Administration of fibronectin-based scaffolding domain proteins for treating overweight, obesity and related health conditions

Abstract

Herein, the tenth ( 10 Methods of improving glycemic control in a human patient, treating, preventing, or reducing overweight or obesity and associated complications, and treating or preventing type II diabetes are provided by administering to the human patient a polypeptide comprising a Fn3 domain. In some embodiments, the polypeptide is administered (or is for administration) according to a specific clinical dosing regimen (e.g., at a specific dose and according to a specific schedule).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 484,286 (filed February 10, 2023) and U.S. Provisional Application No. 63 / 588,035 (filed October 5, 2023), the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Myostatin, also known as growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-β (TGF-β) superfamily of secreted growth factors. Myostatin expression is primarily restricted to skeletal muscle and adipose tissue and has been shown to be a negative regulator of skeletal muscle development (Lee LS, Immunol. Endocr. Metab. Agents Med. Chem. 2010;10:183-194). Findings from both genetic and pharmacological studies indicate that myostatin regulates energy metabolism, and its inhibition can significantly alleviate the progression of metabolic diseases, including overweight, obesity, and related health conditions. For example, myostatin-null mice exhibit reduced body fat accumulation compared to age-matched wild-type mice (McPherron & Lee, J. JCI 109:595, 2002). This reduction in body fat is a manifestation of a reduction in the number and size of adipocytes, suggesting a critical role for myostatin in adipogenesis and myogenesis. Furthermore, increases in skeletal muscle mass and strength are associated with metabolic adaptations that positively impact body composition, energy expenditure, glucose homeostasis, and insulin requirements.

[0003] Over the past two decades, recombinant DNA technology has led to the discovery of numerous protein therapeutics. For example, anti-myostatin Adnectins have been described that effectively inhibit myostatin activity in vitro and in vivo (U.S. Patent Nos. 8,933,199, 8,993,265, 8,853,154, and 9,493,546). These anti-myostatin Adnectins are useful in treating disorders, diseases, and conditions where inhibition of myostatin activity is beneficial, including, for example, muscle-wasting diseases, metabolic disorders, and conditions that cause muscle atrophy.

[0004] Overweight and obesity are complex disease processes that have reached pandemic proportions. Since 1975, global obesity rates have nearly tripled, primarily due to increasingly sedentary lifestyles and unhealthy dietary habits (Boutari, C. and Mantzoros, C., Metabolism. 2022 Aug;133:155-217). Specifically, global obesity rates have increased by approximately 2 percentage points per decade (NCD Risk Factor Collaboration [NCD-RisC] Worldwide trends in body-mass index, underweight, overweight, and obesity from 1975 to 2016: a pooled analysis of 2,416 population-based measurement studies in 128.9 million children, adolescents, and adults. Lancet [London, England] 2017;390:2627-2642). According to the WHO, there are approximately 2 billion overweight adults and approximately 650 million obese adults. Assuming these rates continue at similar rates, it is predicted that by 2025, 2.7 billion adults will be overweight and over 1 billion will be obese (Boutari, C. and Mantzoros, 2022).

[0005] Overweight and obesity, and their associated complications, such as type 2 diabetes, hypertension, dyslipidemia, and coronary heart disease, are major risk factors for overall mortality (Apovian et al. 2015). However, treating obesity is challenging, and the treatment of obesity or overweight and their associated complications, particularly type 2 diabetes, represents a significant unmet medical need. Therefore, it is an object of the present invention to provide improved methods for treating patients with overweight, obesity, and associated complications. Summary of the Invention

[0006] Herein, the tenth ( 10 Methods of improving glycemic control, treating, preventing, or reducing obesity and associated complications, and treating or preventing type II diabetes in a human patient are provided by administering to the human patient a polypeptide comprising a Fn3 domain. In some embodiments, the polypeptide is administered (or is for administration) according to a specific clinical dosing regimen (e.g., at a specific dose and according to a specific schedule).

[0007] In one aspect, a method for improving glycemic control in a human patient is provided, the method comprising administering to a human subject a fibronectin type III fragment of the tenth amino acid sequence ( 10 The method comprises administering to the patient a polypeptide comprising a Fn3 domain.

[0008] In another aspect, a method of treating, preventing, or alleviating obesity and associated complications in a human patient is provided, the method comprising administering to a human subject a fibronectin type III fragment of the tenth amino acid sequence ( 10 The method comprises administering to the patient a polypeptide comprising a Fn3 domain.

[0009] In a further aspect, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering to a subject a fibronectin type III fragment of the tenth amino acid sequence (SEQ ID NO: 1) that binds to myostatin. 10The method comprises administering to the patient a polypeptide comprising a Fn3 domain.

[0010] In one embodiment, the patient's body mass index (BMI) is 30 kg / m 2 In another embodiment, the patient with at least one weight-related complication has a BMI of 25 kg / m or greater. 2 More than 30kg / m 2 In another embodiment, the patient's BMI is less than 25 kg / m 2 is less than.

[0011] In one embodiment, the patient is overweight, with or without associated health conditions. In another embodiment, the patient is obese. In another embodiment, the patient has overweight or obesity-related complications. Overweight and obesity-related complications include, but are not limited to, type 2 diabetes, impaired glucose tolerance, prediabetes, insulin resistance, hypertension, dyslipidemia, increased waist circumference, cardiovascular disease, non-alcoholic fatty liver disease, obstructive sleep apnea, physical dysfunction, osteoarthritis, osteoporosis, kidney disease, sex hormone(s) disorder, endocrine reproductive disorders such as polycystic ovary syndrome or male hypogonadism, stroke, and gallstones. In one embodiment, the patient has type II diabetes.

[0012] In one embodiment, the polypeptide is administered to the patient as a pharmaceutical formulation. 10 Any suitable formulation containing a polypeptide comprising a Fn3 domain can be utilized in accordance with the methods described herein. In one embodiment, the formulation comprises (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 about 20 to about 60 mM, and (iv) a pharmaceutically acceptable aqueous carrier, wherein the pH of the formulation is in the range of about 6.5 to about 7.8.

[0013] In one embodiment, the concentration of the polypeptide in the formulation is about 10 mg / mL to 200 mg / mL, about 10 mg / mL to 150 mg / mL, or about 10 mg / mL to 85 mg / mL.

[0014] In one embodiment, the disaccharide is present in a weight (w / w) ratio of protein to sugar of at least 5:1. In another embodiment, the formulation comprises about 5% to about 30% disaccharide. In another embodiment, the concentration of the disaccharide is about 150 mM to about 800 mM, or about 300 to about 700 mM. In another embodiment, the disaccharide is trehalose, and the formulation comprises about 5 to about 30% trehalose, about 15% to about 25% trehalose, or about 20% to about 25% trehalose. In another embodiment, the disaccharide is trehalose dihydrate, and the concentration of trehalose dihydrate in the formulation is about 150 mM to about 800 mM, about 300 to about 700 mM, 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 mM, about 625 mM, about 650 mM, about 675 mM, or about 700 mM.

[0015] In one embodiment, histidine is present at a concentration of at least 20 mM.

[0016] In one embodiment, the viscosity of the formulation is about 5-20 cps, about 5-15 cps, or about 7-12 cps.

[0017] In one embodiment, the pH is about 6.6 to 7.6, about 6.8 to 7.4, or about 7.0 to 7.3.

[0018] In one embodiment, the formulation comprises a surfactant at a concentration of about 0.01% to 0.5%.

[0019] In one embodiment, the formulation comprises a chelating agent, the concentration of the chelating agent is about 0.01 mM to about 0.5 mM or about 0.05 mM to 0.2 mM, and the chelating agent is selected from the group consisting of DPTA, EDTA, and EGTA.

[0020] In one embodiment, the formulation comprises about 10-140 mg / mL of polypeptide, about 5-25% trehalose dihydrate, about 20-30 mM histidine, and a pharmaceutically acceptable aqueous carrier, and the pH of the formulation is about 6.8-7.3.

[0021] In one embodiment, the formulation comprises about 10-140 mg / mL of 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, and the pH of the formulation is about 6.8-7.3.

[0022] In one embodiment, the formulation comprises about 10-140 mg / mL of polypeptide, about 600 mM trehalose dihydrate, 25-30 mM histidine, and a pharmaceutically acceptable aqueous carrier, and the pH of the formulation is about 7.0-7.3.

[0023] In one embodiment, the formulation comprises about 10-140 mg / mL of 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, and the pH of the formulation is about 7.0-7.3.

[0024] In one embodiment, the formulation comprises about 10-75 mg / mL of polypeptide, about 5-25% trehalose dihydrate, about 20-30 mM histidine, and a pharmaceutically acceptable aqueous carrier, and the pH of the formulation is about 6.8-7.3.

[0025] In one embodiment, the formulation comprises about 10-75 mg / mL of 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, and the pH of the formulation is about 6.8-7.3.

[0026] In one embodiment, the formulation comprises about 10-75 mg / mL of polypeptide, about 600 mM trehalose dihydrate, about 30 mM histidine, about 0.05 mM DTPA, about 0.02% polysorbate 80, and a pharmaceutically acceptable aqueous carrier, and the pH of the formulation is about 7.1.

[0027] In one embodiment, the polypeptide is administered as a unit dosage form containing about 1.0 mL or less of a formulation, the formulation comprising: (i) about 10-75 mg / mL of 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 6.8-7.3.

[0028] The tenth amino acid of type III fibronectin ( 10 Any suitable polypeptide comprising a Fn3 domain can be used in accordance with the methods described herein. 10 The Fn3 domain comprises BC, DE, and FG loops, wherein at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively. 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11. An exemplary polypeptide is BHV-2000 (also known as "Taldef Globep Alfa," "RO7239361," and "BMS-986089"). BHV-2000 has a high affinity (K d = 0.17-0.45 nM) and inhibited intracellular myostatin and GDF-11 second messenger signaling (IC 50=0.06-1 nM and 0.09-0.7 nM) anti-myostatin Adnectin (a modified scaffold based on the tenth fibronectin type III domain). BHV-2000 comprises SEQ ID NO: 78. Thus, in another embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 78.

[0029] The peptide or a formulation comprising the peptide can be administered to a patient by any suitable means. In one embodiment, the peptide is formulated for intravenous administration. In one embodiment, the peptide is formulated for subcutaneous administration.

[0030] The effectiveness of the treatment methods described herein can be assessed using any suitable means. In one embodiment, central obesity is reduced.

[0031] In one embodiment, the method improves glycemic control, hi one embodiment, improved glycemic control is achieved by improving insulin sensitivity.

[0032] In one embodiment, treatment improves the Diabetes Treatment Satisfaction Questionnaire (DTSQ) score, hi another embodiment, treatment improves the Impact of Weight on Quality of Life (IWQOL) score.

[0033] In one embodiment, treatment results in a reduction in total body fat mass (FM) of at least 5 kg compared to baseline. For example, in one embodiment, treatment results in a reduction in total body fat mass (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, 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, 10.7, 10.8, 10.9, 11 kg or more. In one embodiment, treatment results in a reduction of total body FM of at least 5 kg compared to baseline by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0034] In one embodiment, treatment results in at least a 5% reduction in total FM compared to baseline. For example, in one embodiment, treatment results in at least a 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 reduction in total FM compared to baseline. In one embodiment, treatment results in at least a 5% reduction in total FM compared to baseline by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0035] In one embodiment, treatment results in at least a 5% reduction in body weight compared to baseline. For example, in one embodiment, treatment results in at least a 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 reduction in body weight. In one embodiment, treatment results in at least a 5% reduction in body weight compared to baseline by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0036] In one embodiment, treatment results in at least a 2% increase in lean body mass (LM) compared to baseline. For example, in one embodiment, treatment results in at least a 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. In one embodiment, treatment results in at least a 2% increase in body LM compared to baseline by week 6, week 8, week 10, week 12, week 16, week 20, week 24, week 36, week 48, or week 60. In another embodiment, treatment results in at least a 25% increase in body LM compared to baseline by week 8, week 10, week 12, week 16, week 20, week 24, week 36, week 48, or week 60.

[0037] 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 measurement, DXA, hydrostatic weighing, air displacement plethysmography (Bod Pod), bioelectrical impedance analysis (BIA), bioimpedance spectroscopy (BIS), or electrical impedance electromyography (EIM), 3-D body scanner, multicompartment model, and / or magnetic resonance spectroscopy (MRI).

[0038] In one embodiment, treatment reduces waist circumference (WC) and / or waist-to-hip ratio compared to baseline. For example, in one embodiment, treatment reduces WC by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% compared to baseline. In another embodiment, treatment reduces 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. In another embodiment, treatment reduces 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 by 12 weeks, 15 weeks, 18 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0039] In one embodiment, treatment reduces WC and reduces total body weight compared to baseline. For example, in one embodiment, treatment reduces WC by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% compared to baseline, and reduces 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, treatment reduces 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 reduces 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, treatment results in a reduction in WC of 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm or more and a reduction in total body weight of at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% by 12, 15, 18, 24, 36, 48, or 60 weeks compared to baseline.

[0040] In another embodiment, treatment reduces waist-to-hip ratio compared to baseline. In one embodiment, treatment reduces waist circumference (WC) and / or waist-to-hip ratio compared to baseline by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0041] In one embodiment, treatment reduces subcutaneous adipose tissue and abdominal visceral adipose tissue compared to baseline. For example, in one embodiment, treatment reduces subcutaneous adipose tissue and abdominal visceral adipose tissue by 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 compared to baseline. In one embodiment, treatment reduces subcutaneous adipose tissue and abdominal visceral adipose tissue by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks compared to baseline.

[0042] In one embodiment, the treatment improves the diabetic condition. For example, in one embodiment, the treatment improves the patient's HbA1c. In another embodiment, the treatment results in an improvement as assessed by Homeostasis Model Assessment (HOMA). In another embodiment, the treatment results in an improvement as assessed by Quantitative Insulin Sensitivity Test Index (QUICKI). In another embodiment, the treatment results in an improvement as assessed by Matsuda Index. In one embodiment, the treatment improves the diabetic condition by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks compared to baseline.

[0043] In one embodiment, 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. In one embodiment, 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 by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks compared to baseline.

[0044] In one embodiment, treatment reduces insulin levels compared to baseline, hi one embodiment, treatment reduces insulin levels by 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% compared to baseline. In one embodiment, treatment reduces insulin levels by 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% by 8 weeks, 10 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks or 60 weeks compared to baseline.

[0045] In one embodiment, treatment reduces leptin levels compared to baseline, hi one embodiment, treatment reduces leptin levels by 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% compared to baseline. In one embodiment, 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 levels by week 8, week 10, week 12, week 24, week 36, week 48, or week 60 compared to baseline. In one embodiment, treatment results in an improvement in grip strength, e.g., as assessed by dynamometry. In one embodiment, treatment results in an improvement in grip strength by week 24, week 36, week 48, or week 60 compared to baseline.

[0046] In one embodiment, the peptide or formulation thereof is administered as a monotherapy according to the methods described herein. In another embodiment, the peptide or formulation thereof is administered in combination with one or more additional therapeutic agents. In one embodiment, the peptide or formulation thereof and the one or more therapeutic agents are co-administered at the same time (e.g., simultaneously). In another embodiment, the peptide or formulation thereof is administered before the administration of one or more additional therapeutic agents. In another embodiment, the peptide or formulation thereof is administered after the administration of one or more additional therapeutic agents.

[0047] The polypeptide (anti-myostatin Adnectin molecule) is administered to the target population at any suitable dose. In one embodiment, the polypeptide is administered as repeated subcutaneous injections. In one embodiment, the polypeptide is administered at a weekly dosing interval. In one embodiment, the polypeptide is administered at a biweekly dosing interval. In one embodiment, the polypeptide is administered at a monthly dosing interval.

[0048] In one embodiment, the polypeptide is administered at a dose of about 5 to 200 mg. For example, the polypeptide may be administered at a dose of 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, g, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74mg, 75m g, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 22mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93mg, 94mg, 95mg, 96mg, 97mg, 98mg, 99mg , 100mg, 101mg, 102mg, 103mg, 104mg, 105mg, 106mg, 107mg, 108mg, 109mg, 110mg, 111mg, 112mg, 113mg, 114mg, 115mg, 116mg, 117mg, 118mg, 119mg , 120mg, 121mg, 122mg, 123mg, 124mg, 125mg, 126mg, 127mg, 128mg, 129mg, 130mg, 131mg, 132mg, 133mg, 134mg, 135mg, 136mg, 137mg, 138mg, 139mg , 140mg, 141mg, 142mg, 143mg, 144mg, 145mg, 146mg, 147mg, 148mg, 149mg, 150mg, 151mg, 152mg, 153mg, 154mg, 155mg, 156mg, 157mg, 158mg, 159mg , 160mg, 161mg, 162mg, 163mg, 164mg, 165mg, 166mg, 167mg, 168mg, 169mg, 160mg, 171mg, 172mg, 173mg, 174mg, 175mg, 176mg, 177mg, 178mg, 179mg,It can be administered in doses of 170 mg, 181 mg, 182 mg, 183 mg, 184 mg, 185 mg, 186 mg, 187 mg, 188 mg, 189 mg, 180 mg, 191 mg, 192 mg, 193 mg, 194 mg, 195 mg, 196 mg, 197 mg, 198 mg, 199 mg, or 200 mg.

[0049] In one embodiment, a method for improving glycemic control in a human patient is provided, the method comprising administering to a subject a therapeutically effective amount of fibronectin type III fragment containing the tenth amino acid residue ( 10 administering to the patient a polypeptide comprising a Fn3 domain, 10 The Fn3 domain comprises BC, DE, and FG loops, wherein at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively, and the polypeptide is administered weekly or once every two weeks at a dose of 5-200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections). In one embodiment, 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO:8.

[0050] In one embodiment, a method of improving glycemic control in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0051] In one embodiment, a method of improving glycemic control in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 78, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0052] In one embodiment, a method of improving glycemic control in a human patient is provided, the method comprising administering BHV-2000 to the patient, wherein the BHV-2000 is administered at a dose of 5-200 mg once a week or once every two weeks. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0053] In one embodiment, a method is provided for treating, preventing, or alleviating overweight or obesity and associated complications in a human patient, the method comprising administering to a human subject a fibronectin type III fragment of the tenth amino acid sequence ( 10 administering to the patient a polypeptide comprising a Fn3 domain, 10 The Fn3 domain comprises BC, DE, and FG loops, wherein at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively, and the polypeptide is administered weekly or once every two weeks at a dose of 5-200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections). In one embodiment, 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO:8.

[0054] In one embodiment, a method of treating, preventing, or alleviating overweight or obesity and associated complications in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0055] In one embodiment, a method of treating, preventing, or alleviating overweight or obesity and associated complications in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 78, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0056] In one embodiment, a method of treating, preventing, or alleviating overweight or obesity and associated complications in a human patient is provided, the method comprising administering BHV-2000 to the patient, wherein the BHV-2000 is administered once a week or once every two weeks at a dose of 5-200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0057] In one embodiment, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering to a subject a fibronectin type III fragment of the tenth amino acid sequence (SEQ ID NO: 1) that binds to myostatin. 10 administering to the patient a polypeptide comprising a Fn3 domain, 10 The Fn3 domain comprises BC, DE, and FG loops, wherein at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively, and the polypeptide is administered weekly or once every two weeks at a dose of 5-200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections). In one embodiment, 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO:8.

[0058] In one embodiment, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0059] In one embodiment, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 78, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0060] In one embodiment, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering BHV-2000 to the patient, wherein the BHV-2000 is administered at a dose of 5 to 200 mg once a week or once every two weeks. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0061] Furthermore, the 10th amino acid of fibronectin type III ( ) binds to myostatin, such as BHV-2000. 10 Kits are provided that contain a pharmaceutical formulation comprising a polypeptide comprising a Fn3) domain in a therapeutically effective amount adapted for use in the methods described herein.

[0062] In one embodiment, a kit for improving glycemic control in a human patient is provided, the kit comprising: (a) The 10th amino acid of fibronectin type III ( 10 a dose of a polypeptide comprising a Fn3) domain, 10a dose of a polypeptide wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively; (b) instructions for using the polypeptide in a method according to any one of the preceding claims; and Includes:

[0063] In another embodiment, a kit for treating, preventing, or reducing overweight or obesity and associated complications in a human patient is provided, comprising: (a) The 10th amino acid of fibronectin type III ( 10 a dose of a polypeptide comprising a Fn3) domain, 10 a dose of a polypeptide wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively; (b) instructions for using the polypeptide in a method according to any one of the preceding claims; and Includes:

[0064] In another embodiment, a kit for treating or preventing type II diabetes in a human patient is provided, comprising: (a) The 10th amino acid of fibronectin type III ( 10 a dose of a polypeptide comprising a Fn3) domain, 10 a dose of a polypeptide wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively; (b) instructions for using the polypeptide in a method according to any one of the preceding claims; and Includes:

[0065] In one embodiment, 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In one embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11. In one embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 78. In one embodiment, the polypeptide is used for administration as a unit dosage form containing about 1.0 mL or less of a formulation, the formulation comprising (i) about 10-75 mg / mL of 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 6.8-7.3.

[0066] We also investigated the myostatin-binding 10th ( 10 Use of a polypeptide comprising a Fn3 domain is provided, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0067] Furthermore, the tenth (10th) amino acid sequence of fibronectin type III that binds to myostatin was identified for the treatment, prevention, or mitigation of obesity and related complications in human patients. 10 Use of a polypeptide comprising a Fn3 domain is provided, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0068] Furthermore, the tenth (10th) amino acid sequence of fibronectin type III that binds to myostatin for the treatment or prevention of type II diabetes in human patients is disclosed. 10Use of a polypeptide comprising a Fn3 domain is provided, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0069] In one embodiment, the polypeptide is a fibronectin type III polypeptide having the tenth amino acid sequence ( 10 Fn3) domain, 10 The Fn3 domain comprises BC, DE, and FG loops, wherein at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively. In some embodiments, 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11. In another embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 78. In another embodiment, the polypeptide is used for administration as a unit dosage form comprising a formulation of about 1.0 mL or less, the formulation comprising (i) about 10-75 mg / mL of 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 6.8-7.3. [Brief explanation of the drawings]

[0070] [Figure 1] Schematic diagram showing the study design. Baseline was the initiation of treatment with vehicle or taldef globep. [Figure 2] Figure 1 shows baseline changes in total body weight (TBW) and body composition in C57BL / 6J mice treated with taldef-globep versus vehicle at week 8. Error bars represent standard deviation. [Figure 3]1 is a series of bar graphs showing the change from baseline in fat mass in the taldef-globep and vehicle groups. Error bars represent standard deviation. [Figure 4] 1 is a set of bar graphs showing the change from baseline in lean mass in the taldef-globep and vehicle groups. [Figure 5A] 1 is a bar graph showing insulin after 8 weeks of vehicle or taldef globep treatment. [Figure 5B] 1 is a bar graph showing leptin levels after 8 weeks of vehicle or taldef globep treatment. [Figure 6]

[0023] Figure 1 is a line graph showing the correlation between change in TBW and waist circumference (WC) across an adult anti-obesity drug trial. The dotted line represents the identity line. The size of each data point is proportional to the square root of the sample size. DETAILED DESCRIPTION OF THE INVENTION

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and compositions are described herein.

[0072] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0073] The term "about," particularly with respect to a particular quantity or numerical value, is meant to encompass deviations within plus or minus ten percent (±10%) (e.g., ±5%).

[0074] I. Polypeptides and Myostatin-Binding Adnectin Molecules Herein, the tenth ( 10Methods for improving glycemic control, treating, preventing, or reducing obesity and associated complications, and treating or preventing type II diabetes in a human patient are provided by administering to the human patient a polypeptide comprising a Fn3) domain.

[0075] As used herein, "full-length myostatin" 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 biologically active fragments of 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 share 100% sequence identity among many species, including human, mouse, chicken, pig, turkey, and rat (Lee et al., PNAS 2001;98:9306). The sequence of human prepromyostatin is as follows: MQKLQLCVYIYLFMLIVAGPVDLNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 1)

[0076] The sequence of human promyostatin is as follows: NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 2)

[0077] The sequence of mature myostatin (conserved in human, mouse, rat, chicken, turkey, dog, horse and pig) is as follows: DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 3)

[0078] As used herein, "fibronectin-based scaffold" or "FBS" protein or moiety refers to a protein or moiety based on fibronectin type III ("Fn3") repeats. Fibronectin has 18 Fn3 repeats, and although the sequence homology between the repeats is low, they all share a high degree of similarity in tertiary structure. For reviews, see Bork et al., Proc. Natl. Acad. Sci. 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). The Fn3 domain is small, monomeric, soluble, and stable. The lack of disulfide bonds makes it stable under reducing conditions. From N- to C-terminus, the Fn3 domain contains beta strand or beta-like strand A, loop AB, beta strand or beta-like strand B, loop BC, beta strand or beta-like strand C, loop CD, beta strand or beta-like strand D, loop DE, beta strand or beta-like strand E, loop EF, beta strand or beta-like strand F, loop FG, and beta strand or beta-like strand G. Seven antiparallel beta strands are arranged as two beta sheets that form a stable core, while two "faces" are formed by the loops connecting the beta strands or beta-like strands. Loops AB, CD, and EF are located on one face (the "south pole"), and loops BC, DE, and FG are located on the opposite face (the "north pole").

[0079] Adnectins are composed of the tenth human fibronectin type III domain ( 10 Fn3) are a class of therapeutic FBS proteins with high-affinity, specific target-binding properties. [ka]

[0080] Therefore, as used herein, " 10Fn3 domain" or " 10 Fn3 part" or " 10 The "Fn3 molecule" refers to the wild-type 10 It refers to Fn3 and biologically active variants thereof, for example, biologically active variants that specifically bind to a target, such as a target protein.

[0081] As used herein 10 A "region" of an Fn3 domain (or portion or molecule) refers to any of the loops (AB, BC, CD, DE, EF, and FG), the β-strands (A, B, C, D, E, F, and G), the N-terminus (corresponding to amino acid residues 1-7 of SEQ ID NO:1), or the C-terminus (corresponding to amino acid residues 93-94 of SEQ ID NO:1).

[0082] The "scaffolding field" refers to 10 This refers to any non-loop region of the Fn3 domain. The scaffold region includes the A, B, C, D, E, F, and G β-strands, as well as the N-terminal region (amino acids corresponding to residues 1-7 of SEQ ID NO: 1) and the C-terminal region (amino acids corresponding to residues 93-94 of SEQ ID NO: 1).

[0083] The term "anti-myostatin Adnectin" refers to an Adnectin that binds to and antagonizes myostatin and is a human wild-type 10 at least one derived from the Fn3 domain (SEQ ID NO: 1) 10 Anti-myostatin Adnectins refer to protein molecules that contain an Fn3 domain. Anti-myostatin Adnectins can further include additional protein domains (e.g., an Fc domain) and can also refer to multimers of polypeptides, such as dimers, tetramers, and hexamers.

[0084] As used herein, a "polypeptide" refers to any sequence of two or more amino acids, regardless of length, post-translational modification, or function. "Polypeptide," "peptide," and "protein" are used interchangeably herein. Polypeptides include natural amino acids and unnatural amino acids, such as those described in U.S. Pat. No. 6,559,126, incorporated herein by reference. Polypeptides can also be modified by any of a variety of standard chemical methods (e.g., amino acids can be modified with protecting groups, carboxy-terminal amino acids can be made terminal amide groups, amino-terminal residues can be modified with groups that increase lipophilicity, for example, or polypeptides can be chemically glycosylated or otherwise modified to enhance stability or in vivo half-life). Modifications of polypeptides can include attaching another structure, such as a cyclic compound or other molecule, to the polypeptide and can also include polypeptides containing one or more amino acids in an altered configuration (i.e., R or S, or L or D). The peptides of the present 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 Adnectins" or "myostatin Adnectins."

[0085] As used herein, a "polypeptide chain" refers to a polypeptide in which each of its domains is linked to other domain(s) by peptide bond(s), rather than by non-covalent interactions or disulfide bonds.

[0086] 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 substances that would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide is purified (1) to greater than 95% by weight, and most preferably greater than 99% by weight, of the polypeptide as determined by the Lowry method; (2) to a degree sufficient to obtain at least residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue staining or, preferably, silver staining. Since at least one component of the polypeptide's natural environment is absent, an isolated polypeptide includes the polypeptide in situ within a recombinant cell. Ordinarily, however, an isolated polypeptide will be prepared by at least one purification step.

[0087] As used herein, "percent (%) amino acid sequence identity" is defined as the percentage (%) of amino acid residues in a candidate sequence that are identical to the amino acid residues in a selected sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR™) software. Those skilled in the art can easily determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. For example, the % amino acid sequence identity of a given amino acid sequence A with or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a particular % amino acid sequence identity with or against a given amino acid sequence B) is calculated as 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches in a program alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity between A and B will not be equal to the % amino acid sequence identity between B and A.

[0088] As used herein, "conservative substitution" refers to the replacement of an amino acid residue with another amino acid residue without altering the overall structure and function of the peptide, including, but not limited to, the replacement of an amino acid with an amino acid having similar properties (e.g., polarity, hydrogen-bonding ability, acidity, basicity, shape, hydrophobicity, aromaticity, etc.). Exemplary conservative substitutions include those that meet the criteria defined for permissible point mutations in Dayhoff et al., Atlas of Protein Sequence and Structure, 5:345-352 (1978 and Supp.). Conservative substitutions include, for example, 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. "Substitution" or "modification," as used herein, includes those amino acids that have been altered or modified from natural amino acids. Thus, in the context of the present invention, a conservative substitution should be understood as recognized in the art as the substitution of one amino acid for another amino acid with similar properties.

[0089] As used herein, the term "Adnectin binding site" refers to a site or portion of a protein (e.g., myostatin) that interacts with or binds to a particular Adnectin (e.g., due to the epitope recognized by an antibody). Adnectin binding sites can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Adnectin binding sites formed by contiguous amino acids are typically retained by exposure to denaturing solvents, while Adnectin binding sites formed by tertiary folding are typically lost by treatment with denaturing solvents.

[0090] The terms "specifically bind," "specific binding," "selective binding," and "selectively bind," as used interchangeably herein, refer to an Adnectin that exhibits affinity for myostatin but does not significantly bind (e.g., binds less than about 10%) to a different polypeptide as measured by techniques available in the art, such as, but not limited to, Scatchard analysis and / or competitive binding assays (e.g., competitive ELISA, BIACORE assay). The terms are also applicable, for example, when the binding domain of an Adnectin of the invention is specific for myostatin.

[0091] As used herein, the term "preferentially binds" refers to a situation in which the Adnectins of the invention bind to myostatin at least about 20% more than they bind to a different polypeptide, as measured by techniques available in the art, such as, but not limited to, Scatchard analysis and / or competitive binding assays (e.g., competitive ELISA, BIACORE assay).

[0092] As used herein, the term "cross-reactive" refers to an Adnectin that binds to two or more different proteins that have the same or very similar Adnectin binding sites.

[0093] As used herein, "K" D As used herein, the term "desired K" is intended to refer to the dissociation equilibrium constant of a particular Adnectin-protein (e.g., myostatin) interaction or the affinity of the Adnectin for a protein (e.g., myostatin) as measured using a surface plasmon resonance assay or a cell binding assay. D " refers to the K of an Adnectin sufficient for its intended purpose. D For example, the desired K D is the K of an Adnectin required to elicit a functional effect in an in vitro assay, such as a cell-based luciferase assay. D It may refer to.

[0094] As used herein, "k" ass The term "binding rate constant" is intended to refer to the binding rate constant of an Adnectin when it binds to an Adnectin / protein complex.

[0095] As used herein, "k" diss The term "dissociation rate constant" is intended to refer to the dissociation rate constant when an Adnectin dissociates from an Adnectin / protein complex.

[0096] As used herein, "IC 50 The term "inhibitory" refers to the concentration of Adnectin that inhibits a response to 50% of the maximal inhibitory response, i.e., midway between the maximal inhibitory response and the untreated response, in either an in vitro or in vivo assay.

[0097] As used herein, the term "myostatin activity" refers to one or more of the growth-regulating 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 regulate the production of muscle-specific enzymes (e.g., creatine kinase), stimulate myoblast proliferation, and regulate the differentiation of preadipocytes into adipocytes. Myostatin activity can be measured using art-recognized methods, such as those described herein.

[0098] 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 invention to neutralize or antagonize the activity of myostatin in vivo or in vitro. As used herein with respect to the activity of the Adnectins of the invention, the term "inhibition" or "neutralization" refers to the ability to substantially antagonize, prohibit, prevent, suppress, slow, disrupt, eliminate, halt, reduce, or reverse the progression or severity of whatever is being inhibited, including, but not limited to, a biological activity or property, disease, or condition. Inhibition or neutralization is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0099] For example, an anti-myostatin Adnectin in a pharmaceutical formulation may reduce circulating levels of biologically active myostatin normally present in a vertebrate subject, or reduce circulating levels of biologically active myostatin in a subject with a disease that results in elevated circulating levels of myostatin. Reduction of myostatin activity can be determined using in vitro assays described herein, e.g., binding assays.

[0100] The term "PK" is an acronym for "pharmacokinetics" and encompasses the characteristics of a compound, including, for example, absorption, distribution, metabolism, and excretion by a subject. As used herein, a "PK-modulating protein" or a "PK moiety" refers to any protein, peptide, or moiety that, when fused with or administered together with a biologically active molecule, affects the pharmacokinetic properties of the biologically active molecule. Examples of PK-modulating proteins or PK moieties include PEG, human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005 / 0287153 and 2007 / 0003549, PCT Publication Nos. WO2009 / 083804 and WO2009 / 133208), human serum albumin, Fc or Fc fragments and variants thereof, and sugars (e.g., sialic acid).

[0101] The "half-life" of an amino acid sequence or compound can generally be defined as the time it takes for the serum concentration of the polypeptide to decrease by 50% in vivo, for example, due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. Half-life can be determined by any method known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to those skilled in the art and may generally include, for example, administering an appropriate dose of an amino acid sequence or compound of the present invention to a subject, periodically collecting blood or other samples from the subject, determining the level or concentration of the amino acid sequence or compound of the present invention in the blood samples, and calculating the time until the level or concentration of the amino acid sequence or compound of the present invention decreases by 50% compared to the initial level at the time of administration from the data (plots of the data) thus obtained. See, for example, standard handbooks such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0102] The half-life is t 1 / 2 -alpha, t 1 / 2 It can be expressed using parameters such as beta, HL_Lambda_z, and area under the curve (AUC). As used herein, "prolonged half-life" refers to the prolongation of any one of these parameters, any two of these parameters, any three of these parameters, or all four of these parameters. In particular, "prolonged half-life" refers to the prolongation of t 1 / 2 - t with or without prolongation of alpha and / or AUC 1 / 2-Refers to the extension of Beta and / or HL_Lambda_z.

[0103] The terms "mpk", "mg / kg" or "mg / kg" refer to milligrams per kilogram. All terms are used interchangeably throughout this disclosure.

[0104] A. Exemplary Molecules Polypeptides (e.g., anti-myostatin Adnectin molecules) that can be used in the formulations provided herein include an Fn3 domain ( 10 Fn3) (SEQ ID NO: 1).

[0105] In some embodiments, the anti-myostatin Adnectin in the pharmaceutical formulation comprises the BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively.

[0106] In some embodiments, the anti-myostatin Adnectin in the formulation comprises BC, DE and FG loops set forth in SEQ ID NOs: 5, 6 and 7, respectively, wherein the BC loop comprises one, two or three amino acid substitutions, such as conservative amino acid substitutions, that enable the anti-myostatin Adnectin to maintain binding to myostatin.

[0107] In some embodiments, the anti-myostatin Adnectin in the formulation comprises a BC, DE, and FG loop set forth in SEQ ID NOs: 5, 6, and 7, respectively; 10 At least one of the BC, DE and FG loops of the Fn3 domain has one amino acid substitution relative to the respective BC, DE and FG loop of SEQ ID NOs:5, 6 and 7.

[0108] In some embodiments, the anti-myostatin Adnectin in the formulation comprises a BC, DE, and FG loop set forth in SEQ ID NOs: 5, 6, and 7, respectively; 10One loop from the BC, DE or FG loop of the Fn3 domain has one amino acid substitution relative to the respective BC, DE or FG loop of SEQ ID NOs:5, 6 and 7.

[0109] In some embodiments, the anti-myostatin Adnectin in the formulation comprises BC, DE, and FG loops set forth in SEQ ID NO: 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; (ii) the leucine at position 4 of the BC loop (SEQ ID NO: 5) is substituted with an amino acid selected from the group consisting of 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; and (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.

[0110] In some embodiments, the anti-myostatin Adnectin in the formulation comprises BC, DE, and FG loops set forth in SEQ ID NO: 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; and (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.

[0111] In some embodiments, the anti-myostatin Adnectin in the formulation comprises BC, DE, and FG loops 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, and (iii) the glutamine at position 7 of the BC loop (SEQ ID NO: 5) is (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, E, F, H, I, K, L, M, P, R, S, T, V 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.

[0112] In some embodiments, the anti-myostatin Adnectin in the formulation comprises BC, DE, and FG loops set forth in SEQ ID NO: 5, 6, or 7, respectively, and 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.

[0113] In some embodiments, the anti-myostatin Adnectin in the formulation comprises BC, DE, and FG loops 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, and (iii) 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. (iv) the aspartic acid at position 4 in 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; (v) the threonine at position 5 in 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, P, Q, R, S, V, W or Y; (vi) the glycine at position 6 in 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, P, Q, R, S, V, W or Y; , F, H, I, K, L, M, N, Q, R, S, T, V, W or Y; (vii) 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; (viii) 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; (ix) a) 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, (x) the tyrosine at position 10 of the FG loop (SEQ ID NO:7) is substituted with the amino acid F or W, or (ix) 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.

[0114] In some embodiments, the anti-myostatin Adnectin in the formulation comprises BC, DE, and FG loops 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 A, (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; (vii i) 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.

[0115] In some embodiments, the anti-myostatin Adnectin in the formulation comprises BC, DE, and FG loops set forth in SEQ ID NO: 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; and (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.

[0116] In some embodiments, the anti-myostatin Adnectin comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:8. EVVAATPTSLLISWSLPHQGKANYYRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTDTGYLKYKPISINYRT (SEQ ID NO: 8)

[0117] In some embodiments, the polypeptide in the formulation binds to myostatin and comprises an amino acid sequence that 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. 10 For example, in some embodiments, 10 The non-ligand binding sequence of Fn3, i.e., 10 Fn3 scaffolding" is also 10 Fn3 may be modified under conditions that maintain its ligand binding function and / or structural stability. 10 Fn3 scaffold has been reported. In some embodiments, one or more of Asp7, Glu9 and Asp23 are replaced with another amino acid, such as a non-negatively charged amino acid residue (e.g., Asn, Lys, etc.). These mutants have a neutral pH compared to wild type. 10 It has been reported to have an effect of increasing the stability of Fn3 (see, e.g., PCT Publication No. WO02 / 04523). It can be either beneficial or neutral. 10 Various additional modifications to the Fn3 scaffold have been described, see, e.g., Batori et al., Protein Eng., 15(12):1015-1020 (December 2002) and Koide et al., Biochemistry, 40(34):10326-10333 (August 28, 2001).

[0118] In certain embodiments, the amount of the polypeptide in the formulation 10 The Fn3 domain comprises SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In one embodiment, the polypeptide in the formulation 10 The Fn3 domain comprises SEQ ID NO:10.

[0119] B. Extension sequence In certain embodiments, the polypeptide (e.g., anti-myostatin Adnectin molecule) in the formulation is modified to include an N-terminal extension sequence and / or a C-terminal extension sequence. For example, the MG sequence is defined in SEQ ID NO: 4. 10The M may be placed at the N-terminus of Fn3. Typically, M is cleaved, leaving a G at the N-terminus. In some embodiments, an anti-myostatin Adnectin may comprise the amino acid sequence of SEQ ID NO: 8 and an N-terminal extension sequence shown in Table 1. Additionally, M, G, or MG may be placed at the N-terminus of any of the N-terminal extension sequences shown in Table 1. In some embodiments, the anti-myostatin Adnectin in the formulation may be truncated at the threonine corresponding to T94 in SEQ ID NO: 4. Alternatively, a C-terminal extension sequence may be added after the C-terminal residue of SEQ ID NO: 8. Exemplary C-terminal extension sequences are shown in Table 1. [Table 1]

[0120] In certain embodiments, the C-terminal extension sequence (also referred to as the "tail") comprises E and D residues and can be 8-50, 10-30, 10-20, 5-10, and 2-4 amino acids in length. In some embodiments, the tail sequence comprises an ED-based linker, whose 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 sequence may also comprise additional amino acid residues, such as, for example, EI, EID, ES, EC, EGS, and EGC. Such sequences are based in part on known Adnectin tail sequences, such as EIDKPSQ (SEQ ID NO: 20), with residues D and K removed. In exemplary embodiments, the ED-based tail comprises an E, I, or EI residue before the ED repeat.

[0121] C. Anti-myostatin Adnectin Immunoglobulin Fc Fusion In one aspect, a formulation is provided comprising a polypeptide (e.g., an anti-myostatin Adnectin) or a fragment or variant thereof fused to an immunoglobulin Fc domain. As used herein, a "functional Fc region" is an Fc domain or fragment thereof that retains the ability to bind to FcRn. In some embodiments, a functional Fc region binds to FcRn but does not have an effector function. The ability of an 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 C1q 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), and the like. Such effector functions generally require combining an Fc region with a binding domain (e.g., an anti-myostatin Adnectin) and can be assessed using a variety of assays known in the art for assessing such antibody effector functions.

[0122] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region due to 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 the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or the Fc region of a parent polypeptide. A variant Fc region herein preferably has at least about 80% sequence identity, most preferably at least about 90% sequence identity, and even more preferably at least about 95% sequence identity to a native-sequence Fc region and / or the Fc region of a parent polypeptide.

[0123] In an exemplary embodiment, the Fc domain is derived from the IgG1 subclass, although other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used. The sequence of a human IgG1 immunoglobulin Fc domain is shown below. [ka]

[0124] The core hinge sequence is underlined, and the CH2 and CH3 regions are in normal text. It should be understood that the C-terminal lysine is optional.

[0125] Fusions can be formed by attaching an anti-myostatin Adnectin to either end of an Fc molecule, i.e., an Fc-anti-myostatin Adnectin or an anti-myostatin Adnectin-Fc configuration. In certain embodiments, the Fc and anti-myostatin Adnectin are fused via a linker. Exemplary linker sequences are GAGGGGSG (SEQ ID NO:40), EPKSSD (SEQ ID NO:41), D, ESPKAQASSVPTAQPQAEGLA (SEQ ID NO:42), ELQLEESAAEAQDGELD (SEQ ID NO:43), GQPDEPGGS (SEQ ID NO:44), GGSGSGSGSGSGS (SEQ ID NO:45), ELQLEESAAEAQEGELE (SEQ ID NO:46), GSGSG (SEQ ID NO:47), GSGC (SEQ ID NO:48), AGGGGSG (SEQ ID NO:49), GSGS (SEQ ID NO:50), QPDEPGGS (SEQ ID NO:51), GSGSGS (SEQ ID NO:52), TVAAPS (SEQ ID NO:53), KAGGGGSG (SEQ ID NO:54), 4), KGSGSGSGSGSGS (SEQ ID NO: 55), KQPDEPGGS (SEQ ID NO: 56), KELQLEESAAEAQDGELD (SEQ ID NO: 57), KTVAAPS (SEQ ID NO: 58), KAGGGGSGG (SEQ ID NO: 59), KGSGSGSGSGSGSSG (SEQ ID NO: 60), KQPDEPGGSG (SEQ ID NO: 61), KELQLEESAAEAQDGELDG (SEQ ID NO: 62), KTVAAPSG (SEQ ID NO: 63), AGGGGSGG (SEQ ID NO: 64), AGGGGSG (SEQ ID NO: 65), GSGSGSGSGSSGSG (SEQ ID NO: 66), QPDEPGGSG (SEQ ID NO: 67) and TVAAPSG (SEQ ID NO: 68).

[0126] In some embodiments, the Fc region used in the anti-myostatin Adnectin fusion comprises the hinge region of an Fc molecule. As used herein, the "hinge" region comprises the core hinge residues spanning positions 1-16 of SEQ ID NO:39 of an IgG1 Fc region (DKTHTCPPCPAPELLG, SEQ ID NO:69).

[0127] In certain embodiments, the anti-myostatin Adnectin-Fc fusion in the formulation adopts a multimeric structure (e.g., a dimer) due in part to cysteine ​​residues at positions 6 and 9 of SEQ ID NO: 39 within the hinge region. In other embodiments, a hinge region as used herein may further include residues from the CH1 and CH2 regions adjacent to the core hinge sequence as set forth in SEQ ID NO: 39. In yet other embodiments, the hinge sequence is GSTHTCPPCPAPELLG (SEQ ID NO: 70).

[0128] In some embodiments, the hinge sequence may include substitutions that confer desirable pharmacokinetic, biophysical and / or biological properties. Some exemplary hinge sequences are: [ka] In one embodiment, residue P at position 18 of SEQ ID NO: 39 is substituted with S to eliminate Fc effector function, and this substitution is exemplified in hinges having any one of SEQ ID NOs: 72, 73, or 75. In another embodiment, residues DK at positions 1-2 of SEQ ID NO: 39 are substituted with GS to remove a potential clip site, and this substitution is exemplified in SEQ ID NO: 73. In another embodiment, C at position 103 of SEQ ID NO: 76, which corresponds to the heavy chain constant region of human IgG1 (i.e., domains CH1-CH3), is substituted with S to prevent inappropriate cysteine ​​bond formation in the absence of light chains, and this substitution is exemplified in SEQ ID NOs: 71-73. [ka]

[0129] In certain embodiments, anti-myostatin Adnectin-Fc fusions can have the following configurations: 1) anti-myostatin Adnectin-hinge-Fc or 2) hinge-Fc anti-myostatin Adnectin. Accordingly, any anti-myostatin Adnectin of the present invention can be fused to an Fc region comprising a hinge sequence according to these configurations. In some embodiments, a linker can be used to connect the anti-myostatin Adnectin to the hinge-Fc portion; for example, exemplary fusion proteins can have the following configurations: 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 can be positioned at the N-terminus of the fusion polypeptide. For example, if the fusion is produced in a mammalian system, a leader sequence such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 77) can be added to the N-terminus of the fusion molecule. If the fusion is produced in E. coli, the fusion sequence is preceded by a methionine.

[0130] In one embodiment, the polypeptide is an Fc anti-myostatin Adnectin construct known as BHV- 2000. In one embodiment, the Fc anti-myostatin Adnectin construct comprises the following amino acid sequence: [ka] The hinge region is underlined, the linker is in italics, the leader sequence is in bold, and the anti-myostatin Adnectin sequence is underlined and italics.

[0131] In one embodiment, the formulation comprises an Fc anti-myostatin Adnectin construct comprising the following amino acid sequence: GVSDVPRDLEVVAATPTSLLISWSLPHQGKANYYRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTDTGYLKYKPISINYRTEIEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 79)

[0132] The Fc domain consists of the human IgG1 CH2 and CH3 regions, as follows: VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO: 80) and the hinge sequence DKTHTCPPCPAPELLG (SEQ ID NO: 69).

[0133] II. Stable Formulations The polypeptides described herein can be administered as a pharmaceutical formulation.

[0134] A "stable" formulation or drug product is one in which the anti-myostatin Adnectin therein essentially retains its physical and chemical stability and integrity upon storage. The stability of an anti-myostatin Adnectin molecule formulation can be measured at a selected temperature and after a selected period of time. For example, an increase in aggregate formation after lyophilization and storage is an indicator of instability of a lyophilized anti-myostatin Adnectin molecule formulation. Retention of the original clarity, color, and odor during storage, in addition to aggregate formation, are indicators used to monitor the stability of an anti-myostatin Adnectin molecule solution. HMW species are multimers (i.e., tetramers, hexamers, etc.) that have a higher molecular weight than the monomer or dimer forms of the anti-myostatin Adnectin molecule. Typically, a "stable" drug product is one in which the formulation exhibits less than about a 5%, preferably less than about a 3%, increase in aggregation, as measured by the increase in the percentage of high molecular weight species (% HMW), upon storage at 2-8°C for one year. Preferably, the manufactured drug product contains less than about 25% HMW species, preferably less than about 15% HMW species, more preferably less than about 10% HMW species, and most preferably less than about 5% HMW species.

[0135] The "shelf life" of a pharmaceutical product, e.g., a protein, including an anti-myostatin Adnectin, is the length of time the product can be stored before degradation occurs. For example, shelf life may be defined as the time until 0.1%, 0.5%, 1%, 5%, or 10% of the product is degraded.

[0136] The terms "lyophilization" and "freeze-drying" are used interchangeably herein and refer to a material that is first frozen and then dehydrated by reducing the surrounding pressure to allow the frozen water within the material to sublimate.

[0137] A "reconstituted" formulation is prepared by dissolving the lyophilized formulation in an aqueous carrier such that the anti-myostatin Adnectin molecule is soluble in the reconstituted formulation. The reconstituted formulation is suitable for intravenous (IV) or subcutaneous (SC) administration to a patient in need thereof.

[0138] An "isotonic" formulation is one that has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of approximately 250-350 mOsmol / kgH2O. The term "hypertonic" is used to describe formulations that have an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using a vapor pressure or ice osmometer.

[0139] The term "buffer" refers to one or more components that, when added to an aqueous solution, can protect the solution from pH fluctuations upon addition of acid or alkali, or upon dilution with a solvent. Pharmaceutically acceptable buffers include, but are not limited to, histidine, TRIS (tris(hydroxymethyl)aminomethane), citrate, succinate, glycolate, and the like, as described herein.

[0140] The term "pKa" refers to the negative logarithm (p) of the ionization (acid dissociation) constant (Ka) of an acid, which is equal to the pH value at which equal concentrations of the buffer's acid and conjugate base are present (at which half of the acid molecules are ionized). A buffering system is most effective when the p of a buffer equals the pH of the solution being buffered.

[0141] An "acid" is a substance that produces hydrogen ions in aqueous solution. "Pharmaceutically acceptable acids" include inorganic and organic acids that are non-toxic at the concentrations and methods of formulation.

[0142] A "base" is a substance that produces hydroxide ions in aqueous solution. "Pharmaceutically acceptable base" includes inorganic and organic bases that are non-toxic at the concentrations and methods of formulation.

[0143] "Preservatives" are substances that inhibit bacterial activity and can optionally be added to the formulations herein. Adding a preservative can, for example, facilitate the manufacture of multi-use (multi-dose) formulations. Potential preservatives include, for example, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0144] "Surfactants" are surface-active molecules that contain both hydrophobic portions (eg, alkyl chains) and hydrophilic portions (eg, carboxyl and carboxylate groups). Suitable surfactants for use in the formulations of the present invention include polysorbates (e.g., polysorbate 20 or 80), poloxamers (e.g., poloxamer 188), sorbitan esters and derivatives, Triton, sodium lauryl sulfate, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetadine, lauryl-, linoleyl-, or stearyl-sarcosine, linoleyl-, myristyl-, or cetyl-betaine, lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl betaine (e.g., lauramidopropyl), myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl-, or sodium methyl cocoyl-taurate, and the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.).

[0145] "Drug substance" refers to the starting material utilized in the formulation of a final pharmaceutical product. A typical anti-myostatin Adnectin drug substance has a protein concentration of 10 mg / mL-200 mg / mL, a pH of 6.6-7.6, and a % HMW species of less than 5%.

[0146] "Formulated bulk solution" refers to the final formulation before filling into a container, such as a formulated solution before filling into vials for lyophilization, or a formulated solution before filling into syringes for IV and / or SC injection.

[0147] "Drug product" refers to a final formulation packaged in a container that can be reconstituted before use, e.g., with a lyophilized drug product, further diluted before use, e.g., with a liquid drug product, or used as is, e.g., with an SC solution drug product.

[0148] A. Exemplary Formulations In some embodiments, the concentration of the polypeptide (e.g., anti-myostatin Adnectin) in the formulation is between about 10 mg / mL and 200 mg / mL. In other embodiments, the concentration of the polypeptide in the formulation is between about 10 mg / mL and 140 mg / mL. In other embodiments, the concentration of the polypeptide in the formulation is at 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 more. In certain embodiments, the concentration of the polypeptide 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 / mL. In certain embodiments, the concentration of the polypeptide in the formulation is 10.7 mg / mL, 21.4 mg / mL, 50.0 mg / mL, or 71.4 mg / mL.

[0149] The stabilizing sugar in the formulation is a disaccharide having a protein to sugar weight ratio (w / w) of at least 5:1. In some embodiments, the protein to sugar weight ratio is about 5:1 to 10:1. In some embodiments, the protein to sugar ratio is about 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the protein to sugar ratio is about 6.75:1.

[0150] In some embodiments, the formulation contains about 5% to about 30% disaccharides. In some embodiments, the formulation contains about 10% to about 28% disaccharides. In some embodiments, the formulation contains about 15% to about 25% disaccharides. In some embodiments, the formulation contains about 20% to about 25% disaccharides. In some embodiments, the formulation contains about 18%, 19%, 20%, 21%, 22%, 23%, 24%, or about 25% disaccharides.

[0151] In some embodiments, the sugar concentration in the formulation is about 150 mM to about 800 mM. In some embodiments, the sugar concentration in the formulation is about 300 to about 700 mM. In other embodiments, the sugar concentration 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 mM, about 625 mM, about 650 mM, about 675 mM, or about 700 mM.

[0152] 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.

[0153] In some embodiments, the disaccharide is trehalose dihydrate. In some embodiments, the concentration of trehalose dihydrate in the formulation is about 150 mM to about 800 mM. In some embodiments, the concentration of trehalose dihydrate in the formulation is about 300 to about 700 mM. In other embodiments, the concentration of 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 mM, 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.

[0154] The stabilizing sugar in the formulation is used in an amount not greater than that which would result in an undesirable or unsuitable viscosity for administration via an SC syringe. In some embodiments, the viscosity of the formulation is about 5-20 cps. In some embodiments, the viscosity of the formulation is about 7-12 cps. In some embodiments, the viscosity is about 7-10 cps. In some embodiments, the viscosity of the formulation is less than 8 cps.

[0155] The buffering agent in the formulation is present in an amount of at least 20 mM, preferably about 20 mM to 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 contains about 30 mM histidine.

[0156] The pH of the formulation is maintained in the range of about 6.5 to about 7.8. In certain embodiments, the pH is maintained in the range of about pH 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.

[0157] Aqueous carriers used in the formulations herein are pharmaceutically acceptable (safe and non-toxic for human administration) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0158] The formulation may further comprise a surfactant to further reduce the formation of visible particles. Preferred surfactants include poloxamer and polysorbate at concentrations of about 0.01% to 0.5%. In some embodiments, the surfactant concentration is about 0.02% to 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.

[0159] The formulation may further comprise a chelating agent at a concentration of about 0.01 mM to about 0.5 mM, preferably about 0.05 mM to 0.2 mM. Preferred chelating agents include, but are not limited to, DPTA, EDTA, and EGTA. In one embodiment, the chelating agent in the formulation is DPTA at a concentration of about 0.05 mM.

[0160] Optionally, preservatives may be added to the formulations herein to reduce bacterial action, for example, to facilitate the manufacture of multi-use (multi-dose) formulations.

[0161] In some embodiments, the formulations described herein comprise: (i) about 10 to 140 mg / mL of a polypeptide; (ii) about 5-25% trehalose dihydrate, and (iii) containing about 20 to 30 mM histidine; The pH of the formulation is about 6.8 to 7.3.

[0162] In some embodiments, the formulations described herein comprise essentially (i) about 10 to 140 mg / mL of a polypeptide; (ii) about 5-25% trehalose dihydrate, and (iii) about 20-30 mM histidine; The pH of the formulation is about 6.8 to 7.3.

[0163] In some embodiments, the formulations described herein comprise: (i) about 10 to 140 mg / mL of a polypeptide; (ii) about 5-25% trehalose dihydrate; (iii) about 20-30 mM histidine; (iv) about 0.02 to 0.06 mM DTPA, and (v) containing about 0.01 to 0.05% polysorbate 80; The pH of the formulation is about 6.8 to 7.3.

[0164] In some embodiments, the formulations described herein comprise essentially (i) about 10 to 140 mg / mL of a polypeptide; (ii) about 5-25% trehalose dihydrate; (iii) about 20-30 mM histidine; (iv) about 0.02 to 0.06 mM DTPA, and (v) containing about 0.01 to 0.05% polysorbate 80; The pH of the formulation is about 6.8 to 7.3.

[0165] In some embodiments, the formulation comprises: (i) about 10 to 75 mg / mL of a polypeptide; (ii) about 600 mM trehalose dihydrate, and (iii) containing 25 to 30 mM histidine; The pH of the formulation is about 7.0 to 7.3.

[0166] In some embodiments, the formulation comprises essentially (i) about 10 to 75 mg / mL of a polypeptide; (ii) about 600 mM trehalose dihydrate, and (iii) containing 25 to 30 mM histidine; The pH of the formulation is about 7.0 to 7.3.

[0167] In some embodiments, the formulation comprises: (i) about 10 to 75 mg / mL of a polypeptide; (ii) about 600 mM trehalose dihydrate; (iii) about 25-30 mM histidine; (iv) about 0.02 to 0.06 mM DTPA, and (v) containing about 0.01 to 0.05% polysorbate 80; The pH of the formulation is about 7.0 to 7.3.

[0168] In some embodiments, the formulation comprises essentially (i) about 10 to 75 mg / mL of a polypeptide; (ii) about 600 mM trehalose dihydrate; (iii) about 25-30 mM histidine; (iv) about 0.02 to 0.06 mM DTPA, and (v) about 0.01 to 0.05% polysorbate 80; The pH of the formulation is about 7.0 to 7.3.

[0169] In some embodiments, the formulation comprises: (i) about 10 to 75 mg / mL of a polypeptide; (ii) about 600 mM trehalose dihydrate; (iii) about 30 mM histidine; (iv) about 0.05 mM DTPA, and (v) about 0.02% polysorbate 80; The pH of the formulation is about 7.1.

[0170] In some embodiments, the formulation comprises essentially (i) about 10 to 75 mg / mL of a polypeptide; (ii) about 600 mM trehalose dihydrate; (iii) about 30 mM histidine; (iv) about 0.05 mM DTPA, and (v) about 0.02% polysorbate 80; The pH of the formulation is about 7.1.

[0171] In one embodiment, the formulation comprises: (i) about 10.7 mg / mL of polypeptide; (ii) about 600 mM trehalose dihydrate; (iii) about 30 mM histidine; (iv) about 0.05 mM DTPA, and (v) comprising or consisting essentially of about 0.02% polysorbate 80; The pH of the formulation is about 7.1.

[0172] In one embodiment, the formulation comprises: (i) about 21.4 mg / mL of polypeptide; (ii) about 600 mM trehalose dihydrate; (iii) about 30 mM histidine; (iv) about 0.05 mM DTPA, and (v) comprising or consisting essentially of about 0.02% polysorbate 80; The pH of the formulation is about 7.1.

[0173] In one embodiment, the formulation comprises: (i) about 50 mg / mL of a polypeptide; (ii) about 600 mM trehalose dihydrate; (iii) about 30 mM histidine; (iv) about 0.05 mM DTPA, and (v) comprising or consisting essentially of about 0.02% polysorbate 80; The pH of the formulation is about 7.1.

[0174] In one embodiment, the formulation comprises: (i) about 71.4 mg / mL of polypeptide; (ii) about 600 mM trehalose dihydrate; (iii) about 30 mM histidine; (iv) about 0.05 mM DTPA, and (v) comprising or consisting essentially of about 0.02% polysorbate 80; The pH of the formulation is about 7.1.

[0175] The recommended storage conditions for liquid formulations are 2-8°C, with a recommended shelf life of at least 12 months. To ensure efficacy and safety over the course of a pharmaceutical composition's shelf life, the stability of the composition is tested. Typically, stability testing includes, but is not limited to, testing the composition's identity, purity, and potency. Stability is tested both at the intended storage temperature and at elevated temperatures. Purity testing may include, but is not limited to, SDS-PAGE, CE-SDS, isoelectric focusing, immunoelectrophoresis, Western blot, reverse-phase chromatography, size-exclusion chromatography (SEC), ion exchange, and affinity chromatography. Other tests include, but are not limited to, appearance (e.g., color and clarity), particulates, pH, protein concentration, moisture, and reconstitution time.

[0176] The degradation profile during the stability time course, particularly with regard to purity and potency, is closely related to the composition and / or formulation of the pharmaceutical product. In particular, the degradation profile can change significantly with the appropriate selection of formulation. A typical degradation profile of FBS-derived protein molecule products includes the formation of covalent and non-covalent high-molecular-weight aggregates, fragments, deamidation, and oxidation products. In particular, deamidation and oxidation products, as well as other acidic substances, are typically generated during stability testing. In some cases, acidic species limit the acceptable storage period of a pharmaceutical composition. For example, the formation of acidic species due to deamidation can be examined by imaging capillary isoelectric focusing (icIEF). In other cases, the formation of high-molecular-weight aggregates limits the acceptable storage period of a pharmaceutical composition. Aggregate formation can be examined by, for example, SEC (size exclusion chromatography), DLS, MFI, SDS-PAGE, or CE-SDS.

[0177] For example, an anti-myostatin Adnectin formulation with pharmaceutically acceptable stability can be one that exhibits less than about 10%, preferably less than about 5%, and more preferably less than about 2% percent aggregates as determined using SEC analysis when stored for at least about 3 months, preferably about 6 months, more preferably about 12 months or more, e.g., 18 months or more, e.g., at least 24 or 36 months, at a temperature of about 5±3° C. or 25±2° C. Additionally or alternatively, a stable anti-myostatin Adnectin formulation of the invention can be one that exhibits less than 15%, preferably less than 10%, more preferably less than 8%, and most preferably less than 5% percent change in major isoforms as determined using icIEF analysis when stored for at least about 3 months, preferably about 6 months, more preferably about 12 months or more.

[0178] B. Preparation of Formulations The manufacturing process developed for SC formulations typically involves the concentration of the drug substance, optionally using diafiltration (buffer exchange) and ultrafiltration units, followed by the incorporation of sugars, chelating agents, and surfactants, followed by sterile filtration and filling into vials or syringes. Protein purification is the first step after production in a fermentation bioreactor. The protein is purified using multiple column and filtration steps and concentrated into a formulation buffer using tangential flow filtration. The concentrated drug substance is diluted with the formulation buffer to the target concentration, and this solution is sterile filtered and filled into sterile vials / syringes for patient use. Those skilled in the art will recognize the need to overfill containers to compensate for vial, needle, and syringe holdup during preparation and injection. For example, a 5-10% excess of drug is incorporated into each vial of a liquid formulation to compensate for withdrawal losses and ensure that the required (labeled) dose of drug can be withdrawn from the vial.

[0179] Binds to myostatin 10 Preparation of a unit dosage form of a formulation comprising a Fn3 domain-containing polypeptide (also referred to herein as an "anti-myostatin Adnectin") syringe involves protein production in a recombinant cell line, purification via multiple column steps, concentration, and buffer exchange into a formulation buffer using tangential flow filtration. The concentrated protein for tangential flow filtration is further processed by diluting it to the desired protein concentration with a formulation buffer, and the diluted product is loaded into a 1 mL syringe (e.g., insulin syringe, tuberculin syringe, BioPak syringe, NeoPak syringe) after filtration. In one embodiment, the syringe is then equipped with an UltraSafe Passive needle guard.

[0180] A 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 in a volume of 0.7 mL. In some embodiments, the unit dosage form contains a compound that binds to myostatin. 10 The unit dosage form contains 5 to 100 mg of a polypeptide comprising an Fn3 domain. In some embodiments, the unit dosage form contains 7.5 mg, 15 mg, 35 mg, or 50 mg of an anti-myostatin Adnectin.

[0181] In some embodiments, formulations are manufactured as disclosed herein and stored in bulk in 12 L FFTp bags at -60°C, for example, at a polypeptide concentration of 85-150 mg / mL. In some embodiments, formulations are stored at -60°C at a polypeptide concentration of 85 mg / mL. The bulk formulation is then thawed and diluted to the appropriate polypeptide concentration for preparation of unit dosage forms. In some embodiments, the polypeptide concentration of the formulation in the unit dosage form is about 10 mg / mL to about 140 mg / mL. In some embodiments, the polypeptide concentration of the formulation in the unit dosage form is about 10 mg / mL to about 75 mg / mL. In certain embodiments, the polypeptide 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.

[0182] C. Administration Pharmaceutical preparations comprising the polypeptides described herein can be administered to subjects at risk of or exhibiting the pathologies described herein.The preparations described herein are particularly useful for peripheral systemic delivery by intravenous, intraperitoneal or subcutaneous injection.In a preferred embodiment, the preparation is delivered by subcutaneous injection.

[0183] Therapeutic effective dose refers to the dose that produces the therapeutic effect of its administration.The effective amount of pharmaceutical composition used for treatment depends, for example, on the situation and purpose of treatment.Those skilled in the art will understand that the appropriate dosage level for treatment will vary depending on the molecule to be delivered, the indication that the binding agent molecule is used, the route of administration, and the patient's physical size (weight, body surface area or organ size) and condition (age and general health).

[0184] The exact dosage is determined based on the factors related to the subject that needs to be treated, and can be confirmed by standard techniques.Dosage and administration are adjusted to provide a sufficient level of active compound or maintain desired effect.Factors that can be considered include the severity of disease state, the overall health of the subject, the age, weight and sex of the subject, the time and frequency of administration, the combination of drug(s), reaction sensitivity and response to therapy.

[0185] The polypeptide (anti-myostatin Adnectin molecule) is administered to the target population at any suitable dose. In one embodiment, the polypeptide is administered as repeated subcutaneous injections. In one embodiment, the polypeptide is administered at a weekly dosing interval. In one embodiment, the polypeptide is administered at a biweekly dosing interval. In one embodiment, the polypeptide is administered at a monthly dosing interval.

[0186] In one embodiment, the polypeptide is administered at a dose of about 5 to 200 mg. For example, the polypeptide may be administered at a dose of 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, g, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74mg, 75m g, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 22mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93mg, 94mg, 95mg, 96mg, 97mg, 98mg, 99mg , 100mg, 101mg, 102mg, 103mg, 104mg, 105mg, 106mg, 107mg, 108mg, 109mg, 110mg, 111mg, 112mg, 113mg, 114mg, 115mg, 116mg, 117mg, 118mg, 119mg , 120mg, 121mg, 122mg, 123mg, 124mg, 125mg, 126mg, 127mg, 128mg, 129mg, 130mg, 131mg, 132mg, 133mg, 134mg, 135mg, 136mg, 137mg, 138mg, 139mg , 140mg, 141mg, 142mg, 143mg, 144mg, 145mg, 146mg, 147mg, 148mg, 149mg, 150mg, 151mg, 152mg, 153mg, 154mg, 155mg, 156mg, 157mg, 158mg, 159mg , 160mg, 161mg, 162mg, 163mg, 164mg, 165mg, 166mg, 167mg, 168mg, 169mg, 160mg, 171mg, 172mg, 173mg, 174mg, 175mg, 176mg, 177mg, 178mg, 179mg,It can be administered in doses of 170 mg, 181 mg, 182 mg, 183 mg, 184 mg, 185 mg, 186 mg, 187 mg, 188 mg, 189 mg, 180 mg, 191 mg, 192 mg, 193 mg, 194 mg, 195 mg, 196 mg, 197 mg, 198 mg, 199 mg, or 200 mg.

[0187] In one embodiment, the therapeutic dose level(s) and dosing interval(s) are determined by the pharmacokinetic:pharmacodynamic (PK / PD) relationship of the anti-myostatin Adnectin in the target population, as determined in controlled clinical trials. Desired pharmacodynamic changes include, but are not limited to, a decrease in free (unbound) myostatin, improvements in body composition and total body weight.

[0188] The frequency of administration depends on the pharmacokinetic:pharmacodynamic parameters of the binding agent molecule in the formulation used. Typically, the composition is administered until a dosage that achieves the desired effect is reached. Thus, the composition can be administered as a single dose or multiple doses (at the same or different concentrations / dosages) over time. Further adjustment of the appropriate dosage is routinely performed. The appropriate dosage can be confirmed through the use of appropriate dose-response data. For example, the anti-myostatin Adnectin can be administered less frequently (e.g., every two weeks or monthly). Furthermore, adjustments for age, weight, general health, sex, timing of administration, drug interactions, and disease severity may be necessary, as known in the art and can be confirmed by routine experimentation by one of ordinary skill in the art. The anti-myostatin Adnectin can be suitably administered to a patient at once or over a series of treatments.

[0189] III. Method Herein, the tenth ( 10Methods for improving glycemic control, treating, preventing, or reducing obesity and associated complications, and treating or preventing type II diabetes in a human patient are provided by administering to the human patient a polypeptide comprising a Fn3) domain, wherein the polypeptide is administered (or is for administration) according to a specific clinical dosing regimen (e.g., at a specific dose, according to a specific schedule).

[0190] As used herein, the term "subject" or "patient" refers to a human patient (eg, an obese patient, a patient with obesity-related complications, and / or a patient with type II diabetes).

[0191] The terms "mpk", "mg / kg" or "mg / kg" refer to milligrams per kilogram. All terms are used interchangeably throughout this disclosure.

[0192] The terms "individual," "subject," and "patient," used interchangeably herein, refer to animals, preferably mammals (including non-primates and primates) or birds, including, but not limited to, mice, monkeys, humans, livestock mammals (e.g., cows, pigs, sheep), sport mammals (e.g., horses), and pet mammals (e.g., dogs, cats). Preferably, the term refers to humans. The term also refers to birds, including, but not limited to, chickens and turkeys.

[0193] The term "therapeutically effective amount" refers to at least the minimal dose of a drug required to produce a therapeutic effect in a subject, but less than a toxic dose.

[0194] As used herein, the term "pediatric" patient refers to a human patient classified by a physician or caregiver as belonging to a category other than adulthood, including, for example, neonates (both preterm and full-term), infants, children, and adolescents. Typically, a pediatric patient is a patient under the age of 18 (<18 years of age).

[0195] As used herein, the term "adult" patient is a human patient who has been classified by a physician or caregiver based, e.g., on age, developmental status, physiological characteristics, etc., as someone who is not, e.g., a neonate, infant, child, or adolescent. Typically, an adult patient is a patient who is 18 years of age or older (age ≧18 years).

[0196] As used herein, the phrase "body mass index (BMI)" refers to a person's weight in kilograms (or pounds) divided by the square of their height in meters (or feet). A high BMI can indicate high body fat. BMI screens for weight categories that may lead to health problems, but does not diagnose an individual's body fat or health status.

[0197] The term "obesity" is based on body mass index (BMI) for both youth and adults, but the definitions are not directly comparable. Among adults, there are set cut points based on health risk, but among children, the definition is statistical and based on comparison to a reference population. BMI is calculated by dividing weight in kilograms by height in meters squared and rounding to the nearest whole number. Obesity in adults is defined as a BMI of 30 kg / m2 or greater. Obesity in youth is defined as a BMI at or above the age- and sex-specific 95th percentile of the 2000 CDC growth charts.

[0198] The term "overweight" or "overweight state" is based on a BMI of ≥ 25 to < 30 kg / m2. Overweight states can also be associated with at least one additional risk factor for fatal disease (e.g., stroke, heart failure, sudden death), such as diabetes, hypertension, or a family history of premature coronary artery disease.

[0199] Although different subjects may have the same BMI, the ratio of fat to muscle mass is different, so BMI is not necessarily an appropriate indicator for classifying overweight and obesity.A high percentage of muscle mass may result in a high BMI even if the percentage of fat is low.In this case, a subject may be erroneously determined to be overweight or obese based on BMI classification.In addition to BMI, indicators such as waist circumference and body mass index are also used.In clinical trials, DXA and MRI are often used to quantify muscle mass, fat, and fat distribution.

[0200] The term "body composition" is used herein to refer to the proportion of fat and muscle in the human body. Because muscle tissue takes up less space in the body than fat tissue, body composition and weight determine lean mass. Two people of the same sex and weight may appear completely different from each other due to their different body compositions.

[0201] "Lean body mass" (LBM) is a component of body composition and is calculated by subtracting body fat mass from body weight. Total body mass is the sum of lean and fat mass. Lean body mass is body weight minus body fat mass. Lean body mass plus fat mass equals body weight.

[0202] The percentage of total body mass that is lean body mass is not usually given, but is typically between 60 and 90%. Instead, the complement, body fat percentage, is calculated, which is typically between 10 and 40%. Because body fat is less relevant to metabolism, lean body mass (LBM) is described as a better indicator than total body mass for determining appropriate drug levels and assessing metabolic disorders.

[0203] The term "fat mass" refers to the portion of the body that is composed entirely of fat, which can be measured by dual-energy absorptiometry (DXA), MRI, or bioelectrical impedance analysis.

[0204] "Central obesity" refers to:

[0205] Obesity is defined as a condition characterized by abnormal or excessive fat accumulation in adipose tissue. Both the absolute amount of excess fat and its regional distribution among different adipose tissues play important roles in determining the health effects of obesity. Obesity is classified into central / android obesity and peripheral / guinoid obesity, with android obesity typically occurring more frequently in men, while guinoid obesity is more prevalent in women.

[0206] There is strong evidence in the literature that not all obesity is associated with a worse metabolic profile and increased cardiovascular risk. In fact, body fat distribution (i.e., the relative abundance of abdominal and peripheral fat mass) has been considered a better indicator of metabolic and cardiovascular risk than obesity itself. In men, who tend to accumulate adiposity in the trunk, elevated BMI is associated with increased CV risk, whereas in women, BMI is generally a poor indicator / surrogate of cardiovascular risk. Trunk fat mass is divided into subcutaneous fat (SC) (within the abdominal wall) and visceral adipose tissue (VAT) (within the abdominal cavity). Subcutaneous and visceral fat differ significantly in their anatomical structure, cellular composition, endocrine function, and cellular regulation. Compared to SC, VAT exhibits significant cellular, vascular, innervated, and infiltrated inflammatory and immune cells, which increases metabolic activity and the release of inflammatory cytokines, directly and indirectly affecting the risk of insulin resistance, type 2 diabetes, and cardiovascular disease. On the other hand, subcutaneous fat mass, particularly in the thighs and buttocks, is associated with the constitutive secretion of adiponectin, which contributes to insulin sensitivity, anti-inflammatory properties, and anti-atherogenic effects. Low levels of inflammation are associated with muscle wasting, which may further impair insulin sensitivity and increase the relative risk of developing type 2 diabetes. Therefore, even in the absence of overt obesity (i.e., BMI < 30 kg / m²), an imbalance between central and peripheral fat (central obesity) may be associated with significant insulin resistance, metabolic changes, and systemic low-level inflammation, which collectively accelerate atherosclerosis.

[0207] In clinical practice, anthropometric measurements such as waist circumference or waist-hip ratio are widely used to estimate abdominal obesity, but these measurements are associated with inaccurate results due to their inability to distinguish between visceral and subcutaneous abdominal fat. More advanced techniques, such as computed tomography (CT) or dual-energy X-ray absorptiometry (DXA), can assess / measure fat mass more directly. CT is advantageous for distinguishing between VAT and SAT, whereas DXA is useful for assessing the distribution of body fat mass among defined anatomical regions (arms, legs, trunk) and helps distinguish between male and female obesity.

[0208] Type 2 diabetes, also known as "type II diabetes," formerly called "non-insulin-dependent diabetes" or "adult-onset diabetes," accounts for 90-95% of all diabetes cases and involves individuals with insulin resistance and usually relative (rather than absolute) insulin deficiency. At least initially, and often throughout their lives, these patients may not require insulin treatment for survival.

[0209] A variety of causes lead to type 2 diabetes. While the specific etiology is unknown, autoimmune destruction of B cells does not occur, and patients do not have any 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 based on conventional weight standards may have increased body fat, primarily in the abdominal region. Type 2 diabetes often goes undiagnosed for many years because hyperglycemia develops gradually and is often not severe enough in the early stages for patients to notice typical diabetic symptoms. Nevertheless, even undiagnosed patients are at increased risk of developing macrovascular and microvascular complications.

[0210] The term "complications of obesity or overweight" refers to serious chronic disorders associated with diabetes, including, but not limited to, type 2 diabetes, impaired glucose tolerance, prediabetes, insulin resistance, hypertension, dyslipidemia, increased waist circumference, cardiovascular disease, non-alcoholic fatty liver disease, obstructive sleep apnea, physical disability, osteoarthritis, osteoporosis, renal disease, sex hormone(s) disorders, endocrine reproductive disorders such as polycystic ovary syndrome or male hypogonadism, stroke, and gallstones.

[0211] "Glucose intolerance" is defined as the inability to properly metabolize glucose.

[0212] "Insulin sensitivity" refers to the body's sensitivity to the effects of insulin. A person who is said to be insulin sensitive needs less insulin to lower their blood sugar levels than a person who is less sensitive. Insulin sensitivity varies from person to person, and doctors can perform tests to determine an individual's sensitivity to insulin.

[0213] "Insulin resistance" is defined as a state of resistance to insulin, resulting in reduced insulin hormone efficacy, reduced glucose uptake in muscle tissue, impaired glucose oxidation and glycogen synthesis, and insufficient suppression of hepatic glucose production in the liver. In obesity, increased visceral fat mass, accompanied by elevated plasma free fatty acids (FFA) due to enhanced lipolytic activity, reduces insulin resistance through impaired insulin action (Reaven, GM, et al., (1988), Diabetes. 37:1020-1024), a mechanism known as lipotoxicity (DeFronzo RA (2004), Int. J. Clin. Pract. Suppl.; (143):9-21). As used herein, the terms "improving insulin sensitivity" and "treating / reducing insulin resistance" are intended to be synonymous.

[0214] High plasma free fatty acid (FFA) concentrations in skeletal muscle cells reduce insulin-stimulated intracellular glucose transport via the Glut4 transporter (see, e.g., Dresner A., ​​et al. (1999), J. Clin. Invest. 103(2):253-259). In hepatocytes, transient increases in FFAs stimulate gluconeogenesis and hepatic glucose release, and insulin secretion from β cells is enhanced in response to chronic high levels, while chronic high levels have an inhibitory effect (see, e.g., Boden G. (1997), Diabetes. 46(1):3-10). As a result of insulin resistance and lipotoxicity, increased insulin is required to stimulate glucose uptake from adipose and muscle cells and glycogen synthesis in the liver (see, e.g., Boden G. (1997), Diabetes. 46(1):3-10). Overproduction of insulin by pancreatic beta cells is a physiological response to insulin resistance and can lead to impaired beta cell function and ultimately to prediabetes and type 2 diabetes (Donath MY, et al., (2005), Diabetes, 54:S108-S113.).

[0215] The term "improved insulin sensitivity" refers to whole-body responsiveness to glucose, which can be measured by the insulin sensitivity index (which measures the ability of endogenous insulin to lower extracellular glucose by inhibiting hepatic glucose release and promoting peripheral glucose consumption) and the glucose clamp technique (the effect of changes in insulin concentration on glucose clearance, i.e., glucose uptake rate divided by plasma glucose concentration per unit body surface area).

[0216] "Prediabetes" is a pre-diabetic stage in which blood glucose levels are abnormally high (e.g., 100-125 mg / dL).

[0217] Impaired fasting glucose and impaired glucose tolerance are two aspects of prediabetes that share a similar clinical definition (blood glucose levels too high for their respective conditions), but are physiologically distinct. Insulin resistance, metabolic syndrome (or syndrome X), and prediabetes are closely related and have overlapping aspects.

[0218] Herein, the tenth ( 10 Methods of improving glycemic control, treating, preventing, or reducing obesity and associated complications, and treating or preventing type II diabetes in a human patient are provided by administering to the human patient a polypeptide comprising a Fn3 domain. In some embodiments, the polypeptide is administered (or is for administration) according to a specific clinical dosing regimen (e.g., at a specific dose and according to a specific schedule).

[0219] In one aspect, a method for improving glycemic control in a human patient is provided, the method comprising administering to a human subject a fibronectin type III fragment of the tenth amino acid sequence ( 10 The method includes administering to a patient a polypeptide comprising a Fn3 domain, wherein the polypeptide is administered at a dose of 5-200 mg once a week or once every two weeks. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0220] In one embodiment, a method for improving glycemic control in a human patient is provided, the method comprising administering to a subject a therapeutically effective amount of fibronectin type III fragment containing the tenth amino acid residue ( 10 administering to the patient a polypeptide comprising a Fn3 domain, 10The Fn3 domain comprises BC, DE, and FG loops, wherein at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively, and the polypeptide is administered weekly or once every two weeks at a dose of 5-200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections). In one embodiment, 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO:8.

[0221] In one embodiment, a method of improving glycemic control in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0222] In one embodiment, a method of improving glycemic control in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 78, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0223] In one embodiment, a method of improving glycemic control in a human patient is provided, the method comprising administering BHV-2000 to the patient, wherein the BHV-2000 is administered at a dose of 5-200 mg once a week or once every two weeks. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0224] In another aspect, a method of treating, preventing, or alleviating overweight or obesity and associated complications in a human patient is provided, the method comprising: 10The method includes administering to a patient a polypeptide comprising a Fn3 domain, wherein the polypeptide is administered at a dose of 5-200 mg once a week or once every two weeks. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0225] In one embodiment, a method is provided for treating, preventing, or alleviating overweight or obesity and associated complications in a human patient, the method comprising administering to a human subject a fibronectin type III fragment of the tenth amino acid sequence ( 10 administering to the patient a polypeptide comprising a Fn3 domain, 10 The Fn3 domain comprises BC, DE, and FG loops, wherein at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively, and the polypeptide is administered weekly or once every two weeks at a dose of 5-200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections). In one embodiment, 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO:8.

[0226] In one embodiment, a method of treating, preventing, or alleviating overweight or obesity and associated complications in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0227] In one embodiment, a method of treating, preventing, or alleviating overweight or obesity and associated complications in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 78, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0228] In one embodiment, a method of treating, preventing, or alleviating overweight or obesity and associated complications in a human patient is provided, the method comprising administering BHV-2000 to the patient, wherein the BHV-2000 is administered once a week or once every two weeks at a dose of 5-200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0229] In a further aspect, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering to a subject a fibronectin type III fragment of the tenth amino acid sequence (SEQ ID NO: 1) that binds to myostatin. 10 The method includes administering to a patient a polypeptide comprising a Fn3 domain, wherein the polypeptide is administered at a dose of 5-200 mg once a week or once every two weeks. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0230] In one embodiment, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering to a subject a fibronectin type III fragment of the tenth amino acid sequence (SEQ ID NO: 1) that binds to myostatin. 10 administering to the patient a polypeptide comprising a Fn3 domain, 10 The Fn3 domain comprises BC, DE, and FG loops, wherein at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively, and the polypeptide is administered weekly or once every two weeks at a dose of 5-200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections). In one embodiment, 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO:8.

[0231] In one embodiment, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0232] In one embodiment, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering to the patient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 78, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0233] In one embodiment, a method of treating or preventing type II diabetes in a human patient is provided, the method comprising administering BHV-2000 to the patient, wherein the BHV-2000 is administered at a dose of 5 to 200 mg once a week or once every two weeks. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0234] In one embodiment, the patient's body mass index (BMI) is 30 kg / m 2 In another embodiment, the patient's BMI is ≥ 25 and < 30 kg / m 2 In another embodiment, the patient's BMI is <25.

[0235] In one embodiment, the patient is overweight. In another embodiment, the patient is obese. In another embodiment, the patient has obesity-related complications. Exemplary obesity-related complications include, but are not limited to, type 2 diabetes, impaired glucose tolerance, prediabetes, insulin resistance, hypertriglyceridemia, physical disability, osteoporosis, kidney disease, obstructive sleep apnea, sex hormone disorders, endocrine reproductive disorders such as polycystic ovary syndrome or male hypogonadism, osteoarthritis, gastrointestinal cancer, dyslipidemia, hypertension, heart failure, coronary heart disease, stroke, gallstones, and altered gonadal hormone profiles. In one embodiment, the patient has type 2 diabetes.

[0236] The peptide or a formulation comprising the peptide can be administered to a patient by any suitable means. In one embodiment, the peptide is formulated for intravenous administration. In one embodiment, the peptide is formulated for subcutaneous administration.

[0237] IV. Results The effectiveness of the treatment methods described herein can be assessed using any suitable means.

[0238] In one aspect, a method for improving glycemic control in human patients is provided.Glycemic control is an important goal in the management of patients, such as patients with diabetes, and remains the main therapeutic goal in preventing organ damage or other complications caused by diabetes (see, for example, 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(Supplement1):S13-S27).Hemoglobin A1c (HbA1c) is the gold standard for monitoring glycemic control and serves as a surrogate indicator of diabetes-related complications. Among patients with type 2 diabetes, poor glycemic control is a major public health problem and a significant risk factor for the progression of diabetic complications (see, e.g., Koro CE, et al., Diabetes Care. 2004; 27(1):17-20; Yakubu A, et al., Int. J. Sci. Health Res. 2020; 5(4):207-214; and Digssie A, et al., Metab Open. 2020; 8:100056). Strictly maintaining a patient's blood glucose levels within the normal or near-normal range is crucial to minimize diabetic complications (e.g., Mariye T, et al., Endocrinol. Metab. Open Access. 2020; 4(1):1-7). Accordingly, patients treated according to the methods disclosed herein experience an improvement in at least one sign of glycemic control, as assessed, for example, by HbA1c.

[0239] In another aspect, a method for treating, preventing, or alleviating obesity and related complications in a patient is provided. Symptoms of obesity include, but are not limited to, difficulty sleeping, sleep apnea, daytime sleepiness, back and / or joint pain, excessive sweating, heat intolerance, skin fold infections, fatigue, depression, and shortness of breath (dyspnea). Signs of obesity include, but are not limited to, acanthosis nigricans (i.e., a skin disorder characterized by hyperkeratosis and hyperpigmentation in skin folds and underarms), stretch marks (due to swelling and rupture of elastic fibers in the skin), varicose veins in cases of obesity due to endocrinological changes (Cushing's syndrome), swelling and varicose veins in the lower extremities, a body mass index (BMI) of 30 kg / m2 or greater, a waist circumference of more than 94 cm for men and more than 88 cm for women, and high blood pressure of more than 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.

[0240] In a further aspect, a method for treating or preventing type 2 diabetes is provided. Symptoms of type 2 diabetes include, but are not limited to, increased thirst, frequent urination, increased hunger, unintentional weight loss, fatigue, blurred vision, slow wound healing, frequent infections, numbness or tingling in the hands and feet, and darkening of the skin, such as underarms and neck. Factors that increase the risk of type 2 diabetes include, but are not limited to, overweight or obesity, fat distribution primarily in the abdomen (rather than the waist and thighs), physical inactivity, family history, race and ethnicity, blood lipid levels, age, prediabetes, pregnancy-related risk factors, and polycystic ovary syndrome. Potential and frequent complications of diabetes include cardiovascular disease, nerve damage (neuropathy) in the extremities, other nerve disorders, kidney disease, eye disorders, skin disorders, delayed healing, hearing loss, sleep apnea, and dementia. Accordingly, patients treated according to the methods disclosed herein experience improvement in at least one or more symptoms of type 2 diabetes.

[0241] In one embodiment, the method improves glycemic control, hi one embodiment, improved glycemic control is achieved by improving insulin sensitivity.

[0242] In one embodiment, treatment improves the Diabetes Treatment Satisfaction Questionnaire (DTSQ) score, hi another embodiment, treatment improves the Impact of Weight on Quality of Life (IWQOL) score.

[0243] In one embodiment, central obesity is reduced.

[0244] In one embodiment, treatment results in a reduction in total body fat mass (FM) of at least 5 kg compared to baseline. For example, in one embodiment, treatment results in a reduction in total body fat mass (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, 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, 10.7, 10.8, 10.9, 11 kg or more. In one embodiment, treatment results in a reduction of total body FM of at least 5 kg compared to baseline by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0245] In one embodiment, treatment results in at least a 5% reduction in total FM compared to baseline. For example, in one embodiment, treatment results in at least a 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 reduction in total FM compared to baseline. In one embodiment, treatment results in at least a 5% reduction in total FM compared to baseline by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0246] In one embodiment, treatment results in at least a 5% reduction in body weight compared to baseline. For example, in one embodiment, treatment results in at least a 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 reduction in body weight. In one embodiment, treatment results in at least a 5% reduction in body weight compared to baseline by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0247] In one embodiment, treatment results in at least a 2% increase in lean body mass (LM) compared to baseline. For example, in one embodiment, treatment results in at least a 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. In one embodiment, treatment results in at least a 2% increase in body LM compared to baseline by week 6, week 8, week 10, week 12, week 16, week 20, week 24, week 36, week 48, or week 60. In another embodiment, treatment results in at least a 25% increase in body LM compared to baseline by week 8, week 10, week 12, week 16, week 20, week 24, week 36, week 48, or week 60.

[0248] 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 measurement, DXA, hydrostatic weighing, air displacement plethysmography (Bod Pod), bioelectrical impedance analysis (BIA), bioimpedance spectroscopy (BIS), or electrical impedance electromyography (EIM), 3-D body scanner, multicompartment model, and / or magnetic resonance spectroscopy (MRI).

[0249] In one embodiment, treatment reduces waist circumference (WC) and / or waist-to-hip ratio compared to baseline. For example, in one embodiment, treatment reduces WC by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% compared to baseline. In another embodiment, treatment reduces 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. In another embodiment, treatment reduces 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 by 12 weeks, 15 weeks, 18 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0250] In one embodiment, treatment reduces WC and reduces total body weight compared to baseline. For example, in one embodiment, treatment reduces WC by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% compared to baseline, and reduces 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, treatment reduces 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 reduces 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, treatment results in a 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm or more decrease in WC and at least a 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% decrease in total body weight compared to baseline by 12, 15, 18, 24, 36, 48, or 60 weeks.

[0251] In one embodiment, treatment reduces subcutaneous adipose tissue and abdominal visceral adipose tissue compared to baseline. For example, in one embodiment, treatment reduces subcutaneous adipose tissue and abdominal visceral adipose tissue by 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 compared to baseline. In one embodiment, treatment reduces subcutaneous adipose tissue and abdominal visceral adipose tissue by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks compared to baseline.

[0252] In one embodiment, the treatment improves the diabetic condition. For example, in one embodiment, the treatment improves the patient's HbA1c. In another embodiment, the treatment results in an improvement as assessed by Homeostasis Model Assessment (HOMA). In another embodiment, the treatment results in an improvement as assessed by Quantitative Insulin Sensitivity Test Index (QUICKI). In another embodiment, the treatment results in an improvement as assessed by Matsuda Index. In one embodiment, the treatment improves the diabetic condition by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks compared to baseline.

[0253] In one embodiment, 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. In one embodiment, 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 by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks compared to baseline.

[0254] In one embodiment, treatment reduces insulin levels compared to baseline, hi one embodiment, treatment reduces insulin levels by 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% compared to baseline. In one embodiment, treatment reduces insulin levels by 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% by 8 weeks, 10 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks or 60 weeks compared to baseline.

[0255] In one embodiment, treatment reduces leptin levels compared to baseline, hi one embodiment, treatment reduces leptin levels by 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% compared to baseline. In one embodiment, 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 levels compared to baseline by 8 weeks, 10 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks.

[0256] In one embodiment, treatment improves grip strength, e.g., as assessed by dynamometry, hi one embodiment, treatment improves grip strength by 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks compared to baseline.

[0257] V. Kit Furthermore, the 10th amino acid of fibronectin type III ( ) binds to myostatin, such as BHV-2000. 10 Kits are provided that include pharmaceutical preparations containing a polypeptide comprising a Fn3 domain in a therapeutically effective amount suitable for use in the methods described herein.The kits can also optionally include instructions, including, for example, an administration schedule, to enable a medical professional (e.g., a doctor, a nurse, or a patient) to administer the preparation to a patient in need thereof.The kits can also include a syringe.

[0258] In one embodiment, a kit for improving glycemic control in a human patient is provided, the kit comprising: (a) The 10th amino acid of fibronectin type III ( 10 a dose of a polypeptide comprising a Fn3) domain, 10 a dose of a polypeptide wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively; (b) instructions for using the polypeptide in a method according to any one of the preceding claims.

[0259] In another embodiment, a kit for treating, preventing, or alleviating obesity and associated complications in a human patient is provided, comprising: (a) The 10th amino acid of fibronectin type III ( 10 a dose of a polypeptide comprising a Fn3) domain, 10a dose of a polypeptide wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively; (b) instructions for using the polypeptide in a method according to any one of the preceding claims.

[0260] In another embodiment, a kit for treating or preventing type II diabetes in a human patient is provided, comprising: (a) The 10th amino acid of fibronectin type III ( 10 a dose of a polypeptide comprising a Fn3) domain, 10 a dose of a polypeptide wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively; (b) instructions for using the polypeptide in a method according to any one of the preceding claims.

[0261] In one embodiment, 10 The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In one embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11. In one embodiment, the polypeptide is used for administration as a unit dosage form containing about 1.0 mL or less of a formulation, the formulation comprising (i) about 10-75 mg / mL of 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 6.8-7.3.

[0262] We also investigated the myostatin-binding 10th amino acid sequence of fibronectin type III (FNIII) for improving glycemic control in human patients. 10 Use of a polypeptide comprising a Fn3 domain is provided, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0263] Furthermore, the tenth (10th) amino acid sequence of fibronectin type III that binds to myostatin was identified for the treatment, prevention, or mitigation of obesity and related complications in human patients. 10 Use of a polypeptide comprising a Fn3 domain is provided, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0264] Furthermore, the tenth (10th) amino acid sequence of fibronectin type III that binds to myostatin for the treatment or prevention of type II diabetes in human patients is disclosed. 10 Use of a polypeptide comprising a Fn3 domain is provided, wherein the polypeptide is administered once a week or once every two weeks at a dose of 5 to 200 mg. In one embodiment, the polypeptide is administered by subcutaneous injection (e.g., repeated subcutaneous injections).

[0265] In one embodiment, the polypeptide is a fibronectin type III polypeptide having the tenth amino acid sequence ( 10 Fn3) domain, 10 The Fn3 domain comprises BC, DE, and FG loops, wherein at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively. In some embodiments, 10The Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11. In another embodiment, the polypeptide is used for administration as a unit dosage form containing about 1.0 mL or less of a formulation, the formulation comprising (i) about 10-75 mg / mL of 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 6.8-7.3.

[0266] The following examples are illustrative only and should not be construed as limiting the scope of the disclosure, as many variations and equivalents will be apparent to those of ordinary skill in the art upon reading this disclosure. The contents of all references, Genbank entries, patents, and published patent applications cited throughout this application are hereby incorporated by reference. [Example]

[0267] Example 1: Clinical Development Program A clinical development program, including a series of preclinical and clinical trials, will be conducted to evaluate the potential of anti-myostatin Adnectins to treat overweight, obesity, and related health conditions. The development plan will adhere to internationally recognized standards for the proper conduct of animal and human studies in accordance with Good Clinical Practice and Good Laboratory Practice. Studies will be conducted to meet current regulatory guidelines for the development of investigational drugs intended for use in weight management.

[0268] Clinical trials are designed to evaluate the effects of anti-myostatin Adnectins on individuals with overweight, obesity, and related health conditions. The trials are generally double-blind and placebo-controlled. The trials include a 6- to 24-month primary treatment period during which participants receive periodic administration of the blinded study drug or matching placebo. Candidates are screened for eligibility to participate in the treatment phase through a medical history, physical examination, laboratory tests, and clinical assessments. During the trial, participants periodically return to the study site for pre-specified and / or emergent assessments of clinical, pharmacological, quality, and other measures. Participants typically receive counseling regarding physical activity and healthy eating as part of the clinical trial. The trials are developed, organized, and conducted by trained professionals with valid qualifications in conducting human trials and caring for individuals with overweight, obesity, and related health conditions.

[0269] The primary goals of the program include evaluation of the safety, tolerability, and pharmacokinetics of the investigational drug across a range of doses (usual range is 5-200 mg every 1-2 weeks), dosing intervals, treatment duration, and possibly various modes of administration (e.g., subcutaneous or intramuscular injection). Key efficacy outcomes utilized within the program include endpoints consistent with current standard measures of beneficial effect, as defined by sources such as expert opinion, guidance from major regulatory authorities and other competent authorities, and current case law.

[0270] Primary efficacy endpoints for the treatment of weight-related disorders, such as obesity and overweight, include changes in total body weight, changes in body composition (e.g., fat mass, lean mass, etc.), and changes in glycemic control. These outcomes are measured by validated laboratory and imaging techniques (e.g., magnetic resonance imaging [MRI] and dual-energy x-ray absorptiometry [DEXA]). Additionally, beneficial effects are measured by employing validated patient-reported outcome measures (e.g., questionnaires) to inform changes in quality of life in target populations.

[0271] The intended target population is individuals living with overweight, obesity, and / or related health conditions. The program includes adults 18 years of age and older, but may also be expanded, data and regulations permitting, to include individuals under the age of 18 living with overweight, obesity, and related health conditions.

[0272] Example 2: Taldef-globep alfa reduces fat and increases muscle in an obese mouse model Obesity is a disease with excess or abnormal adipose tissue as its primary etiological factor. Currently approved anti-obesity medications (AOMs) achieve a reduction in total body weight (TBW) based on a combined reduction in fat mass and lean muscle mass. However, the loss of lean muscle mass due to AOM may have long-term adverse health effects.

[0273] The purpose of this study was to evaluate the ability of the anti-myostatin Adnectin taldef globep alfa (BHV-2000, also known as RO7239361 and BMS-986089), an anti-myostatin adnectin, to improve body composition in a high-fat diet (HFD)-induced obese mouse model. Briefly, 8-week-old C57BL / 6J male mice were divided into groups based on diet: standard (STD) or HFD, and the timing of intervention (week 0 (HFD), week 4 (HFD4), or week 8 (HFD8) on HFD). Figure 1 shows an outline of the study. Across all treatment groups, taldef globep (100 mg / kg) or vehicle was administered subcutaneously (SC) twice weekly for 8 weeks. Baseline and post-treatment assessments included (A) body composition by EchoMRI™ and (B) measurement of metabolic markers, including a glucose tolerance test (GTT) and insulin and leptin levels.

[0274] As supported by Figure 2, consistent differences were observed in changes in fat mass and lean mass over time between the vehicle and taldef-globep groups. Specifically, after 8 weeks of treatment, all taldef-globep-treated groups demonstrated decreased fat mass and increased lean mass compared to vehicle. Changes in TBW were generally similar over time between the vehicle and taldef-globep groups (Figure 2). Eight-week-old Black mice are in late adolescence / early adulthood and have not yet fully grown. Therefore, some increases in TBW, fat mass, and lean mass over time can be attributed to the normal maturation process, as indicated by the changes in TBW and body composition seen in Group 1 (vehicle on STD) (Figure 2). At 8 weeks of treatment, HFD8 mice in Group 9 (taldef-globep) showed a significant decrease in fat mass and a significant increase in lean mass compared to mice in Group 8 (vehicle on HFD8). Specifically, after 8 weeks of treatment, mice in Group 9 experienced an 11% decrease in baseline fat mass and a 25% increase in lean mass, significantly greater changes in body composition than mice in Group 8 (+31% and +8%, respectively, P ≤ 0.001) (see Figures 2, 3, and 4). Taldef-globep-treated mice consistently experienced greater decreases in insulin and leptin levels than vehicle-treated mice (Figure 5). After 7 weeks of treatment, glucose tolerance did not improve in the intervention group compared with the vehicle group (data not shown). Food intake generally remained consistent over time between taldef-globep-treated and vehicle-treated mice (data not shown).

[0275] In conclusion, in an era of unprecedented reductions in total body weight (TBW) due to AOM, careful consideration of changes in body composition, including the impact of lean muscle mass loss, is increasingly important. In this diet-induced obese mouse model, taldef globebep monotherapy significantly reduced adipose tissue and increased lean muscle mass compared to the vehicle group. These data support further development of taldef globebep as a potential drug candidate with the potential to provide differentiated benefits to individuals living with overweight and obesity.

[0276] Example 3: Correlation between total weight loss and change in waist circumference Obesity is a disease characterized by excess or abnormal adipose tissue, a key contributor to its pathogenesis. Current guidelines from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency recommend change in baseline TBW as the primary efficacy assessment in registrational clinical trials for overweight and obesity. Visceral adipose tissue mass and anthropometric measures of central obesity (e.g., waist-to-height ratio) have proven to be better predictors of cardiometabolic risk than TBW and body mass index (BMI). Furthermore, changes in anthropometric measures of central obesity correlate with changes in visceral adipose tissue. The purpose of this study was to investigate the relationship between changes in TBW and WC in adults undergoing AOM in pivotal trials of drugs recently approved by or under review by the FDA. This analysis included six phase 3 clinical trials conducted in adults living with overweight and obesity, including: (1) the semaglutide STEP1 trial (NCT03548935) (see Wilding JPH, et al. N.Eng.l J.Med.2021;384(11):989-1002), (2) the naltrexone / bupropion COR-I trial (NCT00532779) (Greenway FL, et al. Lancet.2010;376(9741):595-605), and (3) the lorcaserin BLOOM trial (NCT00395135) (Smith SR, et al. N. Engl. J. Med. 2010;363(3):245-256), (4) the phentermine / topiramate CONQUER trial (NCT00553787) (Gadde KM, et al. Lancet. 2011;377(9774):1341-1352), (5) the liraglutide SCALE trial (NCT01272219) (Pi-Sunyer X, et al. N. Engl. J. Med. 2015;373(1):11-22), and (6) the tirzepatide SURMOUNT-1 trial (NCT04184622) (Jastreboff AN, et al. N. Engl. J. Med. 2022;387(3):205-216). Placebo results were not included in the analysis. Correlations were estimated using Pearson correlation coefficients with weights proportional to the number of subjects in each dose group.

[0277] The present analysis included a total of 9938 participants from six clinical trials with study periods ranging from 52 to 72 weeks (Table 2). [Table 2] * Participants with available data for TBW and WC endpoints were included. BID: twice daily, QD: daily, QW: weekly, SC: subcutaneous injection, SR: sustained release.

[0278] Based on the eligibility criteria, the minimum BMI across all studies was 27 kg / m 2 Participants were predominantly female, accounting for 67.1% to 85% of participants. The mean age ranged from 43.8 to 51.1 years, with a minimum age of 18 years across all studies based on eligibility criteria. The mean TBW reduction ranged from 5.0% to 20.9%, and the corresponding mean reduction in WC ranged from 5.0 to 18.5 cm. There was a strong linear relationship between change in TBW and change in WC (Figure 6). Specifically, a 5% reduction in TBW was associated with approximately a 5 cm reduction in WC (r = 0.997).

[0279] In conclusion, in an era of unprecedented declines in TBW due to AOM, it is increasingly important to carefully consider changes in body composition, including changes in lean muscle mass. This analysis suggests that changes in WC strongly correlate with AOM-induced changes in TBW in adults participating in a phase 3 obesity trial. Anthropometric measures of central obesity, such as WC, which are excellent estimates of visceral adipose tissue mass, may serve as important indicators of treatment response to AOM.

[0280] Sequence overview SEQ ID NO: 1 Human prepromyostatin MQKLQLCVYIYLFMLIVAGPVDLNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYT GIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS

[0281] SEQ ID NO: 2 Human promyostatin NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 2)

[0282] SEQ ID NO: 3 Mature myostatin DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS

[0283] SEQ ID NO:4 Wild-type human fibronectin type III domain ( 10 Fn3) [ka]

[0284] SEQ ID NO:5 Anti-myostatin Adnectin BC Loop SWSLPHQGKAN

[0285] SEQ ID NO:6 Anti-myostatin Adnectin DE Loop PGRGVT

[0286] SEQ ID NO:7 Anti-myostatin Adnectin FG Loop TVTDTGYLKYKP

[0287] SEQ ID NO:8 Anti-myostatin Adnectin Core EVVAATPTSLLISWSLPHQGKANYYRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTDTGYLKYKPISINYRT

[0288] SEQ ID NO:9 Anti-myostatin Adnectin core with His6 tagged N-terminal (AdNT1) (underlined) and C-terminal (AdCT1) (italicized) terminal sequences (SEQ ID NO: 38) [ka]

[0289] SEQ ID NO: 10 An N-terminal extension sequence (GVSDVPRDL (SEQ ID NO: 12)) followed by an anti-myostatin Adnectin core sequence, followed by a C-terminal tail (EI) [ka]

[0290] SEQ ID NO: 11 Exemplary reader: "AdNT1" MGVSDVPRDL

[0291] SEQ ID NO: 12 Exemplary reader: "AdNT2" GVSDVPRDL

[0292] SEQ ID NO: 13 Exemplary reader: "AdNT3" VSDVPRDL

[0293] SEQ ID NO: 14 Exemplary reader: "AdNT4" SDVPRDL

[0294] SEQ ID NO: 15 Exemplary reader: "AdNT5" DVPRDL

[0295] SEQ ID NO: 16 Exemplary reader: "AdNT6" VPRDL

[0296] SEQ ID NO: 17 Exemplary reader: "AdNT7" PRDL

[0297] Exemplary reader: "AdNT8" RDL

[0298] Exemplary reader: "AdNT9" DL

[0299] SEQ ID NO: 20 Exemplary tail: "AdCT1" EIDKPSQ

[0300] Exemplary tail: "AdCT2" EI

[0301] SEQ ID NO: 22 Exemplary tail: "AdCT3" EIEPKSS

[0302] SEQ ID NO: 23 Exemplary tail: "AdCT4" EIDKPC

[0303] SEQ ID NO: 24 Exemplary tail: "AdCT5" EIDKP

[0304] SEQ ID NO: 25 Exemplary tail: "AdCT6" EIDK

[0305] SEQ ID NO: 26 Exemplary tail: "AdCT7" EIDKPS

[0306] SEQ ID NO: 27 Exemplary tail: "AdCT8" EIEKPSQ

[0307] SEQ ID NO: 28 Exemplary tail: "AdCT9" EIDKPSQLE

[0308] SEQ ID NO: 29 Exemplary tail: "AdCT10" EIEDEDEDEDED

[0309] SEQ ID NO: 30 Exemplary tail: "AdCT11" EGSGS

[0310] SEQ ID NO: 31 Exemplary tail: "AdCT12" EIDKPCQ

[0311] SEQ ID NO: 32 Exemplary tail: "AdCT13" GSGC

[0312] SEQ ID NO: 33 Exemplary tail: "AdCT14" EGSGC

[0313] SEQ ID NO: 34 Exemplary tail: "AdCT15" EIDKPCQLE

[0314] SEQ ID NO: 35 Exemplary tail: "AdCT16" EIDKPSQHHHHHH

[0315] SEQ ID NO: 36 Exemplary tail: "AdCT17" GSGCHHHHHH

[0316] SEQ ID NO: 37 Exemplary tail: "AdCT18" EGSGCHHHHHH

[0317] SEQ ID NO: 38 Tag: "T1" HHHHHH

[0318] SEQ ID NO: 39 Human IgG1 immunoglobulin Fc domain [ka]

[0319] SEQ ID NO: 40 Linker GAGGGGSG

[0320] SEQ ID NO: 41 Linker EPKSSD

[0321] SEQ ID NO: 42 Linker ESPKAQASSVPTAQPQAEGLA

[0322] SEQ ID NO: 43 Linker ELQLEESAAEAQDGELD

[0323] SEQ ID NO: 44 Linker GQPDEPGGS

[0324] SEQ ID NO: 45 Linker GGSGSGSGSGSGS

[0325] SEQ ID NO: 46 Linker ELQLEESAAEAQEGELE

[0326] SEQ ID NO: 47 Linker GSGSG

[0327] SEQ ID NO: 48 Linker GSGC

[0328] SEQ ID NO: 49 Linker AGGGGSG

[0329] SEQ ID NO:50 Linker GSGS

[0330] SEQ ID NO:51 Linker QPDEPGGS

[0331] SEQ ID NO:52 Linker GSGSGS

[0332] SEQ ID NO:53 Linker TVAAPS

[0333] SEQ ID NO:54 Linker KAGGGGSG

[0334] SEQ ID NO: 55 Linker KGSGSGSGSGSGS

[0335] SEQ ID NO:56 Linker KQPDEPGGS

[0336] SEQ ID NO:57 Linker KELQLEESAAEAQDGELD

[0337] SEQ ID NO:58 Linker KTVAAPS

[0338] SEQ ID NO:59 Linker KAGGGGSGG

[0339] SEQ ID NO: 60 Linker KGSGSGSGSGSGSG

[0340] SEQ ID NO: 61 Linker KQPDEPGGSG

[0341] SEQ ID NO: 62 Linker KELQLEESAAEAQDGELDG

[0342] SEQ ID NO: 63 Linker KTVAAPSG

[0343] SEQ ID NO: 64 Linker AGGGGSGG

[0344] SEQ ID NO: 65 Linker AGGGGSG

[0345] SEQ ID NO: 66 Linker GSGSGSGSGSGSG

[0346] SEQ ID NO: 67 Linker QPDEPGGSG

[0347] SEQ ID NO: 68 Linker TVAAPSG

[0348] SEQ ID NO: 69 hinge DKTHTCPPCPAPELLG

[0349] SEQ ID NO: 70 Hinge (core hinge region is underlined) GSTHTCPPCPAPELLG

[0350] SEQ ID NO: 71 Hinge (core hinge region is underlined) [ka]

[0351] SEQ ID NO:72 Hinge (core hinge region is underlined) [ka]

[0352] SEQ ID NO: 73 Hinge (core hinge region is underlined) [ka]

[0353] SEQ ID NO:74 Hinge (core hinge region is underlined) [ka]

[0354] SEQ ID NO: 75 Hinge (core hinge region is underlined) [ka]

[0355] SEQ ID NO:76 [ka]

[0356] SEQ ID NO:77 leader METDTLLLWVLLLWVPGSTG

[0357] SEQ ID NO:78 Anti-myostatin Adnectin Fc fusion DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPELQLEESAAEAQEGELEGVSDVPRDLEVVAATPTSLLISWSLPHQGKANYYRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTDTGYLKYKPISINYRTEI

[0358] SEQ ID NO:79 Anti-myostatin Adnectin Fc fusion GVSDVPRDLEVVAATPTSLLISWSLPHQGKANYYRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTDTGYLKYKPISINYRTEIEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0359] SEQ ID NO: 80 VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0360] SEQ ID NO: 81 Exemplary N-Terminal Leader Sequences METDTLLLWVLLLWVPGSTG

[0361] SEQ ID NO:82 TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCTTCCTCAGACAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGAGGCTCTGCACCAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGAGCTGCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGAAGGAGAACTGGAAGGCGTGAGCGACGTGCCAGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGCTTGCTGATCAGCTGG TCTCTGCCGCACCAAGGTAAAGCCAATTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTCGTGGTGTTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTTATGCTGTCACTGTTACTGATACAGGGTACCTCAAGTACAAACCAATTTCCATTAATTACCGGACCGAAATT

[0362] sequence number83 GGCGTGAGCGACGTGCCCCGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGCTTGCTGATCAGCTGGTCTCTGCCGCACCAAGGTAAAGCCAATTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTCGTGGTGTTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGTTACTGATACAGGGTACCTCAAGTACAAACCAATTTCCATTAATTACCGGACCGAAATTGAGCCTAAGAGCTCCGACAAAACCCACACATGCCCACCTTGTCCAGCCCCCGAACTGCTGGGCGGCCCTTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAA

Claims

1. A method for improving glycemic control in a human patient, comprising administering to a subject a fibronectin type III fragment of the tenth amino acid sequence ( １０ administering to said patient a polypeptide comprising a Fn3) domain.

2. A method for treating, preventing, or alleviating overweight or obesity and associated complications in a human patient, comprising administering to a subject a therapeutically effective amount of fibronectin type III 10 ( １０ administering to said patient a polypeptide comprising a Fn3) domain.

3. A method for treating or preventing type II diabetes in a human patient, comprising administering to a subject a therapeutically effective amount of fibronectin type III 10 ( １０ administering to said patient a polypeptide comprising a Fn3) domain.

4. 10. The method of any one of the preceding claims, wherein the patient has a BMI of 30 kg / m or greater.

5. The patient's BMI is 25 kg / m ２ More than 30kg / m ２ The method according to any one of claims 1 to 3, wherein the

6. The method of any one of claims 1 to 3, wherein the patient has a BMI of less than 25.

7. The polypeptide is (i) at least 10 mg / mL of said polypeptide; (ii) disaccharides at a concentration of at least 5%; (iii) a histidine buffer at a concentration of about 20 to about 60 mM; and (iv) a pharmaceutically acceptable aqueous carrier and administering the compound as a formulation comprising 10. The method of any one of the preceding claims, wherein the pH range of the formulation is from about 6.5 to about 7.

8.

8. 8. The method of claim 7, wherein the polypeptide concentration in the formulation is about 10 mg / mL to 200 mg / mL, about 10 mg / mL to 150 mg / mL, or about 10 mg / mL to 85 mg / mL.

9. 9. The method of claim 7 or 8, wherein the disaccharides are present in a weight (w / w) ratio of protein to sugar of at least 5:

1.

10. The method of any one of claims 7 to 9, wherein the formulation comprises from about 5% to about 30% of the disaccharide.

11. 11. The method of any one of claims 7 to 10, wherein the concentration of the disaccharide is from about 150 mM to about 800 mM, or from about 300 to about 700 mM.

12. 12. The method of any one of claims 7 to 11, wherein the disaccharide is trehalose and the formulation comprises about 5 to about 30% trehalose, about 15% to about 25% trehalose, or about 20% to about 25% trehalose.

13. 13. The method of any one of claims 7 to 12, wherein the disaccharide is trehalose dihydrate and the concentration of trehalose dihydrate in the formulation is about 150 mM to about 800 mM, about 300 to about 700 mM, 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 mM, about 625 mM, about 650 mM, about 675 mM, or about 700 mM.

14. 14. The method of any one of claims 7 to 13, wherein the histidine is present at a concentration of at least 20 mM.

15. 15. The method of any one of claims 7 to 14, wherein the viscosity of the formulation is about 5 to 20 cps, about 5 to 15 cps, or about 7 to 12 cps.

16. 16. The method of any one of claims 7 to 15, wherein the pH is about 6.6 to 7.6, about 6.8 to 7.4, or about 7.0 to 7.

3.

17. The method of any one of claims 7 to 16, wherein the formulation comprises a surfactant at a concentration of about 0.01% to 0.5%.

18. 18. The method of any one of claims 7 to 17, wherein the formulation comprises a chelating agent, the chelating agent having a concentration of about 0.01 mM to about 0.5 mM or about 0.05 mM to 0.2 mM, and the chelating agent is selected from the group consisting of DPTA, EDTA, and EGTA.

19. The formulation comprises: (a) about 10 to 140 mg / mL of the polypeptide; about 5-25% trehalose dihydrate, about 20-30 mM histidine, and a pharmaceutically acceptable aqueous carrier; The pH of the formulation is about 6.8 to 7.

3. (b) about 10 to 140 mg / mL of the polypeptide; about 5-25% trehalose dihydrate, about 20-30 mM histidine, about 0.02 to 0.06 mM DTPA, about 0.01-0.05% polysorbate 80, and a pharmaceutically acceptable aqueous carrier; The pH of the formulation is about 6.8 to 7.

3. (c) about 10 to 140 mg / mL of the polypeptide; about 600 mM trehalose dihydrate, 25-30 mM histidine, and a pharmaceutically acceptable aqueous carrier; The pH of the formulation is about 7.0 to 7.

3. (d) about 10 to 140 mg / mL of the polypeptide; about 600 mM trehalose dihydrate, 25-30 mM histidine, about 0.02 to 0.06 mM DTPA, about 0.01-0.05% polysorbate 80, and a pharmaceutically acceptable aqueous carrier; The pH of the formulation is about 7.0 to 7.

3. (e) about 10 to 75 mg / mL of the polypeptide; about 5-25% trehalose dihydrate, about 20-30 mM histidine, and a pharmaceutically acceptable aqueous carrier; The pH of the formulation is about 6.8 to 7.

3. (f) about 10 to 75 mg / mL of the polypeptide; about 5-25% trehalose dihydrate, about 20-30 mM histidine, about 0.02 to 0.06 mM DTPA, about 0.01-0.05% polysorbate 80, and a pharmaceutically acceptable aqueous carrier; the pH of the formulation is about 6.8 to 7.3; or (g) about 10 to 75 mg / mL of the polypeptide; about 600 mM trehalose dihydrate, about 30 mM histidine, about 0.05 mM DTPA, approximately 0.02% polysorbate 80, a pharmaceutically acceptable aqueous carrier; The pH of the formulation is about 7.

1. The method according to any one of claims 7 to 18.

20. The polypeptide is administered in a unit dosage form containing about 1.0 mL or less of the formulation, the formulation comprising: (i) about 10 to 75 mg / mL of the polypeptide; (ii) about 5-25% trehalose dihydrate; (iii) about 20-30 mM histidine; (iv) about 0.02 to 0.06 mM DTPA; (v) about 0.01-0.05% polysorbate 80, and (vi) comprising a pharmaceutically acceptable aqueous carrier; The pH of the formulation is about 6.8 to 7.

3. The method according to any one of claims 7 to 10.

21. The aforementioned １０ The method of any one of the preceding claims, wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively.

22. The aforementioned １０ 10. The method of any one of the preceding claims, wherein the Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO:

8.

23. 10. The method of any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

11.

24. The method of any one of claims 1 to 23, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

78.

25. 10. The method of any one of the preceding claims, wherein the polypeptide is administered as repeated subcutaneous injections.

26. 10. The method of any one of the preceding claims, wherein the polypeptide is administered at weekly dosing intervals.

27. 26. The method of any one of claims 1 to 25, wherein the polypeptide is administered at a dosing interval of once every two weeks.

28. 26. The method of any one of claims 1 to 25, wherein the polypeptide is administered at monthly dosing intervals.

29. 10. The method of any one of the preceding claims, wherein the polypeptide is administered in a dose of about 5 to 200 mg.

30. 10. The method of any one of the preceding claims, wherein central obesity is reduced.

31. 31. The method of any one of claims 2 and 4 to 30, wherein the overweight or obesity related complication is selected from the group consisting of type 2 diabetes, impaired glucose tolerance, prediabetes, insulin resistance, hypertension, dyslipidemia, increased waist circumference, cardiovascular disease, non-alcoholic fatty liver disease, obstructive sleep apnea, physical disability, osteoarthritis, osteoporosis, kidney disease, sex hormone(s) disorder, endocrine reproductive disorders such as polycystic ovary syndrome or male hypogonadism, stroke, and gallstones.

32. 32. The method according to claim 1, wherein the improved glycemic control is achieved by improving insulin sensitivity. The method described in paragraph .

33. The method of any one of claims 1-2 and 4-32, wherein the patient has type II diabetes.

34. 34. The method of any one of claims 3 to 33, wherein said treatment improves the Diabetes Treatment Satisfaction Questionnaire (DTSQ) score or the Impact of Weight on Quality of Life (IWQOL) score.

35. 10. The method of any one of the preceding claims, wherein said treatment results in a reduction of total fat mass (FM) of at least 5 kg by 48 weeks compared to baseline.

36. 10. The method of any one of the preceding claims, wherein said treatment results in at least a 5% reduction in total fat mass (FM) by 48 weeks compared to baseline.

37. 10. The method of any one of the preceding claims, wherein said treatment results in at least a 5% reduction in body weight compared to baseline by 48 weeks.

38. 10. The method of any one of the preceding claims, wherein said treatment results in at least a 2% increase in lean body mass (LM) by 48 weeks compared to baseline.

39. 37. The method of claim 35 or 36, wherein FM is assessed by dual-energy x-ray absorptiometry (DXA).

40. 39. The method of claim 38, wherein LM is assessed by dual-energy x-ray absorptiometry (DXA).

41. 10. The method of any one of the preceding claims, wherein said treatment reduces waist circumference (WC) and / or waist-to-hip ratio compared to baseline by 48 weeks.

42. 10. The method of any one of the preceding claims, wherein said treatment reduces subcutaneous fat and abdominal visceral adipose tissue by 48 weeks compared to baseline.

43. 10. The method of any one of the preceding claims, wherein said treatment improves the diabetic condition by 48 weeks compared to baseline.

44. 44. The method of claim 43, wherein the improvement in the diabetic condition is assessed by HbA1c, homeostasis model assessment (HOMA), quantitative insulin sensitivity testing index (QUICKI), and / or Matsuda index.

45. 10. The method of any one of the preceding claims, wherein said treatment results in a shift towards 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 by week 48 compared to baseline.

46. 10. The method of any one of the preceding claims, wherein said treatment improves grip strength by 48 weeks compared to baseline as assessed by dynamometry.

47. 1. A kit for improving glycemic control in a human patient, said kit comprising: (a) The tenth amino acid of fibronectin type III ( １０ a dose of a polypeptide comprising a Fn3 domain, １０ a dose of a polypeptide wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively; (b) instructions for using the polypeptide in a method according to any one of the preceding claims; and Includes a kit.

48. 1. A kit for treating, preventing, or alleviating obesity and associated complications in a human patient, said kit comprising: (a) The tenth amino acid of fibronectin type III ( １０ a dose of a polypeptide comprising a Fn3 domain, １０ a dose of a polypeptide wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively; (b) instructions for using the polypeptide in a method according to any one of the preceding claims; and Includes a kit.

49. 1. A kit for treating or preventing type II diabetes in a human patient, said kit comprising: (a) The tenth amino acid of fibronectin type III ( １０ a dose of a polypeptide comprising a Fn3 domain, １０ a dose of a polypeptide wherein the Fn3 domain comprises BC, DE, and FG loops, and at least one of the BC, DE, and FG loops has 0, 1, 2, or 3 amino acid substitutions relative to the individual BC, DE, and FG loops set forth in SEQ ID NOs: 5, 6, and 7, respectively; (b) instructions for using the polypeptide in a method according to any one of the preceding claims; and Includes a kit.

50. The aforementioned １０ The kit of any one of claims 47 to 49, wherein the Fn3 domain comprises the amino acid sequence set forth in SEQ ID NO:

8.

51. The kit of any one of claims 47 to 49, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

11.

52. The kit of any one of claims 47 to 49, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

78.

53. The polypeptide is adapted to be administered as a unit dosage form containing about 1.0 mL or less of the formulation, the formulation comprising: (i) about 10 to 75 mg / mL of the polypeptide; (ii) about 5-25% trehalose dihydrate; (iii) about 20-30 mM histidine; (iv) about 0.02 to 0.06 mM DTPA; (v) about 0.01-0.05% polysorbate 80, and (vi) comprising a pharmaceutically acceptable aqueous carrier; 50. The kit of any one of claims 47 to 49, wherein the pH of the formulation is about 6.8 to 7.3.

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