < 30 kg / m2. Overweight condition can also be associated with at least one additional risk factor for fatal diseases (e.g., stroke, heart failure, sudden death), such as diabetes, hypertension, family history of premature coronary artery disease, etc. Because different subjects can have same BMI, but different percentage of fat and muscle mass, BMI is not always a good index to classify overweight and obesity. A high percentage of muscle mass can lead to a high BMI even with a small percentage of fat. In this case the subject can be wrongly considered overweight or obese, based on the BMI classification. Other indexes in addition to BMI are used, namely waist circumference and a body shape index. Imaging, by DXA and MRI, is often used in clinical trials to quantify the percentage of muscle, fat and the fat distribution.

[0074] The term "body composition" is used herein to describe the percentages of fat and muscle in human bodies. Because muscular tissue takes up less space in our body than fat tissue, body composition, as well as our weight, determines leanness. Two people of same sex and body weight may look completely different from each other because they have a different body composition.

[0075] "Lean body mass" (LBM) is a component of body composition, calculated by subtracting body fat weight from total body weight. Total body weight is lean plus fat. Lean body mass equals body weight minus body fat. Lean body mass plus body fat equals body weight.

[0076] The percentage of total body mass that is lean is usually not quoted, but would typically be 60-90%. Instead, the body fat percentage, which is the complement, is computed, and is typically 10-40%. The lean body mass (LBM) has been described as an index superior to total body weight for prescribing proper levels of medications and for assessing metabolic disorders, as body fat is less relevant for metabolism.

[0077] The term "fat mass" refers to that portion of the human body that is composed strictly of fat. It can be measured with dual energy absorptiometry DXA, MRI or bioelectrical impedance techniques.

[0078] The term "central adiposity" refers to the following:

[0079] The term "type II diabetes" referred as type 2 diabetes, previously referred to as "non- insulin-dependent diabetes" or "adult-onset diabetes," accounts for 90-95% of all diabetes, encompasses individuals who have insulin resistance and usually relative (rather than absolute) insulin deficiency. At least initially, and often throughout their lifetime, these individuals may not need insulin treatment to survive. There are various causes of type 2 diabetes. Although the specific etiologies are not known, autoimmune destruction of B-cells does not occur, and patients do not have any of the other known causes of diabetes. Most, but not all, patients with type 2 diabetes are overweight or obese. Excess weight itself causes some degree of insulin resistance. Patients who are not obese or overweight by traditional weight criteria may have an increased percentage of body fat distributed predominantly in the abdominal region. Type 2 diabetes frequently goes undiagnosed for many years because hyperglycemia develops gradually and, at earlier stages, is often not severe enough for the patient to notice the classic diabetes symptoms. Nevertheless, even undiagnosed patients are at increased risk of developing macrovascular and microvascular complications.

[0080] The term "comorbidities of obesity or overweight" is associated with serious chronic disorders including, but not limited to: type 2 diabetes, glucose intolerance, prediabetes, insulin resistance, hypertension, dyslipidemia, increased waist circumference, cardiovascular disease, non-alcoholic fatty liver disease, obstructive sleep apnea, physical impairment, osteoarthritis, osteoporosis, renal disease, sexual hormone(s) impairment, endocrine reproductive disorders such as polycystic ovary syndrome or male hypogonadism, stroke, and gallstones.

[0081] "Glucose intolerance" is defined as the inability to properly metabolize glucose.

[0082] "Insulin sensitivity" describes how sensitive the body is to the effects of insulin. Someone said to be insulin sensitive will require smaller amounts of insulin to lower blood glucose levels than someone who has low sensitivity. Insulin sensitivity varies from person to person and doctors can perform tests to determine how sensitive an individual is to insulin.

[0083] "Insulin resistance" is defined as a condition of tolerance to insulin, making the hormone less effective, causing decreased glucose uptake in muscle tissue that result in impaired glucose oxidation and glycogen synthesis, and a deficient suppression of hepatic glucose production in the liver. In obese, increased visceral fat mass with elevation of plasma free fatty acid (FFA) caused by the intensified lipolytic activity, worsen insulin resistance through the impairment of insulin action (Reaven, G.M., et al., (1988), Diabetes. 37: 1020-1024), a mechanism known as lipotoxicity (DeFronzo R.A. (2004), Ini. J. Clin. Pract. Suppl:, (143):9-21). Herein the terms "improving insulin sensitivity" and "treating / lowering insulin resistance" shall be construed as equivalent.

[0084] High concentrations of plasma free fatty acid (FFA) in skeletal muscle cells lead to reduction in insulin-stimulated intracellular transport of glucose through the Glut4 transporter (see, e.g., Dresner A., et al. (1999), J. Clin. Invest. 103(2): 253-259), in the hepatocytes lead to enhanced rate of gluconeogenesis and glucose release from the liver and augmented insulin secretion from 0 cells in response to a transient increase of FFA or an inhibition effect in response to chronic elevated levels (see, e.g., Boden G. (1997), Diabetes. 46(l):3-10). As a result of insulin resistance and lipotoxicity, more insulin is needed to induce glucose uptake from fat and muscle cells, and glycogen synthesis in the liver (see, e.g., Boden G. (1997), Diabetes. 46(l):3-10). The overproduction of insulin by pancreatic 0 cells is the physiologic reaction to insulin resistance and can lead to decline of 0 cell function and, eventually to prediabetes and type 2 diabetes (Donath M.Y., et al., (2005), Diabetes, 54: S108-S1 13.).

[0085] The term "improving insulin sensitivity" refers to the systemic responsiveness to glucose, which can be measured by the insulin sensitivity index (which measures the ability of endogenous insulin to lower glucose in extracellular fluids by inhibiting glucose release from the liver and stimulating the peripheral consumption of glucose) and the glucose-clamp technique (which measures the effect of changes in insulin concentration on glucose clearance-glucose uptake rate divided by plasma glucose concentration per unit of body surface area).

[0086] "Prediabetes" is the precursor stage before diabetes mellitus in which blood sugar is abnormally high (e.g., 100-125 mg / dL). Impaired fasting glycemia and impaired glucose tolerance are two aspects of prediabetes that are similar in clinical definition (glucose levels too high for their context) but are physiologically distinct. Insulin resistance, metabolic syndrome (or syndrome X), and prediabetes are closely related to one another and have overlapping aspects.

[0087] Various aspects of the present invention are described in further detail in the following subsections.

[0088] II. Anti-myostatin Adnectins

[0089] Provided herein are methods for treating decreasing body weight and preventing significant loss of lean muscle mass, methods of decreasing body weight and increasing lean muscle mass, methods of treating a metabolic disease, disorder, or condition, methods of increasing the ratio of lean muscle mass to fat, methods of treating, preventing, or reducing overweight or obesity and related comorbidities, methods of treating or preventing type II diabetes, and methods of improving glycemic control using a combination of an effective amount of a polypeptide which binds to myostatin (e.g., an anti-myostatin Adnectin, such as an anti- myostatin Fc-Adnectin) and an effective amount of a GLP-1 receptor agonist (e.g., semaglutide). Various features of the anti-myostatin Adnectin for use in the methods described herein are provided below.

[0090] A. Anti-myostatin Adnectin

[0091] The anti-myostatin Adnectins suitable for use in the methods described herein comprises an Fn3 domain derived from the wild-type tenth module of the human fibronectin type III domain (10Fn3) (SEQ ID NO: 4).

[0092] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, and 7, respectively.

[0093] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, and 7, respectively, wherein the BC loop comprises 1, 2 or 3 amino acid substitutions, such as conservative amino acid substitutions, which allow the anti- myostatin Adnectin to maintain binding to myostatin.

[0094] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, and 7, respectively, wherein at least one loop of the BC, DE, and FG loops of the10Fn3 domain has 1 amino acid substitution relative to the respective BC, DE, and FG loops of SEQ ID NOs: 5, 6, and 7.

[0095] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, and 7, respectively, wherein one loop from the BC, DE, or FG loop of the10Fn3 domain has 1 amino acid substitution relative to the respective BC, DE, or FG loop of SEQ ID NOs: 5, 6, and 7.

[0096] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, or 7, respectively, wherein (i) the serine at position 3 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of A, C, D, F, H, I, K, L, N, Q, R, T, V, W, or Y; (n) the leucine at position 4 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from M or V; (iii) the proline at position 5 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of A, C, D, E, I, K, L, M, N, Q, R, S, T, V, or Y; (vi) the histidine at position 6 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, or Y; (vii) the glutamine at position 7 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; (viii) the glycine at position 8 of the BC loop (SEQ ID NO: 5) is substituted with the amino acid S; (ix) the lysine at position 9 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y; (x) the alanine at position 10 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of C, G, L, M, S, or T; or (xi) the asparagine at position 11 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of A, C, F, H, P, Q, R, S, or Y.

[0097] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, or 7, respectively, wherein (i) the serine at position 3 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of C, F, I, V, W, or Y; (ii) the histidine at position 6 of the BC loop (SEQ ID NO: 6) is substituted with an amino acid selected from the group consisting of C, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, or Y; (iii) the lysine at position 9 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of A, C, G, H, I, L, M, N, Q, R, S, V, W, or Y; (iv) the alanine at position 10 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of G, L, M, or S; or (v) the asparagine at position 11 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of C, H, Q, S, or Y.

[0098] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, or 7, respectively, wherein (i) the serine at position 3 of the BC loop (SEQ ID NO: 5) is substituted with the amino acid F or W; (ii) the histidine at position 6 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of C, F, G, I, K, L, M, N, R, S, T, V, W, or Y; (iii) the glutamine at position 7 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of A, C, E, F, H, I, K, L, M, P, R, S, T, V, or Y; (iii) the lysine at position 9 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of A, C, H, L, M, N, R, V, W, or Y; (iv) the alanine at position 10 of the BC loop (SEQ ID NO: 5) is substituted with the amino acid G or L; or (v) the asparagine at position 11 of the BC loop (SEQ ID NO: 5) is substituted with the amino acid H or Q.

[0099] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, or 7, respectively, wherein the valine at position 5 of the DE loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, I, K, L, M, N, Q, S, or T. In some embodiments, the valine at position 5 of the DE loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of C, E, I, L, M, Q, or T. In some embodiments, the valine at position 5 of the DE loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of C, E, I, L, or M.

[0100] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, or 7, respectively, wherein (i) the valine at position 2 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, F, I, L, M, Q, T, W, or Y; (ii) the threonine at position 3 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, F, G, H, I, K, L, M, N, Q, R, S, V, W, or Y; (iii) the aspartic acid at position 4 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; (iv) the threonine at position 5 of the FG loop (SEQ ID NO: 7) is substituted to with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K,

[0101] L, M, N, P, Q, R, S, V, W, or Y; (v) the glycine at position 6 of the FG loop (SEQ ID NO: 7) is substituted to with an amino acid selected from the group consisting of A, C, D, E, F, H, I, K, L,

[0102] M, N, Q, R, S, T, V, W, or Y; (vi) the tyrosine at position 7 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, F, H, I, L, M, N, P, S, T, V, or W; (vii) the leucine at position 8 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, E, F, H, I, K, M, N, Q, R, S, T, V, W, or Y; (viii) the lysine at position 9 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; (ix) the tyrosine at position 10 of the FG loop (SEQ ID NO: 7) is substituted with the amino acid F or W; or (x) the lysine at position 11 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y.

[0103] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, or 7, respectively, wherein (i) the valine at position 2 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, I, L, or M; (ii) the threonine at position 3 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of C, F, H, I, L, M, Q, R, S, V, W, or Y; (iii) the aspartic acid at position 4 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, E, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; (iv) the threonine at position 5 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, V, W, or Y; (v) the glycine at position 6 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, D, E, F, H, I, L, M, N, Q, S, T, V, W, or Y; (vi) the tyrosine at position 7 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of C, F, I, L, M, P, T, V, or W; (vii) the leucine at position 8 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of C, F, H, I, K, M, N, Q, R, T, V, W, or Y; (viii) the lysine at position 9 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, E, F, G, I, L, M, N, P, Q, R, S, T, V, W, or Y; (ix) the tyrosine at position 10 of the FG loop (SEQ ID NO: 7) is substituted with the amino acid W; or (x) the lysine at position 11 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, D, E, G, H, L, M, N, P, Q, R, S, T, or V.

[0104] In some embodiments, the anti-myostatin Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 5, 6, or 7, respectively, wherein (i) the valine at position 2 of the FG loop (SEQ ID NO: 7) is substituted with the amino acid I; (ii) the threonine at position 3 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of C, F, I, L, M, V, W, or Y; (iii) the aspartic acid at position 4 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, E, F, G, H, I, L, M, N, Q, S, T, or V; (iv) the threonine at position 5 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, D, F, G, I, L, M, N, Q, S, V, W, or Y; (v) the glycine at position 6 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, S, T, or W; (vi) the tyrosine at position 7 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of F, I, V, or W; (vii) the leucine at position 8 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of F, H, I, M, V, W, or Y; (viii) the lysine at position 9 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, C, F, G, I, L, M, T, V, or W; or (x) the lysine at position 11 of the FG loop (SEQ ID NO: 7) is substituted with an amino acid selected from the group consisting of A, G, L, M, P, Q, or R.

[0105] In certain embodiments, the anti-myostatin Adnectin comprises an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence which differs from SEQ ID NO: 8 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., by amino acid modifications, such as amino acid substitutions):

[0106] EWAATPTSLLISWSLPHQGKANYYRITYGETGGNSPVQEFTVPGRGVTA TISGLKPGVDYTITVYAVTVTDTGYLKYKPISINYRT (SEQ ID NO: 8)

[0107] In some embodiments, the anti-myostatin Adnectin contains a10Fn3 domain which binds myostatin comprising an amino acid sequence which is at least 90%, 95%, 98%, 99% or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In some embodiments, the anti-myostatin Adnectin contains a10Fn3 domain which binds myostatin comprising an amino acid sequence which differs from the non-BC, DE, and FG loop regions of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., by amino acid modifications, such as amino acid substitutions). For example, in some embodiments, the non-ligand binding sequences of10Fn3, i.e., the "10Fn3 scaffold", may also be altered provided that the10Fn3 retains ligand binding function and / or structural stability. A variety of mutant10Fn3 scaffolds have been reported. In some embodiments, one or more of Asp 7, Glu 9, and Asp 23 is replaced by another amino acid, such as, for example, a non-negatively charged amino acid residue (e.g., Asn, Lys, etc.). These mutations have been reported to have the effect of promoting greater stability of the mutant10Fn3 at neutral pH as compared to the wild-type form (see, e.g., PCT Publication No. WO 02 / 04523). A variety of additional alterations in the10Fn3 scaffold that are either beneficial or neutral have been disclosed. See, for example, Batori et al., Protein Eng., 15(12): 1015-1020 (December 2002); Koide et al., Biochemistry, 40(34):10326-10333 (Aug. 28, 2001).

[0108] In some embodiments, the10Fn3 domain of the anti-myostatin Adnectin comprises SEQ ID NO: 8. In some embodiments, the10Fn3 domain of the anti-myostatin Adnectin comprises SEQ ID NO: 9. In some embodiments, the10Fn3 domain of the anti-myostatin Adnectin comprises SEQ ID NO: 10.

[0109] In some embodiments, the anti-myostatin Adnectins are modified to comprise an N- terminal extension sequence and / or a C-terminal extension sequence. For example, an MG sequence may be placed at the N-terminus of the10Fn3 defined by SEQ ID NO: 4. The M will usually be cleaved off, leaving a G at the N-terminus. In some embodiments, the anti-myostatin Adnectin may comprise the amino acid sequence of SEQ ID NO: 8, and an N-terminal extension sequence (exemplary leader) as shown in Table 1 (i.e., any one of SEQ ID NOs: 11-17, or RDL or DL). In addition, an M, G or MG may also be placed N-terminal to any of the N-terminal extensions shown in Table 1 (i.e., any one of SEQ ID NOs: 11-17, or RDL or DL). In some embodiments, the anti-myostatin Adnectin may be truncated at the threonine corresponding to T94 of SEQ ID NO: 4. Alternatively, C-terminal extensions may be added after the C-terminal residue of SEQ ID NO: 8. Exemplary C-terminal extension sequences are shown in Table 1 (i.e., any one of SEQ ID NOs: 18-34, or El).

[0110] Table 1: Summary of N-terminal and C-terminal Extension Sequences

[0111] In some embodiments, the C-terminal extension sequences (also called "tails") comprise E and D residues, and may be between 8 and 50, 10 and 30, 10 and 20, 5 and 10, and 2 and 4 amino acids in length. In some embodiments, tail sequences include ED-based linkers in which the sequence comprises tandem repeats of ED. In exemplary embodiments, the tail sequence comprises 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4 or 5 ED repeats. In certain embodiments, the ED- based tail sequences may also include additional amino acid residues, such as, El, EID, ES, EC, EGS, and EGC. Such sequences are based, in part, on known Adnectin tail sequences, such as EIDKPSQ (SEQ ID NO: 18), in which residues D and K have been removed. In exemplary embodiments, the ED-based tail comprises an E, I or El residues before the ED repeats.

[0112] In some embodiments, the anti-myostatin Adnectins comprise a10Fn3 domain fused to an immunoglobulin Fc region, or a fragment or variant thereof. As used herein, a "functional Fc region" is an Fc domain or fragment thereof which retains the ability to bind FcRn. In some embodiments, a functional Fc region binds to FcRn, but does not possess effector function. The ability of the Fc region or fragment thereof to bind to FcRn can be determined by standard binding assays known in the art. In other embodiments, the Fc region or fragment thereof binds to FcRn and possesses at least one “effector function” of a native Fc region. Exemplary "effector functions" include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an anti-myostatin Adnectin) and can be assessed using various assays known in the art for evaluating such antibody effector functions.

[0113] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% sequence identity with a native sequence Fc region and / or with an Fc region of a parent polypeptide, and most preferably at least about 90% sequence identity therewith, more preferably at least about 95% sequence identity therewith.

[0114] In an exemplary embodiment, the Fc domain is derived from an IgGl subclass, however, other subclasses (e.g., IgG2, IgG3, and IgGl) may also be used. Shown below is the sequence of a human IgGl immunoglobulin Fc domain:

[0115] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSIIEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<E YKCKVSNKALPAPIEKHSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 76)

[0116] The core hinge sequence is underlined, and the CH2 and CH3 regions are in regular text. It should be understood that the C-terminal lysine (K) is optional. In some embodiments, the C- terminal “GK” is optional.

[0117] The fusion of the anti-myostatin Adnectin with the Fc region may be formed by attaching an anti-myostatin Adnectin to either end of the Fc molecule, i.e., Fc-anti-myostatin Adnectin or anti-myostatin Adnectin-Fc arrangements. In certain embodiments, the Fc and anti-myostatin Adnectin are fused via a linker. Exemplary linker sequences include GAGGGGSG (SEQ ID NO: 35), EPKSSD (SEQ ID NO: 36), D, ESPKAQASSVPTAQPQAEGLA (SEQ ID NO: 37), ELQLEESAAEAQDGELD (SEQ ID NO: 38), GQPDEPGGS (SEQ ID NO: 39), GGSGSGSGSGSGS (SEQ ID NO: 40), ELQLEESAAEAQEGELE (SEQ ID NO: 41), GSGSG (SEQ ID NO: 42), GSGC (SEQ ID NO: 43), AGGGGSG (SEQ ID NO: 44), GSGS (SEQ ID NO: 45), QPDEPGGS (SEQ ID NO: 46), GSGSGS (SEQ ID NO: 47), TVAAPS (SEQ ID NO: 48), KAGGGGSG (SEQ ID NO: 49), KGSGSGSGSGSGS (SEQ ID NO: 50), KQPDEPGGS (SEQ ID NO: 51), KELQLEESAAEAQDGELD (SEQ ID NO: 52), KTVAAPS (SEQ ID NO: 53), KAGGGGSGG (SEQ ID NO: 54), KGS GSGSGS GSGSG (SEQ ID NO: 55), KQPDEPGGSG (SEQ ID NO: 56), KELQLEESAAEAQDGELDG (SEQ ID NO: 57), KTVAAPSG (SEQ ID NO: 58) AGGGGSGG (SEQ ID NO: 59), AGGGGSG (SEQ ID NO: 60), GSGSGSGSGSGSG (SEQ ID NO: 61), QPDEPGGSG (SEQ ID NO: 62), and TVAAPSG (SEQ ID NO: 63). In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 35-63, or an amino acid sequence which differs from SEQ ID NOs: 64-72 by 1, 2, or 3 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0118] In some embodiments, the Fc region used in the anti-myostatin Adnectin comprises the hinge region of an Fc molecule. In some embodiments, the hinge region comprises the core hinge residues spanning positions 1-16 of SEQ ID NO: 76 (DKTHTCPPCPAPELLG; SEQ ID NO: 64) of the IgGl Fc region. In some embodiments, the hinge region comprises the amino acid of any one of SEQ ID NOs: 64-72, or an amino acid sequence which differs from SEQ ID NOs: 64-72 by 1, 2, or 3 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0119] In certain embodiments, the anti-myostatin Adnectin - Fc region fusion adopts a multimeric structure (e.g., dimer) owing, in part, to the cysteine residues at positions 6 and 9 of SEQ ID NO: 76 within the hinge region. In other embodiments, the hinge region as used herein, may further include residues derived from the CHI and CH2 regions that flank the core hinge sequence, as shown in SEQ ID NO: 76. In yet other embodiments, the hinge sequence is GSTHTCPPCPAPELLG (SEQ ID NO: 65).

[0120] In some embodiments, the hinge sequence may include substitutions that confer desirable pharmacokinetic, biophysical, and / or biological properties. Some exemplary hinge sequences include EPKS SDKTHTCPPCPAPELLGGPS (SEQ ID NO: 66; core hinge region underlined), EPKS SDKTHTCPPCP APELLGGS S (SEQ ID NO 67; core hinge region underlined), EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 68; core hinge region underlined), DKTHTCPPCPAPELLGGPS (SEQ ID NO: 69; core hinge region underlined), and DKTHTCPPCP APELLGGS S (SEQ ID NO: 70; core hinge region underlined). In one embodiment, the residue P at position 18 of SEQ ID NO: 76 has been replaced with S to ablate Fc effector function; this replacement is exemplified in hinges having any one of SEQ ID NOs: 67, 68, and 70. In another embodiment, the residues DK at positions 1-2 of SEQ ID NO: 76 have been replaced with GS to remove a potential clip site; this replacement is exemplified in SEQ ID NO: 68. In another embodiment, the C at position 103 of SEQ ID NO: 79, which corresponds to the heavy chain constant region of human IgGl (i.e., domains CH1-CH3), has been replaced with S to prevent improper cysteine bond formation in the absence of a light chain; this replacement is exemplified in SEQ ID NOs: 66-68. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQS SGLYSLS S WTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 80)

[0121] In certain embodiments, an anti-myostatin Adnectin-Fc fusion may have the following configurations: 1) anti-myostatin Adnectin-hinge-Fc or 2) hinge-Fc-anti-myostatin Adnectin. Therefore, any anti-myostatin Adnectin described herein can be fused to an Fc region comprising a hinge sequence according to these configurations. In some embodiments, a linker may be used to join the anti-myostatin Adnectin to the hinge-Fc moiety, for example, an exemplary fusion protein may have the configuration anti-myostatin Adnectin-linker-hinge-Fc or hinge-Fc-linker- anti-myostatin Adnectin. Additionally, depending on the system in which the fusion polypeptide is produced, a leader sequence may be placed at the N-terminus of the fusion polypeptide. For example, if the fusion is produced in a mammalian system, a leader sequence such as MRAWIFFLLCLAGRALA (SEQ ID NO: 74) or METDTLLLWVLLLWVPGSTG (SEQ ID NO: 75) may be added to the N-terminus of the fusion molecule. If the fusion is produced in E. coli, the fusion sequence will be preceded by a methionine.

[0122] In one embodiment, the polypeptide that binds to myostatin described herein is an Fc- anti-myostatin Adnectin construct known as BHV-2000. BHV-2000, which comprises BC, DE, and FG loop sequences of SEQ ID NOs: 5, 6, and 7, respectively, has the following sequence (hinge region is underlined, the linker is in italics, the leader sequence is in bold, and the anti- myostatin Adnectin sequence is underlined and in italics):

[0123] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSIIEDPEVKFN WYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<C I<VSNI<AL PAPIEKHSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT OK.SLSLSPELOLEESAAEAOEGELEGVSDVP DLEWAATPTSLLISWSLPHQGKANY YRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTDTGYLKYKPISIN YRTE1 (SEQ ID NO: 81). 1 B. Fc engineered Adnectins

[0124] In some embodiments, the polypeptide used in the methods described herein is an anti- myostatin Adnectin with an engineered Fc region, referred to herein as an “anti-myostatin Fc engineered Adnectin.” Accordingly, in some embodiments, the anti-myostatin Adnectin comprises a10Fn3 domain and an engineered Fc region, wherein the engineered Fc region comprises at least one amino acid modification (e.g., amino acid substitution), but no more than 15 amino acid modifications (e.g., amino acid substitutions), relative to a wild-type Fc region (e.g., a wild-type IgGl or IgG4 Fc region) or S228P IgG4 Fc domain. In some embodiments, the Fc region differs from a reference human Fc region (e.g., a wild-type IgGl Fc region or IgG4 Fc region or S228P IgG4 Fc domain) by at least one amino acid, but no more than 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 differences relative to the reference IgGl Fc region or IgG4 Fc region or S228P IgG4 Fc domain). In some embodiments, such engineered Fc regions differ in sequence from wild- type Fc regions and thus do not comprise the amino acid sequence of a wild-type Fc region (e.g., do not comprise the amino acid sequence of a wild- type human IgGl Fc region, human IgG2 Fc region, human IgG3 Fc region, human IgG4 Fc region). In some embodiments, such engineered Fc regions differ in sequence from a S228P IgG4 Fc domain (e.g., a human S228P IgG4 Fc domain).

[0125] In some embodiments, the engineered Fc region of an anti-myostatin Fc engineered Adnectin comprises the amino acid sequence of any one of SEQ ID NOs: 83-91, or an amino acid sequence which is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 83-91, or an amino acid sequence which differs from any one of SEQ ID NOs: 83-91 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0126] In some embodiments, the engineered Fc region of an anti-myostatin Fc engineered Adnectin comprises the amino acid sequence of any one of SEQ ID NOs: 92-101, or an amino acid sequence which is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 92-101, or an amino acid sequence which differs from any one of SEQ ID NOs: 92-101 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., by amino acid modifications, such as amino acid substitutions). In some embodiments, the polypeptide is an Fc engineered variant of the Fc-anti- myostatin Adnectin construct known as BHV-2000, wherein the Fc engineered variant comprises the sequence of SEQ ID NO: 81, with the exception of at least one amino acid modification (e.g., at least one amino acid substitution) but no more than 15 amino acid modifications (e.g., no more than 15 amino acid substitutions) in the Fc region.

[0127] In some embodiments, the anti-myostatin Fc engineered Adnectin binds to active myostatin with a KD of about 5-15 nM, for example, as assessed using biolayer interferometry.

[0128] In some embodiments, the anti-myostatin Fc engineered Adnectin bind with a stronger affinity to FcRn at pH. 6.0 compared to a polypeptide comprising the amino acid sequence of SEQ ID NO: 81, e.g., as assessed by biolayer interferometry.

[0129] In some embodiments, the anti-myostatin Fc engineered Adnectin binds to FcRn at pH 6.0 with a 40-60 fold (e.g., 40-55 fold, 40-50 fold, 40-45 fold, 45-60 fold, 45-55 fold, 45-50 fold, 50-60 fold, 55-60 fold) higher affinity than a polypeptide comprising the amino acid sequence of SEQ ID NO: 81, e.g., as assessed by biolayer interferometry.

[0130] In some embodiments, the anti-myostatin Fc engineered Adnectin has a lower (i.e., weaker) affinity for FcyRI relative to a polypeptide comprising the amino acid sequence of SEQ ID NO: 81, e.g., as assessed by biolayer interferometry.

[0131] In some embodiments, the anti-myostatin Fc engineered Adnectin has a lower (i.e., weaker) affinity for Cl q relative to a polypeptide comprising the amino acid sequence of SEQ ID NO: 81, e.g., as assessed by biolayer interferometry.

[0132] In some embodiments, the anti-myostatin Fc engineered Adnectin inhibits SMAD2 / 3 signaling.

[0133] In some embodiments, the anti-myostatin Fc engineered Adnectin has increased FcRn- mediated recycling relative to a polypeptide comprising the amino acid sequence of SEQ ID NO: 81, e.g., as assessed by the cell-based assay described in Example 6. In some embodiments, the increase in FcRn- mediated recycling over a polypeptide comprising the amino acid sequence of SEQ ID NO: 81 is at least 2-fold, at least 3-rold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12 fold, at least 13 -fold, at least 14-fold, at least 15-fold, about 2-fold to about 15-fold, about 5-fold to about 15-fold, about 8-fold to about 15-fold, about 10-fold to about 15-fold, about 13 -fold to about 15-fold, about 2-fold to about 13-fold, about 5-fold to about 13-fold, about 8-fold to about 13 fold, about 10-fold to about 13-fold, about 2-fold to about 10-fold, about 5-fold to about 10- fold, about 8-fold to about 10-fold, about 2-fold to about 8-fold, about 5-fold to about 8-fold, or about 2-fold to about 5-fold.

[0134] In some embodiments, the anti-myostatin Fc engineered Adnectin has a longer half-life (e.g., in circulation) relative to a polypeptide comprising the amino acid sequence of SEQ ID NO: 81. The half-life of the anti-myostatin Fc engineered Adnectins can be determined using art-recognized methods.

[0135] In some embodiments, the anti-myostatin Fc engineered Adnectin comprises (a) a10Fn3 domain which binds to myostatin described herein (e.g., the10Fn3 domain of BHV-2000 or a variant thereof), and (b) an Fc region which differs in amino acid sequence from a reference human IgGl Fc region comprising the amino acid sequence of SEQ ID NO: 78 or a reference human S228P IgG4 Fc region comprising the amino acid sequence of SEQ ID NO: 79 or wildtype human IgG4 Fc region (SEQ ID NO: 79 without the S228P substitution) by at least one amino acid but no more than 15 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0136] In some embodiments, the10Fn3 domain of the anti-myostatin Fc engineered Adnectin comprises BC, DE, and FG loops comprising the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, or a10Fn3 domain wherein at least one loop of the BC, DE, and FG loops has 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops of SEQ ID NOs: 5, 6, and 7, respectively. Exemplary substitutions to the BC, DE, and / or FG loops of SEQ ID NOs: 5, 6, and 7, respectively, are described supra.

[0137] In some embodiments, the10Fn3 domain of the anti-myostatin Fc engineered Adnectin comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence which is at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 8, or an amino acid sequence which differs from the amino acid sequence of SEQ ID NO: 8 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (e.g., by amino acid modifications, such as amino acid substitutions). In some embodiments, the10Fn3 domain comprises an N-terminal leader sequence and / or a C- terminal tail sequence. In some embodiments, the N-terminal leader sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 11-17, or the amino acid sequence RDL or DL. In some embodiments, the C-terminal leader sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 18-34, or the amino acid sequence El. In some embodiments, the10Fn3 domain comprises both an N-terminal leader sequence and a C-terminal tail sequence. Accordingly, in some embodiments, the10Fn3 domain comprises an N-terminal leader sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 11- 17, RDL, or DL, and the C-terminal leader sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 18-34 or El.

[0138] In some embodiments, the Fc region of the anti-myostatin Fc engineered Adnectin differs from the amino acid sequence of SEQ ID NO: 78 by at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids, but by no more than 15 amino acids (e.g., by amino acid modifications, such as amino acid substitutions). In some embodiments, the Fc region of the anti-myostatin Fc engineered Adnectin differs from the amino acid sequence of SEQ ID NO: 78 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (e.g., by amino acid modifications, such as amino acid substitutions). The difference in amino acid sequences relative to reference sequence SEQ ID NO: 78 may be introduced by site-directed mutagenesis using art-recognized methods, or the Fc region may be chemically synthesized to have the variant sequence.

[0139] In some embodiments, the Fc region of the anti-myostatin Fc engineered Adnectin differs from the amino acid sequence of SEQ ID NO: 79 by at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids, but by no more than 15 amino acids (e.g., by amino acid modifications, such as amino acid substitutions). In some embodiments, the Fc region of the anti-myostatin Fc engineered Adnectin differs from the amino acid sequence of SEQ ID NO: 79 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (e.g., by amino acid modifications, such as amino acid substitutions). The difference in amino acid sequences relative to reference sequence SEQ ID NO: 79 may be introduced by site-directed mutagenesis using art-recognized methods, or the Fc region may be chemically synthesized to have the variant sequence.

[0140] In some embodiments, the Fc region of the anti-myostatin Fc engineered Adnectin comprises an engineered IgGl Fc region. In some embodiments, the engineered IgGl Fc region comprises one or more of the following: an alanine at position 234 (e.g., a L234A substitution), an alanine at position 235 (e.g., a L235A substitution), a leucine at position 248 (e.g., an M248L substitution), a tyrosine at position 252 (e.g., an M252Y substitution), a threonine at position 254 (e.g., a S254T substitution), a glutamic acid at position 256 (e.g., a T256E substitution), a glutamine at position 297 (e.g., a N297Q substitution), an alanine at position 329 (e.g., a P329A substitution), and a serine at position 434 (e.g., a N434S substitution). In some embodiments, the engineered IgGl Fc region comprises an alanine at position 234 and an alanine at position 235 (e.g., a L234A / L235A substitution). In some embodiments, the engineered IgGl Fc region comprises an alanine at position 234, an alanine at position 235, and an alanine at position 329 (e.g., a L234A / L235A / P329A substitution). In some embodiments, the engineered IgGl Fc region comprises an alanine at position 234, an alanine at position 235, an alanine at position 329, a leucine at position 248, and a serine at position 434 (e.g., a L234A / L235A / P329A / M248L / N434S substitution). In some embodiments, the engineered IgGl Fc region comprises an alanine at position 234, an alanine at position 235, an alanine at position 329, a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256 (e.g., a L234A / L235A / P329A / M252Y / S254T / T256E substitution). In some embodiments, the engineered IgGl Fc region comprises a leucine at position 248 and a serine at position 434 (e.g., a M248L / N434S substitution). In some embodiments, the engineered IgGl Fc region comprises a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256 (e.g., a M252Y / S254T / T256E substitution). In some embodiments, the engineered IgGl Fc region comprises a tyrosine at position 252, a threonine at position 254, a glutamic acid at position 256, and a glutamine at position 297 (e.g., a M252Y / S254T / T256E / N297Q substitution). In some embodiments, the engineered IgGl Fc region comprises a leucine at position 248, a glutamine at position 297, and a serine at position 424 (e.g., a N297Q / M248L / N434S substitution). In some embodiments, the engineered IgGl Fc region comprises the amino acid sequence of any one of SEQ ID NOs: 83-89, or an amino acid sequence which is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 83-89, or an amino acid sequence which differs from any one of SEQ ID NOs: 83-89 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0141] In some embodiments, the Fc region of the anti-myostatin Fc engineered Adnectin comprises an engineered IgG4 Fc region. In some embodiments, the engineered IgG4 Fc region comprises one or more of the following: an alanine at position 234 (e.g., a F234A substitution), an alanine at position 235 (e.g., a L235A substitution), a tyrosine at position 252 (e.g., an M252Y substitution), a threonine at position 254 (e.g., a S254T substitution), a glutamic acid at position 256 (e.g., a T256E substitution), and a glutamine at position 297 (e.g., a N297Q substitution). In some embodiments, the engineered IgG4 Fc region comprises an alanine at position 234, an alanine at position 235, a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256 (e.g., a F234A / L235A / M252Y / S254T / T256E substitution). In some embodiments, the engineered IgG4 Fc region comprises a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256, and a glutamine at position 297 (e.g., a M252Y / S254T / T256E / N297Q substitution). In some embodiments, the engineered IgG4 Fc region comprises the amino acid sequence of SEQ ID NO: 90 or 91, or an amino acid sequence which is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90 or 91, or an amino acid sequence which differs from SEQ ID NO: 90 or 91 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0142] In some embodiments, the numbering of positions in the Fc region for embodiments described herein is according to the EU Index.

[0143] In some embodiments, the engineered Fc region of the anti-myostatin Fc engineered Adnectin comprises a hinge sequence, for example, a hinge sequence comprising the amino acid sequence selected from any one of SEQ ID NOs: 64-72, or an amino acid sequence which is at least 90% identical (e.g., at least 95% identical) to an amino acid sequence selected from any one of SEQ ID NOs: 64-72, or an amino acid sequence which differs from any one of SEQ ID NOs: 64-72 by 1, 2, or 3 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0144] In some embodiments, the engineered Fc region of the anti-myostatin Fc engineered Adnectin comprises the amino acid sequence of any one of SEQ ID NOs: 92-99. In some embodiments, the engineered Fc region of the anti-myostatin Fc engineered Adnectin comprises an amino acid sequence which is at least 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 92-99. In some embodiments, the engineered Fc region of the anti-myostatin Fc engineered Adnectin comprises an amino acid sequence which differs from any one of SEQ ID NOs: 92-99 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., by amino acid modifications, such as amino acid substitutions). In some embodiments, the anti-myostatin Fc engineered Adnectin comprises a signal peptide at the N-terminus. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 74 or 75, or an amino acid sequence which differs from SEQ ID NO: 74 or 75 by 1, 2, or 3 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0145] In some embodiments, the engineered Fc region is linked to the10Fn3 domain of the anti- myostatin Fc engineered Adnectin via a linker (e.g., a peptide linker). In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 35-63, or an amino acid sequence which differs from any one of SEQ ID NOs: 35-63 by 1, 2, or 3 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0146] In some embodiments, the Fc region is located N-terminal to the10Fn3 domain and thus has the configuration Fc domain -10Fn3 domain, Fc domain - linker -10Fn3 domain, or signal peptide - Fc domain - linker -10Fn3 domain.

[0147] In some embodiments, the Fc region is located C-terminal to the10Fn3 domain and thus has the configuration10Fn3 domain - Fc region,10Fn3 domain- linker - Fc region, or signal peptide -10Fn3 domain - linker - Fc region.

[0148] In some embodiments, the anti-myostatin Fc engineered Adnectin comprises the amino acid sequence of any one of SEQ ID NO: 102-111, or a polypeptide comprising an amino acid sequence which is at least 90%, 95%, 98%, or 99% identical to any one of SEQ ID NOs: 102- 111, or a polypeptide which differs from the amino acid sequence of any one of SEQ ID NOs: 102-111 by at least one amino acid but no more than 30, 20, or 10 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0149] In some embodiments, the anti-myostatin Fc engineered Adnectin comprises the amino acid sequence of any one of SEQ ID NO: 112-121, or a polypeptide comprising an amino acid sequence which is at least 90%, 95%, 98%, or 99% identical to any one of SEQ ID NOs: 102- 111, or a polypeptide which differs from the amino acid sequence of any one of SEQ ID NOs: 102-111 by at least one amino acid but no more than 30, 20, or 10 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0150] In some embodiments, the anti-myostatin Fc engineered Adnectin comprises the amino acid sequence of any one of SEQ ID NO: 122-131, or a polypeptide comprising an amino acid sequence which is at least 90%, 95%, 98%, or 99% identical to any one of SEQ ID NOs: 102- 111, or a polypeptide which differs from the amino acid sequence of any one of SEQ ID NOs: 102-111 by at least one amino acid but no more than 30, 20, or 10 amino acids (e.g., by amino acid modifications, such as amino acid substitutions).

[0151] III. Compositions and stable formulations of polypeptides that bind to myostatin

[0152] In some embodiments, the polypeptides that bind to myostatin (e.g., anti-myostatin Adnectins) for use in the methods described herein are administered as a composition (e.g., pharmaceutical composition) comprising the anti-myostatin Adnectin and a carrier (e.g., a pharmaceutically acceptable carrier). Suitable carriers for the composition are known in the art, for example, in U.S. Patent No. 8,853,154, the content of which are herein incorporated by reference in its entirety.

[0153] In some embodiments, the anti-myostatin Adnectins described herein are administered to a subject (e.g., a human patient) as a stable formulation comprising the anti-myostatin Adnectin.

[0154] A. Exemplary Formulations

[0155] In some embodiments, the concentration of the anti-myostatin Adnectin in the formulation is between about 10 mg / mL and 200 mg / mL. In other embodiments, the concentration of the anti-myostatin Adnectin in the formulation is between about 10 mg / mL and 140 mg / mL. In other embodiments, the concentration of the anti-myostatin Adnectin in the formulation is least about 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50, mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL or higher. In certain embodiments, the concentration of the anti- myostatin Adnectin in the formulation is at least about 110 mg / mL, 115 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140, mg / mL or 145, mg / m L. In certain embodiments, the concentration of the anti-myostatin Adnectin in the formulation is 10.7 mg / mL, 21.4 mg / mL, 50.0 mg / mL or 71.4 mg / mL.

[0156] The stabilizing sugar in the formulation is a disaccharide in a weight (w / w) ratio of at least 5: 1 protein to sugar. In some embodiments, the protein: sugar weight ratio is between about 5:1 to 10:1. In some embodiments, the proteimsugar ratio is about 6:1, 7: 1, 8:1, 9: 1 or 10: 1. In some embodiments, the proteimsugar ratio is about 6.75:1. In some embodiments, the formulation comprises about 5% to about 30% of the disaccharide. In some embodiments, the formulation comprises about 10% to about 28% of the disaccharide. In some embodiments, the formulation comprises about 15% to about 25% of the disaccharide. In some embodiments, the formulation comprises about 20% to about 25% of the disaccharide. In some embodiments, the formulation comprises about 18%, 19%, 20%, 21%, 22%, 23%, 24% or about 25% of the disaccharide.

[0157] In some embodiments, the concentration of the sugar in the formulation is about 150 mM to about 800 mM. In some embodiments, the concentration of the sugar in the formulation is about 300 to about 700 mM. In other embodiments, the concentration of the sugar in the formulation is about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 575 mM, about 600, about

[0158] 625 mM, about 650 mM, about 675 mM or about 700 mM.

[0159] In some embodiments, the disaccharide is trehalose. In some embodiments, the formulation comprises about 5 to about 30% trehalose. In some embodiments, the formulation comprises about 10% to about 28% trehalose. In some embodiments, the formulation comprises about 15% to about 25% trehalose. In some embodiments, the formulation comprises about 20% to about 25% trehalose. In some embodiments, the formulation comprises about 18%, 19%, 20%, 21%, 22%, 23%, 24% or about 25% trehalose. In one embodiment, the formulation comprises 22% trehalose. In another embodiment, the formulation comprises 23% trehalose.

[0160] In some embodiments, the disaccharide is trehalose dihydrate. In some embodiments, concentration of trehalose dihydrate in the formulation is about 150 mM to about 800 mM. In some embodiments, the concentration of the trehalose dihydrate in the formulation is about 300 to about 700 mM. In other embodiments, the concentration of the trehalose dihydrate in the formulation is about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 575 mM, about 600, about 625 mM, about 650 mM, about 675 mM or about 700 mM. In one embodiment, the concentration of trehalose dihydrate in the formulation is 600 nM.

[0161] The stabilizing sugar in the formulation is employed in an amount no greater than that which may result in a viscosity undesirable or unsuitable for administration via SC syringe. In some embodiments, the viscosity of the formulation is from about 5 to 20 cps. In some embodiments, the viscosity of the formulation is from about 7 to 12 cps. In some embodiments, the viscosity is about 7-10 cps. In some embodiments, the viscosity of the formulation is less than 8 cps.

[0162] The buffering agent in the formulation is present in an amount of at least 20 mM, and is preferably between about 20 mM and about 40 mM. In some embodiments, the buffering agent is histidine at a concentration of about 20 mM, about 25 mM, about 30 mM or about 35 mM. In one embodiment, the formulation comprises about 30 mM histidine.

[0163] The pH of the formulation is maintained at a range from about 6.5 to about 7.8. In certain embodiments, the pH is maintained at a range from about pH from 6.6 to 7.6. In certain embodiments, the pH of the formulation is about 6.8 to 7.4. In certain embodiments, the pH of the formulation is about 7.0 to 7.3. In some embodiments, the pH of the formulation is 6.9, 7.0, 7.1, 7.2 or 7.3. In some embodiments, the pH of the formulation is about 7.1.

[0164] The aqueous carrier used in the formulations herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0165] The formulations may further comprise a surfactant to further reduce the formation of visible particulates. Preferred surfactants include poloxamer and polysorbate at a concentration of between about 0.01% and 0.5%. In some embodiments, the concentration of the surfactant is between about 0.02% and about 0.1%. In one embodiment, the surfactant is poloxamer 188. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. In one embodiment, the surfactant is polysorbate 80.

[0166] The formulations may further comprise a chelator at a concentration between about 0.01 mM and about 0.5 mM, preferably, between about 0.05 mM and 0.2 mM. Preferred chelators include, but are not limited to DPTA, EDTA and EGTA. In one embodiment, the chelator in the formulation is DPTA at a concentration of about 0.05 mM.

[0167] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.

[0168] In some embodiments, the formulation provided herein comprises:

[0169] (i) about 10-140 mg / mL of the anti-myostatin Adnectin; 1 (ii) about 5-25% trehalose dihydrate; and

[0170] (iii) about 20-30 mM histidine, wherein the pH of the formulation is about 6.8 to 7.3.

[0171] In some embodiments, the formulation provided herein consists essentially of:

[0172] (i) about 10-140 mg / mL of the anti-myostatin Adnectin;

[0173] (ii) about 5-25% trehalose dihydrate; and

[0174] (iii) about 20-30 mM histidine, wherein the pH of the formulation is about 6.8 to 7.3.

[0175] In some embodiments, the formulation provided herein comprises:

[0176] (i) about 10-140 mg / mL of the anti-myostatin Adnectin;

[0177] (ii) about 5-25% trehalose dihydrate;

[0178] (iii) about 20-30 mM histidine;

[0179] (iv) about 0.02-0.06 mM DTPA; and

[0180] (v) about 0.01-0.05% polysorbate 80 wherein the pH of the formulation is about 6.8 to 7.3.

[0181] In some embodiments, the formulation provided herein consists essentially of:

[0182] (i) about 10-140 mg / mL of the anti-myostatin Adnectin;

[0183] (ii) about 5-25% trehalose dihydrate;

[0184] (iii) about 20-30 mM histidine;

[0185] (iv) about 0.02-0.06 mM DTPA; and

[0186] (v) about 0.01-0.05% polysorbate 80 wherein the pH of the formulation is about 6.8 to 7.3.

[0187] In some embodiments, the formulation comprises:

[0188] (i) about 10-75 mg / mL of the anti-myostatin Adnectin;

[0189] (ii) about 600 mM trehalose dihydrate; and

[0190] (iii) 25-30 mM histidine, wherein the pH of the formulation is about 7.0 to 7.3.

[0191] In some embodiments, the formulation consists essentially of:

[0192] (i) about 10-75 mg / mL of the anti-myostatin Adnectin;

[0193] (ii) about 600 mM trehalose dihydrate; and

[0194] (iii) 25-30 mM histidine, wherein the pH of the formulation is about 7.0 to 7.3.

[0195] In some embodiments, the formulation comprises:

[0196] (i) about 10-75 mg / mL of the anti-myostatin Adnectin;

[0197] (ii) about 600 mM trehalose dihydrate;

[0198] (iii) 25-30 mM histidine;

[0199] (iv) about 0.02-0.06 mM DTPA; and

[0200] (v) about 0.01-0.05% polysorbate 80, wherein the pH of the formulation is about 7.0 to 7.3.

[0201] In some embodiments, the formulation consists essentially of:

[0202] (i) about 10-75 mg / mL of the anti-myostatin Adnectin;

[0203] (ii) about 600 mM trehalose dihydrate;

[0204] (iii) 25-30 mM histidine;

[0205] (iv) about 0.02-0.06 mM DTPA; and

[0206] (v) about 0.01-0.05% polysorbate 80, wherein the pH of the formulation is about 7.0 to 7.3.

[0207] In some embodiments, the formulation comprises

[0208] (i) about 10-75 mg / mL of the anti-myostatin Adnectin;

[0209] (ii) about 600 mM trehalose dihydrate;

[0210] (iii) about 30 mM histidine;

[0211] (iv) about 0.05 mM DTPA; and

[0212] (v) about 0.02% polysorbate 80, wherein the pH of the formulation is about 7.1.

[0213] In some embodiment, the formulation consists essentially of:

[0214] (i) about 10-75 mg / mL of the anti-myostatin Adnectin;

[0215] (ii) about 600 mM trehalose dihydrate;

[0216] (iii) about 30 mM histidine;

[0217] (iv) about 0.05 mM DTPA; and

[0218] (v) about 0.02% polysorbate 80, wherein the pH of the formulation is about 7.1.

[0219] In one embodiment, the formulation comprises or consists essentially of:

[0220] (i) about 10.7 mg / mL of the anti-myostatin Adnectin; (ii) about 600 mM trehalose dihydrate;

[0221] (iii) about 30 mM histidine;

[0222] (iv) about 0.05 mM DTPA; and

[0223] (v) about 0.02% polysorbate 80, wherein the pH of the formulation is about 7.1.

[0224] In one embodiment, the formulation comprises or consists essentially of:

[0225] (i) about 21.4 mg / mL of the anti-myostatin Adnectin;

[0226] (ii) about 600 mM trehalose dihydrate;

[0227] (iii) about 30 mM histidine;

[0228] (iv) about 0.05 mM DTPA; and

[0229] (v) about 0.02% polysorbate 80, wherein the pH of the formulation is about 7.1.

[0230] In one embodiment, the formulation comprises or consists essentially of:

[0231] (i) about 50 mg / mL of the anti-myostatin Adnectin;

[0232] (ii) about 600 mM trehalose dihydrate;

[0233] (iii) about 30 mM histidine;

[0234] (iv) about 0.05 mM DTPA; and

[0235] (v) about 0.02% polysorbate 80, wherein the pH of the formulation is about 7.1.

[0236] In one embodiment, the formulation comprises or consists essentially of:

[0237] (i) about 71.4 mg / mL of the anti-myostatin Adnectin;

[0238] (ii) about 600 mM trehalose dihydrate;

[0239] (iii) about 30 mM histidine;

[0240] (iv) about 0.05 mM DTPA; and

[0241] (v) about 0.02% polysorbate 80, wherein the pH of the formulation is about 7.1.

[0242] The recommended storage condition for the liquid formulation is from 2-8°C, with a recommended shelf life of at least 12 months. In order to ensure efficacy and safety during the time course of the shelf life of pharmaceutical formulations, the composition is stability tested. Typically, the stability tests include but are not limited to tests regarding identity, purity and potency of the composition. The stability is tested both at the intended storage temperature and at elevated temperature or temperatures. Purity tests may include but are not limited to SDS- PAGE, CE-SDS, isoelectrofocusing, immunoelectrophoresis, Western blot, reversed-phase chromatography, size-exclusion chromatography (SEC), ion exchange and affinity chromatography. Other tests may include, but are not limited to: visual appearance such as color and transparency, particulates, pH, protein concentration measurement, moisture and reconstitution time.

[0243] The degradation profile regarding, in particular, purity and potency, during the stability time course is intimately coupled to the composition and / or the formulation of the pharmaceutical product. In particular, proper choice of formulation may significantly change the degradation profile. Typical degradation profiles for protein molecules products derived from FBS includes the formation of covalent and non-covalent high molecular weight aggregates, fragments, deamidation and oxidation products. Particularly, de-amidation and oxidation products as well as other acidic species usually develop during the time course of the stability testing. In some cases, the acidic species limits the acceptable shelf life of the pharmaceutical composition. The formation of acidic species due to, for example, deamidation, can be tested by, e.g., imaged capillary isoelectrofocusing (icIEF). In other cases, the formation of high molecular weight aggregates limits the acceptable shelf life of the pharmaceutical composition. The formation of aggregates may be tested by for example SEC (size exclusion chromatography), DLS, MFI, SDS-PAGE or CE-SDS.

[0244] For example, an anti-myostatin Adnectin formulation with pharmaceutically acceptable stability can be one wherein, when stored at a temperature of about 5 ± 3°C or 25 ± 2°C for a period of least about 6 weeks, for example, about 3 months, preferably about 6 months, and more preferably about 12 months or longer, such as 18 months or longer, such as for at least 24 or even 36 months, the percentage of aggregates is less than about 10%, preferably less than about 5%, more preferably less than about 2%, when determined using SEC analysis. Additionally or alternatively, a stable anti-myostatin Adnectin formulation of the invention can be one wherein, when stored at a temperature of about 5 ± 3 °C or 25 ± 2°C for a period of at least about 6 weeks, for example, about 3 months, preferably about 6 months, and more preferably about 12 months or longer, the changes of main isoform are less than 15%, preferably less than 10%, more preferably less than 8%, most preferably less than 5%, when determined using icIEF analysis. B. Preparation of the Formulation

[0245] The manufacturing process developed for SC formulations typically involves compounding with sugar, chelating agent and surfactant, followed by aseptic sterile filtration and filling into vials or syringes, optionally preceded by diafiltration (buffer exchange) and concentration of drug substance using an ultrafiltration unit. The protein purification is the first stage after production in the fermentation bioreactor. The protein is purified using multiple column and filtration steps and concentrated using tangential flow filtration into the formulation buffer. The concentrated drug substance is diluted with the formulation buffer at the target concentrations and this solution is sterile filtered and filled into sterile vials / syringes for patient use. One skilled in the art would be aware of the need to overfill the container so as to compensate for vial, needle, syringe hold-up during preparation and injection. For example, a 5- 10% overage of drug product is incorporated into each vial of liquid formulation to account for withdrawal losses and guarantee that required dose (label claim) of drug product can be withdrawn from the vial.

[0246] Preparation of unit dosage forms for the formulation comprising the anti-myostatin Adnectin involves protein production in a recombinant cell line, purification vial multiple column steps, concentration and buffer exchange into formulation buffer using tangential flow filtration. The concentrated protein for the tangential flow filtration is further processed by dilution with formulation buffer to the target protein concentrations and the diluted product, after filtration, is filled into 1 mb syringes (e.g., insulin syringe, tuberculin syringe, BioPak syringe, NeoPak syringe). In one embodiment, the syringes are then equipped with an UltraSafe Passive needle guard.

[0247] The unit dosage form of the formulation typically contains about 0.3 to 1.5 mL of the formulation. In certain embodiments, the unit dosage form contains a volume of 0.3, 0.5, 0.7, 0.8, 1.0, 1.2, 1.4 or 1.4 mL. In certain embodiments, the unit dosage form is provided at a volume of 0.7 mL. In some embodiments, the unit dosage form contains 5-100 mg of the anti- myostatin Adnectin. In some embodiments, the unit dosage form comprises 7.5 mg, 15 mg, 35 mg or 50 mg of the anti-myostatin Adnectin.

[0248] In some embodiments, the formulations are manufactured as disclosed herein and are stored in bulk at -60°C, for example, in 12L FFTp bags at polypeptide concentration of 85-150 mg / mL. In some embodiments, the formulations are stored at -60°C at an anti-myostatin Adnectin concentration of 85 mg / mL. The bulk formulations are then thawed and diluted to the appropriate anti-myostatin Adnectin concentration for preparation of the unit dosage forms. In some embodiments, the anti-myostatin Adnectin concentration of the formulation in the unit dosage form is about 10 mg / mL to about 140 mg / mL. In some embodiments, the anti-myostatin Adnectin concentration of the formulation in the unit dosage form is about 10 mg / mL to about 75 mg / mL. In certain embodiments, the anti-myostatin Adnectin concentration of the formulation in the unit dosage form is 10.7 mg / mL, 20.4 mg / ml, 50 mg / mL or 71.4 mg / mL.

[0249] C. Administration

[0250] A pharmaceutical formulation comprising an anti-myostatin Adnectin described herein can be administered to a subject at risk for or exhibiting pathologies as described herein. The formulations provided herein are particularly useful for peripheral systemic delivery by intravenous, intraperitoneal or subcutaneous injection. In some embodiments, the formulations are delivered by subcutaneous injection.

[0251] A therapeutically effective dose refers to a dose that produces the therapeutic effects for which it is administered. An effective amount of a pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding agent molecule is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient.

[0252] The exact dosage is determined based on factors related to the subject requiring treatment and can be ascertained using standard techniques. Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and sex of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy.

[0253] The polypeptides (anti-myostatin Adnectins) are administered in the target population at any suitable dose. In one embodiment, the polypeptides are administered as repeated subcutaneous injections. In one embodiment, the polypeptides are administered at a dosing interval of once a week. In one embodiment, the polypeptides are administered at a dosing interval of once every two weeks. In one embodiment, the polypeptides are administered at a dosing interval of once a month.

[0254] In one embodiment, the therapeutic dose level(s) and dosing interval(s) are determined by the pharmacokinetic: pharmacodynamic (PK / PD) correlation of the anti-myostatin Adnectin in the target population, as determined in a controlled clinical trial. Desired pharmacodynamic changes include, but are not limited to, reduction in free (unbound) myostatin, improvements in body composition and total body weight.

[0255] The frequency of dosing depends upon the pharmacokinetic: pharmacodynamic parameters of the binding agent molecule in the formulation used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition can therefore, be administered as a single dose or as multiple doses (at the same or different concentrations / dosages) over time. Further refinement of the appropriate dosage is routinely made. Appropriate dosages can be ascertained through use of appropriate dose-response data. For example, the anti-myostatin Adnectin can be administered less frequently (e.g., bi-weekly, or monthly). In addition, as is known in the art, adjustments for age as well as the body weight, general health, sex, time of administration, drug interaction, and the severity of the disease may be necessary, and re ascertainable with routine experimentation by those skilled in the art. The anti-myostatin Adnectin can be suitably administered to the patient at one time or over a series of treatments.

[0256] IV. GLP-1 receptor agonists

[0257] GLP-1 receptor agonists (e.g., FDA-approved GLP-1 receptor agonists) suitable for use in combination with anti-myostatin Adnectins described herein in the methods provided herein mimic the effects of the hormone GLP-1 and lower blood sugar levels and promote weight loss.

[0258] GLP-1 receptor agonists are recommended as an add-on therapy to lifestyle intervention (calorie restriction and exercise) in people with a BMI >30 or a BMI >27 with at least one weight-related comorbidity.

[0259] GLP-1 receptor agonists suitable for use in the methods provided herein include, but are not limited to, Albiglutide (Tanzeum), Dulaglutide (Trulicity®), Exenatide (Byetta®), Exenatide extended-release (Bydureon®), Liraglutide (Victoza®;Saxenda®), Lixisenatide (Adlyxin®), Semaglutide injection (Ozempic®; Wegovy®), Semaglutide tablets (Rybelsus®), Tirzepatide (Mounjaro®; Zepbound®), and maridebart cafraglutide (MariTide, AMG133). In some embodiments, the GLP-1 receptor agonist is Semaglutide injection (Ozempic®;Wegovy®), Semaglutide tablets (Rybelsus®), or Tirzepatide (Mounjaro®; Zepbound®). In some embodiments, the GLP-1 receptor agonist is semaglutide. In one embodiment, the GLP-1 receptor agonist is tirzepatide.

[0260] In some embodiments, the GLP-1 receptor agonist is administered subcutaneously or orally. In some embodiments, the subject is an adult. In some embodiments, the subject is a pediatric patient at least 10 years old. In some embodiments, the subject is a pediatric patient at least 12 years old.

[0261] In some embodiments, the subject is administered the recommended dosage of the GLP-1 receptor agonist. For example, in some embodiments, the method comprises subcutaneous administration of albiglutide once a week at a dose of between 30 mg and 50 mg. In some embodiments, the method comprises subcutaneous administration of dulaglutide once a week at a dosage of between 0.75 mg and 4.5 mg, or about 0.75 mg, about 1.5 mg, about 3.0 mg, or about 4.5 mg, or 0.75 mg, 1.5 mg, 3.0 mg, or 4.5 mg. In some embodiments, the method comprises subcutaneous administration of exentatide twice a day at a dose between 5 mcg and 10 mcg. In some embodiments, the method comprises subcutaneous administration of exenatide extended-release form (Bydureon®) once a week at a dose of 2 mg. In some embodiments, the method comprises subcutaneous administration of liraglutide once a day at a dose between 0.6 mg and 3 mg. In some embodiments, the method comprises subcutaneous administration of liraglutide once a day at a dose of about 0.6 mg, about 1.2 mg, about 1.8 mg, about 2.4 mg, or about 3.0 mg per day. In some embodiments, the method comprises subcutaneous administration of liraglutide once a day at a dose of 0.6 mg, 1.2 mg, 1.8 mg, 2.4 mg, or 3.0 mg per day. In some embodiments, the method comprises subcutaneous administration of lixisenatide once a day at an initial dose of 10 mcg for 14 days followed by a maintenance dose of 20 mcg once a day.

[0262] In some embodiments, the method comprises administration of semaglutide. In some embodiments, the semaglutide is administered subcutaneously once a week at a dose of between 0.25 mg to 1 mg. In some embodiments, the semaglutide is administered subcutaneously once a week at a dose of about 0.25 mg, about 0.5 mg, about 0.75 mg, or about 1 mg. In some embodiments, the semaglutide is administered subcutaneously once a week at a dose of 0.25 mg, 0.5 mg, 0.75 mg, or 1 mg. In other embodiments, the method comprises daily administration of semaglutide orally at a dose of between about 1.5 mg and about 14 mg. In some embodiments, the semaglutide is orally administered once a day at a dose of about 1.5 mg, about 3 mg, about 7 mg, or about 14 mg. In some embodiments, the semaglutide is orally administered once a day at a dose of 1.5 mg, 3 mg, 7 mg, or 14 mg.

[0263] In some embodiments, the method comprises subcutaneous administration of tirzepatide once a week at a dose of between 2.5 mg and 15 mg. In some embodiments, the tirzepatide is administered subcutaneous once a week at a dose of about 2.5 mg, about 5.0 mg, about 7.5 mg, about 10.0 mg, about 12.5 mg, or about 15 mg. In some embodiments, the tirzepatide is administered subcutaneous once a week at a dose of 2.5 mg, 5.0 mg, 7.5 mg, 10.0 mg, 12.5 mg, or 15 mg.

[0264] V. Methods

[0265] Provided herein are methods related to decreasing body weight while increasing lean muscle mass or preventing significant loss of lean muscle mass in subjects being treated with a GLP-1 receptor agonist using an anti-myostatin Adnectin described herein in combination with a GLP-1 receptor agonist (e.g., semaglutide). Such methods are relevant for the treatment of, e.g., metabolic diseases, disorders, or conditions, overweight or obesity and related comorbidities, and type II diabetes. They are also relevant for improving glycemic control, and increasing the ratio of lean muscle mass to fat. In some embodiments, the subject to be treated is a human (e.g., a human patient). In some embodiments, the anti-myostatin Adnectins and GLP-1 receptor agonist are pharmaceutically acceptable to a mammal, in particular a human. A "pharmaceutically acceptable" polypeptide refers to a polypeptide that is administered to an animal without significant adverse medical consequences, such as essentially endotoxin free, or very low endotoxin levels.

[0266] Accordingly, in one aspect, provided herein is a method of decreasing body weight and prevent significant loss of lean muscle mass in a subject (e.g., a human patient), the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) and an effective amount of an GLP-1 receptor agonist (e.g., semaglutide) such that body weight is decreased and significant loss of lean muscle mass is prevented in the subject. In another aspect, provided herein is a method of decreasing body weight and increasing lean muscle mass in a subject (e.g., a human patient), the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) and an effective amount of an GLP-1 receptor agonist (e.g., semaglutide) such that body weight is decreased and lean muscle mass is increased in the subject.

[0267] In another aspect, provided herein is a method of treating a metabolic disease, disorder, or condition in a subject (e.g., a human patient), the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) and an effective amount of an GLP-1 receptor agonist (e.g., semaglutide) such that the metabolic disease, disorder, or condition is treated in the subject. Exemplary metabolic diseases, disorders, or conditions which can be treated include, for example, diabetes (e.g., type II diabetes), prediabetes, hyperglycemia, hyperinsulinemia, hyperlipidemia, insulin resistance, impaired glucose metabolism, obesity, metabolic syndrome, glucose intolerance, prediabetes, hypertension, dyslipidemia, fatty liver disorder, increased waist circumference, cardiovascular disease, non-alcoholic fatty liver disease, obstructive sleep apnea, physical impairment, osteoarthritis, osteoporosis, renal disease, sexual hormone(s) impairment, endocrine reproductive disorders such as polycystic ovary syndrome or male hypogonadism, stroke, and gallstones.

[0268] In another aspect, provided herein is a method of increasing the ratio of lean muscle mass to fat in a subject (e.g., a human patient), the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) and an effective amount of an GLP-1 receptor agonist (e.g., semaglutide) such that the ratio of lean muscle mass to fat is increased in the subject.

[0269] In another aspect, provided herein is a method of treating, preventing, or reducing overweight or obesity and related comorbidities in a subject (e.g., a human patient), comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) and an effective amount of an GLP-1 receptor agonist (e.g., semaglutide) such that overweight or obesity and related comorbidities is treated, prevented, or reduced in the subject. Obesity is a condition in which excess body fat has accumulated to such an extent that health may be negatively affected. It is commonly defined as a body mass index (BMI) of 30 kg / m2or higher which distinguishes it from being overweight as defined by a BMI of 25 kg / m2or higher (see, e.g., World Health Organization (2000) (PDF). Technical report series 894: Obesity: Preventing and managing the global epidemic. Geneva: World Health Organization). Excessive body weight is associated with various diseases, particularly cardiovascular diseases, diabetes mellitus type II, obstructive sleep apnea, certain types of cancer, and osteoarthritis.

[0270] A subject with obesity may be identified, for example, by determining BMI (BMI is calculated by dividing the subject's mass by the square of his or her height), waist circumference and waist-hip ratio (the absolute waist circumference (>102 cm in men and >88 cm in women) and the waist-hip ratio (the circumference of the waist divided by that of the hips of >0.9 for men and >0.85 for women) (see, e.g., Yusuf S, et al., (2004). Lancet 364: 937-52), and / or body fat percentage (total body fat expressed as a percentage of total body weight: men with more than 25% body fat and women with more than 33% body fat are obese; body fat percentage can be estimated from a person's BMI by the following formula: Bodyfat% = (1.2 * BMI) + (0.23 * age) - 5.4 - (10.8 * gender), where gender is 0 if female and 1 if male). Body fat percentage measurement techniques include , for example, computed tomography (CT scan), magnetic resonance imaging (MRI), and dual energy X-ray absorptiometry (DEXA).

[0271] In another aspect, provided herein is a method of treating or preventing type II diabetes in a subject (e.g., a human patient), comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) and an effective amount of an GLP-1 receptor agonist (e.g., semaglutide) such that type II diabetes is treated or prevented in the subject.

[0272] Myostatin is involved in the pathogenesis of type II diabetes mellitus. Myostatin is expressed in adipose tissue and myostatin deficient mice exhibit reduced fat accumulation as they age. Moreover, glucose load, fat accumulation, and total body weight are reduced in myostatin lacking agouti lethal yellow and obese (Lepob / ob) mice (Yen et al., FASEB J. 8:479, 1994; McPherron et al., 2002). As disclosed in US2011 / 0008375, myostatin antagonists can decrease the fat to muscle ratio in an aged mouse model, preserve skeletal muscle mass and lean body mass, and attenuate kidney hypertrophy in STZ-induced diabetic mice. The term “type II diabetes” refers to a chronic, life-long disease that results when the body’s insulin does not work effectively. A main component of type II diabetes is "insulin resistance," wherein the insulin produced by the pancreas cannot connect with fat and muscle cells to allow glucose inside to produce energy, causing hyperglycemia (high blood glucose). To compensate, the pancreas produces more insulin, and cells, sensing this flood of insulin, become even more resistant, resulting in a vicious cycle of high glucose levels and often high insulin levels. The phrase “disorders associated with diabetes” or “diabetes associated disorders” or “diabetes related disorders,” as used herein, refers to conditions and other diseases which are commonly associated with or related to diabetes. Example of disorders associated with diabetes include, for example, hyperglycemia, hyperinsulinaemia, hyperlipidaemia, insulin resistance, impaired glucose metabolism, obesity, diabetic retinopathy, macular degeneration, cataracts, diabetic nephropathy, glomerulosclerosis, diabetic neuropathy, erectile dysfunction, premenstrual syndrome, vascular restenosis, ulcerative colitis, coronary heart disease, hypertension, angina pectoris, myocardial infarction, stroke, skin and connective tissue disorders, foot ulcerations, metabolic acidosis, arthritis, and osteoporosis.

[0273] The efficacy of the combination of anti-myostatin Adnectins and GLP-1 receptor agonist (e.g., semaglutide) in the treatment of metabolic disorders can be determined, for example, by one or more methods of measuring an increase in insulin sensitivity, an increase in glucose uptake by cells from the subject, a decrease in blood glucose levels, increase in muscle mass, and a decrease in body fat.

[0274] For example, in subjects having type II diabetes or who are at risk of developing diabetes, HbAlc levels can be monitored. The term “hemoglobin 1 AC” or “HbAlc” as used herein refers to the product of a non-enzymatic glycation of the hemoglobin B chain. The desired target range of HbAlc levels for people with diabetes can be determined from American Diabetes Association (ADA) guidelines, i.e., the Standards of Medical Care in Diabetes (Diabetes Care 2012;35(Suppl 1): S511-563). Current HbAlc target levels are generally <7.0% for people with diabetes, and people who do not have diabetes typically have HbAlc values of less than 6%. Accordingly, the efficacy of the combination treatment with an anti-myostatin Adnectin described herein and semaglutide can be determined by an observed decrease in the HBAlc level in a subject. In another aspect, provided herein is a method of improving glycemic control in a subject (e.g., a human patient), the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) and an effective amount of an GLP-1 receptor agonist (e.g., semaglutide) such that glycemic control is improved in the subject.

[0275] In another aspect, provided herein is a method of increasing muscle mass in a human patient undergoing treatment with a GLP-1 receptor agonist, the method comprising administering to the patient an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) in addition to the GLP-1 receptor agonist (e.g., semaglutide) such that muscle mass is increased in the patient undergoing treatment with the GLP-1 receptor agonist.

[0276] In another aspect, provided herein is a method of preventing reduction in muscle mass in a human patient undergoing treatment with a GLP-1 receptor agonist, the method comprising administering to the patient an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) in addition to the GLP-1 receptor agonist (e.g., semaglutide) such that the reduction in muscle mass is prevented in the patient.

[0277] In another aspect, provided herein is a method of providing therapeutic weight loss in a subject (e.g., an overweight or obese subject, or a subject with type II diabetes), the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) and an effective amount of an GLP-1 receptor agonist (e.g., semaglutide) such that therapeutic weight loss is achieved in the subject.

[0278] In another aspect, provided herein is a method of inhibiting myostatin activity and increasing GLP-1 receptor activity in a subject, the method comprising the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein) and an effective amount of an GLP-1 receptor agonist (e.g., semaglutide) such that myostatin activity is inhibited and GLP-1 receptor activity is increased in the subject.

[0279] In some embodiments, the polypeptide is administered subcutaneously. In some embodiments, the GLP-1 receptor agonist is administered intravenously. In some embodiments, the GLP-1 receptor agonist is administered orally. In some embodiments, the GLP-1 receptor agonist is administered subcutaneously.

[0280] In some embodiments, the polypeptide, or formulation comprising the polypeptide, and GLP-1 receptor agonist are administered simultaneously. In some embodiments, the first dose of the polypeptide, or formulation comprising the polypeptide, and the first dose of the GLP-1 receptor agonist are administered simultaneously.

[0281] In some embodiments, the polypeptide, or formulation comprising the polypeptide, is administered prior to administration of the GLP-1 receptor agonist.

[0282] In some embodiments, the polypeptide, or formulation comprising the polypeptide, is administered after administration of the GLP-1 receptor agonist.

[0283] In some embodiments, the polypeptide is administered once a week. In some embodiments, the polypeptide is administered once every two weeks. In some embodiments, the polypeptide is administered once a month.

[0284] In some embodiments, the GLP-1 receptor agonist is semaglutide and is administered subcutaneously or orally. In some embodiments, semaglutide is administered subcutaneously once a week. In some embodiments, semaglutide is administered subcutaneously once a week at the recommended dose. In some embodiments, semaglutide is administered orally once a day. In some embodiments, semaglutide is administered once a day at the recommended dose.

[0285] In some embodiments, the methods described herein comprise administering an anti- myostatin Adnectin described herein (e.g., BHV-2000) and semaglutide. In some embodiments, the anti-myostatin Adnectin (e.g., BHV-2000) is administered once a week subcutaneously and semaglutide is administered subcutaneously once a week. In some embodiments, the anti- myostatin Adnectin (e.g., BHV-2000) is administered once a week subcutaneously and semaglutide is administered orally once a day. In some embodiments, the anti-myostatin Adnectin (e.g., BHV-2000) is administered once every two weeks subcutaneously and semaglutide is administered subcutaneously once a week. In some embodiments, the anti- myostatin Adnectin (e.g., BHV-2000) is administered once every two weeks subcutaneously and semaglutide is administered orally once a day. In some embodiments, the anti-myostatin Adnectin (e.g., BHV-2000) is administered once monthly subcutaneously and semaglutide is administered subcutaneously once a week. In some embodiments, the anti-myostatin Adnectin (e.g., BHV-2000) is administered once monthly subcutaneously and semaglutide is administered orally once a day.

[0286] In some embodiments of the methods described herein, the polypeptide (e.g., anti- myostatin Adnectin described herein, such as BHV-2000) is administered at a dose of 5-200 mg (e.g., 5-150 mg, 5-100 mg, 5-50 mg, 5-25 mg, 25-200 mg, 25-150 mg, 25-100 mg, 25-50 mg, 50- 200 mg, 50-150 mg, 50-100 mg, 75-200 mg, 75-150 mg, 75-100 mg, 100-200 mg, 100-150 mg, or 150-200 mg) once every week, once every two weeks, or once monthly (e.g., as described in WO2024 / 168325, the contents of which are herein incorporated by reference in their entirety). In some embodiments, the polypeptide is administered as a subcutaneous injection (e.g., repeated subcutaneous injections).

[0287] VI. Outcomes

[0288] The efficacy of the treatment methods provided herein can be assessed using any suitable means known in the art.

[0289] In one aspect, methods for treating, preventing, or reducing obesity and related comorbidities in a patient are provided. Symptoms of obesity include, but are not limited to, difficulty in sleeping, sleep apnea, daytime drowsiness, back and / or joint pains, excessive sweating, intolerance to heat, infections in skin folds, fatigue, depression, and feeling of shortness of breath (dyspnea). Signs of obesity include, but are not limited to, acanthosis nigricans (i.e., a skin disorder characterized by the presence of hyperkeratosis and hyperpigmentation in the skin folds and armpits), stretch marks (due to distension and rupture of the elastic fibers of the skin), vinous in the case of obesity due to endocrinological alteration (Cushing), swelling and varicose veins in the lower limbs, Body Mass Index (BMI) greater than or equal to 30 kg / m2, waist circumference greater than 94 cm in men and 88 cm in women, and high blood pressure level > 140 / 90 mmHg. Accordingly, patients treated according to the methods disclosed herein experience improvement in at least one or more symptoms or signs of obesity.

[0290] In another aspect, methods for improving glycemic control in a human patient are provided. Glycemic control is a key goal in the management of patients, for example, patients with diabetes, and it remains the main therapeutic target for the prevention of organ damage and other complications arising from diabetes (see, e.g., Imran SA, et al., Can. J. Diabetes. 2018;42:S42-S46 and American Diabetes Association. Classification and diagnosis of diabetes standards of medical care in diabetes, ADA Diabetes Care J. Clin. Appl. Res. Educ. 2018;41 (Supplement 1):S13-S27). Hemoglobin Ale (HbAlc) is the gold standard for monitoring glycemic control and serves as a surrogate for diabetes-related complications. Poor glycemic control is a major public health issue among patients with type 2 diabetes mellitus and a significant risk factor for the progression of diabetic complications (see, e.g., Koro CE, etal., Diabetes Care. 2004;27(l): 17-20; Yakubu A, etal. nt. J. Sci. Health Res. 2020;5(4):207-214; and Digssie A, et al., Metab Open. 2020;8: 100056). To minimize diabetic complications, strictly maintaining a patient’s blood glucose level in the normal or close to the normal range is crucial (see, e.g., Mariye T, etal., Endocrinol. Metab. Open Access. 2020;4(l): 1— 7). In some embodiments, patients treated according to the methods disclosed herein, e.g., patients with type II diabetes, experience improvement in at least one sign of glycemic control, for example, as assessed by HbAlc. Accordingly, methods of treating or preventing type II diabetes are also provided herein. Symptoms of type II diabetes include, but are not limited to, increased thirst, frequent urination, increased hunger, unintended weight loss, fatigue, blurred vision, slow- healing sores, frequent infections, numbness or tingling in the hands or feet, areas of darkened skin, usually in the armpits and neck. Factors that increase the risk of type 2 diabetes include, but are not limited to: being overweight or obese, fat distribution mainly in the abdomen (as opposed to hips and thighs), inactivity, family history, race and ethnicity, blood lipid levels, age, prediabetes, pregnancy-related risks, and polycystic ovary syndrome. Potential complications of diabetes and frequent comorbidities include heart and blood vessel disease, nerve damage (neuropathy) in limbs, other nerve damage, kidney disease, eye damage, skin conditions, slow healing, hearing impairment, sleep apnea, and dementia. Accordingly, patients treated according to the methods disclosed herein experience improvement in at least one or more symptoms of type II diabetes.

[0291] In one embodiment, the method results in an improvement in glycemic control. In one embodiment an improvement in glycemic control is achieved by improving insulin sensitivity. In one embodiment, the treatment results in an improvement in a Diabetes Treatment Satisfaction Questionnaire (DTSQ) score. In another embodiment, the treatment results in an improvement in an Impact of Weight on Quality of Life (IWQOL) score. In one embodiment, central adiposity is reduced. In one embodiment, central adiposity is reduced (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide).

[0292] In one embodiment, one or more cardiovascular events are reduced in patients with obesity or overweight. In one embodiment, one or more cardiovascular events are reduced in patients with obesity or overweight relative to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide).

[0293] In one embodiment, one or more obesity-related malignancies are reduced. In one embodiment, obesity-related malignancies are reduced relative to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide).

[0294] In one embodiment, metabolic dysfunction-associated steatohepatitis (MASH) is reduced. In one embodiment, metabolic dysfunction-associated steatohepatitis (MASH) is reduced relative to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide).

[0295] In one embodiment, the treatment results in a decrease in total body fat mass (FM) of at least 5 kg compared to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide). For example, in one embodiment, the treatment results in a decrease in total body FM of at least 5.1., 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1., 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1., 8.2, 8.3, 8.4, 8.5, 8.6,

[0296] 8.7, 8.8, 8.9, 9, 9.1., 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1., 10.2, 10.3, 10.4, 10.5, 10.6,

[0297] 10.7, 10.8, 10.9, 11 kg or more.

[0298] In one embodiment, the treatment results in an at least 5% decrease in total body FM compared to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide). For example, in one embodiment, the treatment results in an at least 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5% or more decrease in total body FM.

[0299] In one embodiment, the treatment results in an at least 5% decrease in body weight compared to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide). For example, in one embodiment, the treatment results in an at least 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5% or more decrease in body weight.

[0300] In one embodiment, the treatment results in an at least 2% increase in body lean mass (LM) compared to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide, or prior to administration of a polypeptide described herein in a patient already undergoing treatment with a GLP-1 receptor agonist). For example, in one embodiment, the treatment results in at least 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35% or more increase in body LM compared to baseline.

[0301] FM, LM, and body fat can be assessed by any suitable means. In one embodiment, FM is assessed by dual-energy x-ray absorptiometry (DXA). In one embodiment, LM is assessed by DXA. In one embodiment, body fat is assessed by skinfold calipers, body circumference measurements, DXA, hydrostatic weighing, air displacement plethysmography (Bod Pod), bioelectrical impedance analysis (BIA), bioimpedance spectroscopy (BIS), or electrical impedance myography (EIM), a 3-D body scanner, a multi-compartment model, and / or magnetic resonance spectroscopy (MRI).

[0302] In one embodiment, the treatment results in a decrease in waist circumference (WC) and / or waist-to-hip ratio compared to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide). For example, in one embodiment, the treatment results in a decrease in WC by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% compared to baseline. In another embodiment, the treatment results in a decrease in WC by 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm or more compared to baseline.

[0303] In one embodiment, the treatment results in a decrease in WC and a decrease in total body weight compared to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide). For example, in one embodiment, the treatment results in a decrease in WC by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% compared to baseline and a decrease in total body weight by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% compared to baseline. In another embodiment, the treatment results in a decrease in WC by 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm or more compared to baseline and a decrease in total body weight by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% compared to baseline.

[0304] In one embodiment, the treatment results in a decrease in subcutaneous and abdominal visceral adipose tissue compared to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide). For example, in one embodiment, the treatment results in at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6. 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5% or more decrease in subcutaneous and abdominal visceral adipose tissue compared to baseline.

[0305] In one embodiment, the treatment results in an improvement in diabetes status. For example, in one embodiment, the treatment results in an improvement in the patient’s HbAlc. In another embodiment, the treatment results in an improvement as assessed by Homeostatic Model Assessment (HOMA). In another embodiment, the treatment results in an improvement as assessed by quantitative insulin-sensitivity check index (QUICKI). In another embodiment, the treatment results in an improvement as assessed by Matsuda Index.

[0306] In one embodiment, the treatment results in a shift toward normal levels of one or more biomarkers selected from the group consisting of serum lipids, high-sensitivity C-reactive protein (hs-CRP), interleukin 6, leptin, and adiponectin.

[0307] In one embodiment, the treatment results in a reduction in insulin level compared to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide). In one embodiment, the treatment results in a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50% reduction in insulin level compared to baseline.

[0308] In one embodiment, the treatment results in a reduction in leptin level compared to baseline (e.g., prior to initiation of the combination therapy with a polypeptide described herein (e.g., an anti-myostatin Adnectin such as BHV-2000) and GLP-1 receptor agonist such as semaglutide). In one embodiment, the treatment results in a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50% reduction in leptin level compared to baseline.

[0309] In one embodiment, the treatment results in an improvement in hand grip strength, for example, as assessed by dynamometry.

[0310] VII. Kits

[0311] Further provided herein are kits that include a pharmaceutical formulation containing a polypeptide which comprises a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., BHV-2000) and a GLP-1 receptor agonist in therapeutically effective amounts adapted for use in the methods described herein. The kits optionally also can include instructions, e.g., comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the agents to a patient in need thereof. The kit also can include one or more syringes.

[0312] In one embodiment, provided is a kit for decreasing body weight and preventing significant loss of lean muscle mass in a subject, wherein the kit comprises:

[0313] (a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein), wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,

[0314] (b) one or more doses of a dose of a GLP-1 receptor agonist (e.g., semaglutide), and

[0315] (c) instructions for administering the polypeptide and the GLP-1 receptor agonist. In another embodiment, provided is a kit for decreasing body weight and increasing lean muscle mass in a subject, wherein the kit comprises: (a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein), wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,

[0316] (b) one or more doses of a dose of a GLP-1 receptor agonist (e.g., semaglutide), and

[0317] (c) instructions for administering the polypeptide and the GLP-1 receptor agonist. In another embodiment, provided is a kit for treating a metabolic disease, disorder, or condition in a human patient, wherein the kit comprises:

[0318] (a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein), wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,

[0319] (b) one or more doses of a dose of a GLP-1 receptor agonist (e.g., semaglutide), and

[0320] (c) instructions for administering the polypeptide and the GLP-1 receptor agonist. In another embodiment, provided is a kit for increasing the ratio of lean muscle mass to fat in a subject, wherein the kit comprises:

[0321] (a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin (e.g., an anti-myostatin Adnectin described herein), wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,

[0322] (b) one or more doses of a dose of a GLP-1 receptor agonist (e.g., semaglutide), and

[0323] (c) instructions for administering the polypeptide and the GLP-1 receptor agonist.

[0324] In another embodiment, provided is a kit for treating, preventing, or reducing overweight or obesity and related comorbidities in a human patient, wherein the kit comprises: (a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,

[0325] (b) one or more doses of a dose of a GLP-1 receptor agonist, and

[0326] (c) instructions for administering the polypeptide and the GLP-1 receptor agonist.

[0327] In another embodiment, provided is a kit for treating or preventing type II diabetes in a human patient, wherein the kit comprises:

[0328] (a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,

[0329] (b) one or more doses of a dose of a GLP-1 receptor agonist, and

[0330] (c) instructions for administering the polypeptide and the GLP-1 receptor agonist.

[0331] In some embodiments, the polypeptide comprises a10Fn3 domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the polypeptide is BHV-2000.

[0332] In some embodiments, the GLP-1 receptor agonist is selected from Albiglutide (Tanzeum), Dulaglutide (Trulicity®), Exenatide (Byetta®), Exenatide extended-release (Bydureon®), Liraglutide (Victoza®;Saxenda®), Lixisenatide (Adlyxin®), Semaglutide injection (Ozempic®; Wegovy®), Semaglutide tablets (Rybelsus®), Tirzepatide (Mounjaro®; Zepbound®), and maridebart cafraglutide (MariTide, AMG133). In some embodiments, the GLP-1 receptor agonist is tirzepatide. In some embodiments, the GLP-1 receptor agonist is semaglutide. The following examples are merely illustrative and should not be construed as limiting the scope of this disclosure in any way as many variations and equivalents will become apparent to those skilled in the art upon reading the present disclosure. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0333] EXAMPLES

[0334] Example 1. Impact of BHV-2000 complexes on serum half-life and signal transduction at ActRIIb.

[0335] BHV-2000 is an anti-myostatin Adnectin (also known as taldefgrobep alpha, RO7239361, and BMS-986089) which binds to myostatin with low picomolar affinity, forming a stable BHV-2000 / myostatin complex which potently binds activin II receptors and competes with receptor ligands. This Example describes the serum half-life of BHV-2000 / myostatin complexes and the impact of BHV-2000 / myostatin complexes on SMAD2 / 3 signaling.

[0336] As shown in FIG. 1A, after human subjects were administered a single 45 mg dose of the BHV-2000 / myostatin complex, the concentration of the BHV-2000 / myostatin complex was maintained at about 20 nM in plasma for over 166 hours.

[0337] Stable BHV-2000 / myostatin complexes inhibited activin A-mediated signal transduction through ActRIIA / B, as reflected by inhibition of SMAD2 / 3 signaling relative to activin A treatment alone (FIG. IB). EC50 values were 4.687 nM for activin A alone, 50.58 nM for BHV- 2000 / myostatin + activin A, and 100.4 nM for BHV-2000 / GDF-11 complexes + activin A.

[0338] Example 2. Impact of BHV-2000 on myostatin- and ActRII ligand-induced lipid accumulation, SMAD2 / 3 signaling, and mitochondrial content in adipocytes in vitro.

[0339] The impact of BHV-2000 on myostatin- and ActRII ligand- induced lipid accumulation, SMAD2 / 3 signaling, and mitochondrial content in adipocytes was tested.

[0340] Briefly, 3T3-L1 fibroblasts were differentiated into adipocytes, followed by the addition of BHV-2000 to myostatin or a combination of ActRII ligands (combination of myostatin + growth differentiation factor 11 + activin A) (FIG. 2A). Post differentiation, adipocytes were assessed for the following: (i) lipid content and droplet size by BODIPY staining and flow cytometry, lipolysis through free glycerol, fatty acid oxidation via Seahorse Bioanalyzer, (ii) mitochondrial activity by co-staining with MitoTracker™ Green and tetramethylrhodamine methyl ester, (iii) SMAD2 / 3 signaling by enzyme-linked immunosorbent assay (ELISA), and (iv) intracellular metabolite abundance measurement using ThermoFisher Q Extractive Orbitrap Mass Spectrometer from 3T3-L1 adipocytes extracted with -20°C methanol / acetonitrile / water (40 / 40 / 20).

[0341] As shown in FIG. 2B, treatment of adipocytes with BHV-2000 reduced lipid storage relative to the control. BHV-2000 also significantly reduced myostatin-induced lipid storage (FIG. 2B), resulting in smaller intracellular lipid droplets as reflected by the significant increase in intracellular granularity as assessed by SSC (FIG. 2C). Treatment of adipocytes with BHV- 2000 significantly decreased SMAD2 / 3 signaling induced by a combination of ActRII ligands or myostatin as the only ligand to levels observed in the control (FIG. 2D). Furthermore, mitochondrial content was significantly reduced with a combination of ActRII ligands or myostatin alone. However, this reduction was reversed in the presence of BHV-2000 to levels observed in the control (FIG. 2E). These results collectively support a role for activin receptor- mediated signaling in regulating adipose homeostasis, and that inhibiting SMAD signaling with BHV-2000 leads to decreased adipose mass.

[0342] With regard to the abundance of intracellular metabolites, BHV-2000 reversed ActRII ligand-induced alterations in intracellular levels of various metabolites in adipocytes (FIG. 2F). The effects of BHV-2000 on ActRII ligand- induced levels of two representative intracellular metabolites, phosphocreatine and proline, are shown in FIGs. 2G and 2H, respectively. Specifically, while ActRII ligands (combination of myostatin, GDF11, and activin A) increased the relative abundance of phosphocreatine and proline, the increase was not observed when the ActRII ligands were combined with BHV-2000 treatment. Similar results were observed in cells treated with myostatin (rather than the ActRII ligand cocktail) and the combination of myostatin and BHV-2000. Proline buildup in ActRII-stimulated adipocytes suggests decreased proline catabolism in the presence of ActRII ligands, preventing a switch to fat-burning metabolism; proline levels are reduced in the presence of BHV-2000. Example 3. Combination of BHV-2000 and semaglutide on body weight changes in DIO mice

[0343] The effects of the combination of an anti-myostatin Adnectin (BHV-2000) and semaglutide (a GLP-1 receptor agonist) on changes in body weight, fat mass, and lean muscle mass in a diet-induced obesity model (DIO mice) fed a high fat diet.

[0344] Briefly, 6-week-old DIO mice were started on a high fat diet (60% fat) 13 weeks prior to subcutaneous administration of (i) vehicle (n=16), (ii) BHV-2000 (100 mg / kg; n=16), (iii) semaglutide (20 pg / kg; n=16), (iv) semaglutide (40 pg / kg; n=16), (v) BHV-2000 (100 mg / kg) + semaglutide (20 pg / kg) (n=16), or (vi) BHV-2000 (100 mg / kg) + semaglutide (40 pg / kg) (n=16) for 8 weeks, followed by a 4- week rebound observation period with no dosing. Vehicle and BHV-2000 were administered twice a week (16 dosing occasions) and semaglutide was administered once daily. For the BHV-2000 + semaglutide groups, BHV-2000 and semaglutide were co-administered. A schematic of the experimental setup is shown in FIG. 3.

[0345] Body composition (EchoMRI™) and metabolic markers were assessed prior to dosing (baseline), week 4, week 8, and week 12 (4 weeks post-dosing). Insulin tolerance was tested in mice fasted for 4 hours and blood glucose levels were measured at 0, 15, 30, 60, and 120 minutes. For MicroCT, whole-body scans were performed using Quantum GX. Mice were anesthetized with isoflurane and placed on a platform with the detector. MicroCT images were exported into DICOM formats, and adipose tissue depots were differentiated using the muscular abdominal wall based on density. Visceral and subcutaneous adipose depots were manually outlined after thresholding, and imaging processing was performed using Image-J software.

[0346] All mice were in the adolescent stage of growth upon entry into the study and had not yet reached their maximum weight. Therefore, vehicle control animals continued to gain fat and lean mass. After 8 weeks of dose initiation, all BHV-2000 dose groups consistently demonstrated lower fat mass and greater lean mass over time between vehicle and BHV-2000 arms in all test groups.

[0347] Body weight and body composition measurements performed prior to dosing (baseline), Week 4, Week 8, and Week 12 (4 weeks post-dosing). Body weight and percent change in body weight are illustrated in FIGs. 4A and 4B, respectively. Absolute fat mass is illustrated in FIG. 5A, and percent change in absolute fat mass is illustrated in FIGs. 5B and 5C. Percent change in lean muscle mass is illustrated in FIGs. 6A and 6B. NMR body weight, NMR fat mass, and NMR lean mass are summarized in Tables 2-4, respectively.

[0348] Table 2. Descriptive statistics for NMR body weight

[0349] Table 3. Descriptive statistics for NMR fat mass

[0350] Table 4. Descriptive statistics for NMR lean mass As shown in FIGs. 5B, 5C, 6A, and 6B, BHV-2000 monotherapy directly lowered baseline fat mass by -26.8% at Week 8 and increased lean mass by +20.2% while on the HFD. This is compared to a +14.5% increase in fat mass and +5.6% increase in lean mass observed in the vehicle control on a similar diet.

[0351] Both semaglutide treatment arms (20 pg / kg and 40 pg / kg) demonstrated significant reductions in fat mass of -4.3% and -5.6%, respectively. Both semaglutide arms also decreased lean mass relative to the vehicle control, which had an increase in lean mass.

[0352] BHV-2000 monotherapy and combination therapy with semaglutide resulted in greater reductions in fat mass than semaglutide alone. Moreover, BHV-2000 monotherapy increased lean muscle mass, while combination therapy with semaglutide prevented muscle loss observed with semaglutide alone. Specifically, the combination of BHV-2000 with semaglutide yielded a -21.8% loss in fat mass for the 20 pg / kg arm and -24.5% for the 40 pg / kg arm. In contrast to the semaglutide monotherapy arms, the combination of the two agents led to a net increase in lean mass of +19.5% and +16.6 for the 20 pg / kg and 40 pg / kg arms, respectively.

[0353] During the dosing period, the loss of fat mass and gain of lean mass had an overall lowering of total body weight that returned to baseline levels at Week 8, but these lean and fat mass changes were still lower than the vehicle that continued to add both lean and fat mass (FIGs. 4A and 4B).

[0354] As shown in FIG. 5A, following the cessation of dosing while continuing on a HFD, all treated groups observed demonstrated an increase in fat mass, consistent with the development of ADA against BHV-2000.

[0355] Metabolic markers were also assessed between treatment groups. Serum chemistry and serum hormone samples were collected from mice 8 weeks after dose initiation. Concentrations of serum chemistry and hormone parameters are illustrated in FIGs. 7 and 8, respectively. No significant changes in serum chemistry parameters, including no difference in LDL cholesterol were observed between vehicle and BHV-2000 (Ta)-treated mice. Serum hormone levels measured at the end of treatment period in the HFD-fed mice demonstrated a normalization of insulin and leptin levels that were elevated in the overweight and obese mice when comparing vehicle control and BHV-2000-treated mice. Following administration of vehicle or BHV-2000, no meaningful differences in adiponectin and corticosterone were observed between vehicle and test groups. Serum chemistry samples were collected from mice at dose initiation and 6 weeks after dose initiation. Concentrations of baseline serum glucose concentrations are shown in FIG. 9A, and insulin tolerance testing in FIGs. 9B and 9C. Across all groups there were no significant differences in baseline blood glucose levels following a 4 hour fast after 8 weeks of treatment and (FIG. 9 A). FIGs. 9B and 9C show the results of the insulin sensitivity testing. Treatment with BHV-2000 resulted in significant improvement in insulin sensitivity relative to vehicle and commensurate with semaglutide.

[0356] Consistent with body composition changes seen by EchoMRI, MicroCT analysis of mice undergoing the various treatments revealed that, after 8 weeks, BHV-2000 treatment significantly increased lean muscle mass while reducing fat mass, observed both in BHV-2000 monotherapy and in combination with semaglutide. In contrast, treatment with semaglutide alone led to a loss of both lean mass and fat mass, as compared to vehicle treatment. The combination of BHV-2000 and semaglutide led to a similar body composition to the BHV-2000 monotherapy arm. The increases in lean muscle mass in the BHV-2000 arms continued into the post-dosing window.

[0357] In summary, (a) BHV-2000 significantly reduced fat mass and body weight, while increasing lean muscle mass relative to vehicle, (b) BHV-2000 yielded an additive effect to semaglutide-induced fat loss while significantly increasing the amount of lean mass gained relative to semaglutide alone, and (c) improvements in insulin sensitivity with BHV-2000 monotherapy were comparable to semaglutide alone and significantly better than vehicle. These findings support the effectiveness of BHV-2000 as monotherapy and in combination with incretin mimetic agents (such as GLP-1 receptor agonists) used to reduce fat and total body weight while maintaining lean mass in individuals living with overweight and obesity.

[0358] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims. For example, pharmaceutically acceptable salts other than those specifically disclosed in the description and Examples herein can be employed. Furthermore, it is intended that specific items within lists of items, or subset groups of items within larger groups of items, can be combined with other specific items, subset groups of items or larger groups of items whether or not there is a specific disclosure herein identifying such a combination.

[0359] Table 5. Summary of Sequences

Claims

AMENDED CLAIMS received by the International Bureau on 05 November 2025 (05.11 .2025)1. A method of decreasing body weight and preventing significant loss of lean muscle mass in a subject, the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin and an effective amount of an GLP- 1 receptor agonist such that body weight is decreased and significant loss of lean muscle mass is achieved in the subject.

2. A method of decreasing body weight and increasing lean muscle mass in a subject, the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin and an effective amount of a GLP- 1 receptor agonist such that body weight is decreased in lean muscle mass is increased in the subject.

3. A method of treating a metabolic disease, disorder, or condition in a subject, the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin and an effective amount of a GLP- 1 receptor agonist such that a metabolic disease, disorder, or condition is treated in the subject.

4. The method of claim 3, wherein the human patient has a disease or disorder selected from the group consisting of diabetes, prediabetes, hyperglycemia, hyperinsulinemia, hyperlipidemia, insulin resistance, impaired glucose metabolism, obesity, metabolic syndrome, glucose intolerance, prediabetes, hypertension, dyslipidemia, fatty liver disorder, increased waist circumference, cardiovascular disease, non-alcoholic fatty liver disease, obstructive sleep apnea, physical impairment, osteoarthritis, osteoporosis, renal disease, sexual hormone(s) impairment, endocrine reproductive disorders such as polycystic ovary syndrome or male hypogonadism, stroke, and gallstones.

5. A method of increasing the ratio of lean muscle mass to fat in a subject, the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin and an effective amount of a GLP-1 receptor agonist such that the ratio of lean muscle mass to fat is increased in the subject.

6. A method of treating, preventing, or reducing overweight or obesity and related comorbidities in a subject, comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin and an effective amount of a GLP- 1 receptor agonist such that overweight or obesity and related comorbidities is treated, prevented, or reduced in the subject.

7. A method of treating or preventing type II diabetes in a subject, comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin and an effective amount of a GLP-1 receptor agonist such that type II diabetes is treated or prevented in the subject.

8. A method of improving glycemic control in a subject, the method comprising administering to the subject an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin and an effective amount of a GLP- 1 receptor agonist such that glycemic control is improved in the subject.

9. A method of increasing muscle mass in a human patient undergoing treatment with a GLP-1 receptor agonist, the method comprising administering to the patient an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin in addition to the GLP- 1 receptor agonist such that muscle mass is increased in the patient undergoing treatment with the GLP- 1 receptor agonist.

10. A method of preventing reduction in muscle mass in a human patient undergoing treatment with a GLP- 1 receptor agonist, the method comprising administering to the patient an effective amount of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds tomyostatin in addition to the GLP- 1 receptor agonist such that muscle mass reduction is prevented in the patient undergoing treatment with the GLP- 1 receptor agonist.

11. The method of any one of the preceding claims, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively.

12. The method of any one of the preceding claims, wherein the10Fn3 domain comprises the BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively.

13. The method of any one of the preceding claims, wherein the10Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO: 8.

14. The method of any of the preceding claims, wherein the polypeptide comprises the amino acid sequence set forth SEQ ID NO: 12.

15. The method of any of the preceding claims, wherein the polypeptide comprises the amino acid sequence set forth SEQ ID NO: 41.

16. The method of any of the preceding claims, wherein the polypeptide comprises the amino acid sequence set forth SEQ ID NO: 78.

17. The method of any of the preceding claims, wherein the polypeptide comprises the amino acid sequence set forth SEQ ID NO: 81.

18. The method of any of the preceding claims, wherein the polypeptide is taldefgrobep alpha.

19. The method of any one of the preceding claims, wherein the polypeptide is administered subcutaneously.

20. The method of any one of the preceding claims, wherein the polypeptide is administered as a formulation comprising(i) at least 10 mg / mL of the polypeptide;(ii) a disaccharide at a concentration of at least 5%;(iii) a histidine buffer at a concentration of between about 20 to about 60 mM; and(iv) a pharmaceutically acceptable aqueous carrier, wherein the formulation has a pH range of about 6.5 to about 7.8.

21. The method of claim 20, wherein the formulation comprises:(a) about 10-140 mg / mL of the polypeptide; about 5-25% trehalose dihydrate; about 20-30 mM histidine; and a pharmaceutically acceptable aqueous carrier, wherein the pH of the formulation is about 6.8 to 7.3;(b) about 10-140 mg / mL of the polypeptide; about 5-25% trehalose dihydrate; about 20-30 mM histidine; about 0.02-0.06 mM DTPA; about 0.01-0.05% polysorbate 80; and a pharmaceutically acceptable aqueous carrier, wherein the pH of the formulation is about 6.8 to 7.3;(c) about 10-140 mg / mL of the polypeptide; about 600 mM trehalose dihydrate;25-30 mM histidine; and a pharmaceutically acceptable aqueous carrier, wherein the pH of the formulation is about 7.0 to 7.3;(d) about 10-140 mg / mL of the polypeptide;about 600 mM trehalose dihydrate;25-30 mM histidine; about 0.02-0.06 mM DTPA; about 0.01-0.05% polysorbate 80; and a pharmaceutically acceptable aqueous carrier, wherein the pH of the formulation is about 7.0 to 7.3;(e) about 10-75 mg / mL of the polypeptide; about 5-25% trehalose dihydrate; about 20-30 mM histidine; and a pharmaceutically acceptable aqueous carrier, wherein the pH of the formulation is about 6.8 to 7.3;(f) about 10-75 mg / mL of the polypeptide; about 5-25% trehalose dihydrate; about 20-30 mM histidine; about 0.02-0.06 mM DTPA; about 0.01-0.05% polysorbate 80; and a pharmaceutically acceptable aqueous carrier, wherein the pH of the formulation is about 6.8 to 7.3;(g) about 10-75 mg / mL of the polypeptide; about 600 mM trehalose dihydrate; about 30 mM histidine; about 0.05 mM DTPA; about 0.02% polysorbate 80; a pharmaceutically acceptable aqueous carrier,wherein the pH of the formulation is about 7.1.

22. The method of any one of the preceding claims, wherein the polypeptide is administered once a week.

23. The method of any one of claims 1-21, wherein the polypeptide is administered once every two weeks.

24. The method of any one of claims 1-21, wherein the polypeptide is administered once a month.

25. The method of any one of the preceding claims, wherein the GLP-1 receptor agonist is administered subcutaneously or orally.

26. The method of any one of the preceding claims, wherein the GLP-1 receptor agonist is administered subcutaneously once weekly.

27. The method of claim 26, wherein the GLP-1 receptor agonist is administered at a dose of between about 0.25 mg to about 2 mg.

28. The method of any one of claims 1-25, wherein the GLP-1 receptor agonist is administered orally once daily.

29. The method of claim 28, wherein the GLP-receptor agonist is administered at a dose of between about 1.5 mg / day and about 14 mg / day.

30. The method of any one of the preceding claims, wherein the first dose of the polypeptide and the first dose of the GLP-1 receptor agonist are administered simultaneously.

31. The method of any one of claims 1-29, wherein the polypeptide, or formulation comprising the polypeptide, is administered prior to administration of the GLP-1 receptor agonist.

32. The method of any one of claims 1-29 wherein the polypeptide, or formulation comprising the polypeptide, is administered after administration of the GLP-1 receptor agonist.

33. The method of any one of the preceding claims, wherein the GLP-1 receptor agonist is semaglutide.

34. The method of any one of claims 1-32, wherein the GLP-1 receptor agonist is tirzepatide.

35. A kit for decreasing body weight and preventing significant loss of lean muscle mass in a subject, wherein the kit comprises:(a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,(b) one or more doses of a dose of a GLP- 1 receptor agonist, and(c) instructions for administering the polypeptide and the GLP-1 receptor agonist.

36. A kit for decreasing body weight and increasing lean muscle mass in a subject, wherein the kit comprises:(a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,(b) one or more doses of a dose of a GLP- 1 receptor agonist, and(c) instructions for administering the polypeptide and the GLP-1 receptor agonist.

37. A kit for treating a metabolic disease, disorder, or condition in a subject, wherein the kit comprises:(a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,(b) one or more doses of a dose of a GLP- 1 receptor agonist, and(c) instructions for administering the polypeptide and the GLP-1 receptor agonist.

38. A kit for increasing the ratio of lean muscle mass to fat in a subject, wherein the kit comprises:(a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,(b) one or more doses of a dose of a GLP- 1 receptor agonist, and(c) instructions for administering the polypeptide and the GLP-1 receptor agonist.

39. A kit for treating, preventing, or reducing overweight or obesity and related comorbidities in a subject, wherein the kit comprises:(a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,(b) one or more doses of a dose of a GLP- 1 receptor agonist, and(c) instructions for administering the polypeptide and the GLP-1 receptor agonist.

40. A kit for treating or preventing type II diabetes in a subject, wherein the kit comprises:(a) one or more doses of a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin, wherein the10Fn3 domain comprises BC, DE, and FG loops, and wherein at least one loop of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively,(b) one or more doses of a dose of a GLP- 1 receptor agonist, and(c) instructions for administering the polypeptide and the GLP-1 receptor agonist.

41. The kit if any one of claims 35-40, wherein the polypeptide comprises a10Fn3 domain comprising the amino acid sequence of SEQ ID NO: 8.

42. The kit if any one of claims 35-41, wherein the polypeptide comprises a10Fn3 domain comprising the amino acid sequence of SEQ ID NO: 12.

43. The kit of any one of claims 35-42, wherein the polypeptide comprises the amino acid sequence set forth SEQ ID NO: 41.

44. The kit if any one of claims 35-43, wherein the polypeptide comprises a10Fn3 domain comprising the amino acid sequence of SEQ ID NO: 78.

45. The kit if any one of claims 35-44, wherein the polypeptide comprises a10Fn3 domain comprising the amino acid sequence of SEQ ID NO: 81.

46. The kit if any one of claims 35-45, wherein the polypeptide is taldefgrobep alpha.

47. The method of any one of claims 1-15 and 19-34, wherein the polypeptide further comprises an Fc region, wherein the Fc region differs in amino acid sequence from a reference human IgGl Fc region comprising the amino acid sequence of SEQ ID NO: 78 or a reference human S228PIgG4 Fc region comprising the amino acid sequence of SEQ ID NO: 79 by at least one amino acid but no more than 15 amino acids.

48. The method of claim 47, wherein the Fc region differs from the amino acid sequence of SEQ ID NO: 78 by at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids.

49. The method of claim 47 or 48, wherein the Fc region comprises a combination of amino acid substitutions relative to SEQ ID NO: 78 selected from the group consisting of:(a) L234A / L235A / P329A / M248L / N434S,(b) L234A / L235A,(c) L234A / L235A / P329A,(d) L234A / L235A / P329A / M252Y / S254T / T256E,(e) M248L / N434S,(f) M252Y / S254T / T256E,(g) M252Y / S254T / T256E / N297Q, and(h) N297Q / M248L / N434S, wherein the numbering of positions is according to the EU Index.

50. The method of any one of claims 47-49, wherein the Fc region comprises the amino acid sequence of any one of SEQ ID NOs: 84, 83, and 85-89.

51. The method of claim 50, wherein the Fc region further comprises the amino acid sequence of any one of SEQ ID NOs: 71, 64-70, and 72.

52. The polypeptide of any one of claims 47-51, wherein the Fc region comprises the amino acid sequence of any one of SEQ ID NOs: 94, 92, 93, and 95-99.

53. The polypeptide of claim 47, wherein the Fc region differs from the amino acid sequence of SEQ ID NO: 79 by at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids.

54. The polypeptide of claim 47 or 53, wherein the Fc region comprises a combination of amino acid substitutions relative to SEQ ID NO: 79 selected from the group consisting of:(a) F234A / L235A / M252Y / S254T / T256E, and(b) M252Y / S254T / T256E / N297Q, wherein the numbering of positions is according to the EU Index.

55. The polypeptide of any one of claims 47, 53, or 54, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 90 or 91.

56. The polypeptide of claim 55, wherein the Fc region further comprises the amino acid sequence of any one of SEQ ID NOs: 72 and 64-71.

57. The polypeptide of any one of claims 47 and 53-56, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 100 or 101.

58. The polypeptide of any one of claims 47-57, which comprises a signal peptide.

59. The polypeptide of claim 58, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 74 or 75, or an amino acid sequence which differs from SEQ ID NO: 74 or 75 by no more than 3 amino acids.

60. The polypeptide of any one of claims 47-59, which comprises a linker between the10Fn3 domain to the Fc region.

61. The polypeptide of claim 60, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 41, 35-40, and 42-63, or an amino acid sequence which differs from any one of SEQ ID NOs: 41, 35-40, and 42-63 by no more than 1, 2, or 3 amino acids.10062. The polypeptide of any one of claims 47-61, wherein the Fc region is located N-terminal to the10Fn3 domain.

63. The polypeptide of any one of claims 47-62, wherein the configuration of the polypeptide, from N-terminus to C-terminus, is as follows: Fc domain - linker -10Fn3 domain.

64. The polypeptide of claim 63, wherein the configuration of the polypeptide, from N-terminus to C-terminus, is as follows: signal peptide - Fc domain - linker -10Fn3 domain.

65. The polypeptide of any one of claims 47-61, wherein the Fc region is located C-terminal to the10Fn3 domain.

66. The polypeptide of any one of claims 47-61 and 65, wherein the configuration of the 5 polypeptide, from N-terminus to C-terminus, is as follows:10Fn3 domain - linker - Fc region.

67. The polypeptide of claim 66, wherein the configuration of the polypeptide, from N-terminus to C-terminus, is as follows: signal peptide -10Fn3 domain - linker - Fc region.

68. The polypeptide of any one of claims 47-64, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 124, 102-123, and 125-131, or a polypeptide comprising an amino acid sequence which is at least 90%, 95%, 98%, or 99% identical to any one of SEQ ID NOs: 124, 102-123, and 125-131, or a polypeptide which differs from the amino acid sequence of any one of SEQ ID NOs: 124, 102-123, and 125-131 by at least one amino acid but no more than 30, 20, or 10 amino acids.