Methods for preventing and treating heart disease

Abstract

The present disclosure relates to novel uses and methods for preventing and / or treating heart disease using a therapeutically effective amount of an ActRII receptor antagonist, e.g., an ActRII receptor binding molecule, e.g., an ActRII receptor antibody, such as a bimagazumab antibody.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201880020467.9. The original application is the Chinese national phase application of the international application PCT / US2018 / 023390 with the application date of March 20, 2018, which claims the priority of U.S. Provisional Application No. 62 / 476,054 filed on March 24, 2017. Technical Field

[0002] The present disclosure is in the field of type II activin receptor (ActRII) antagonists, which are molecules that can antagonize the binding of activin, growth differentiation factor (GDF), bone morphogenetic protein (BMP) and myostatin to human ActII receptors, such as antagonist antibodies to ActRIIA and / or ActRIIB, such as bimagrumab. Specifically, the present disclosure relates to the prevention and / or treatment of heart failure, including heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF), and to treating structural and / or functional cardiac abnormalities associated with the condition by administering a therapeutically effective amount of an ActRII receptor antagonist to a subject, the condition being such as valvular heart disease, coronary artery disease, hypertension, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic or infectious agents, and hereditary or idiopathic dilated cardiomyopathy. Notably, these conditions include clinical syndromes that often coexist but can occur alone and are sometimes referred to as systolic and / or diastolic heart failure, left-sided and / or right-sided heart failure, and congestive heart failure. Background Art

[0003] Activin receptor type IIB (ActRIIB) is a signaling receptor for various members of the transforming growth factor β (TGF-β) superfamily. Members of this family include activin A, nodal, BMP2, BMP6, BMP7, BMP9, GDF5, GDF8 (myostatin), and GDF11, all of which are involved in the negative regulation of muscle (Akpan et al., 2009).

[0004] Myostatin (GDF8) acts via type II activin receptors (primarily via ActRIIB), and its proposed signaling is via the SMAD 2 / 3 pathway, which is involved in the inhibition of protein synthesis, as well as myocyte differentiation and proliferation. Myostatin inhibition or genetic ablation increases muscle mass and strength (Lee et al. 2005, Lee and McPherron 2001, Whittemore et al. 2003).

[0005] Bimagrumab, also known as BYM338, is a monoclonal antibody developed to competitively bind to the type IIB activin receptor (ActRII) with an affinity greater than that of the natural ligands of the type IIB activin receptor, myostatin or activin. Bimagrumab is disclosed in WO2010 / 125003, which is incorporated herein by reference as if fully set forth. Bimagrumab is a fully human antibody (modified IgG1, 234-235-Ala-Ala, λ2) that binds to the ligand binding domains of ActRIIA and ActRIIB, thereby preventing the binding and subsequent signaling of ligands of ActRIIA and ActRIIB, including myostatin and activin, which act as natural inhibitors of skeletal muscle growth. Bimagrumab cross-reacts with human and mouse ActRIIB and is effective in human, macaque, mouse and rat skeletal muscle cells. ActRIIB is widely distributed in skeletal muscle, adipose tissue and various organs including the heart (Rebbapragada et al., 2003).

[0006] Heart failure is a clinical syndrome in which impaired cardiac function results in insufficient systemic perfusion to meet the metabolic demands of the body. Heart failure is divided into two major categories: (1) heart failure with reduced ejection fraction (HFrEF) (also known as "systolic heart failure") and (2) heart failure with preserved ejection fraction (HFpEF) (also known as "diastolic heart failure"). In HFrEF, reduced myocardial contractility is the primary mechanism that impairs cardiac output and leads to insufficient systemic perfusion. In HFpEF, resting myocardial contractility is generally preserved. However, a variety of other defects in cardiac function, including cardiac reserve and diastolic function, impair the functional performance of the heart, resulting in a similar phenotype to clinical heart failure. Various conditions can damage or weaken the heart and lead to heart failure, including, for example, valvular heart disease, coronary artery disease, hypertension, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic or infectious agents, and genetic and / or idiopathic dilated cardiomyopathy.

[0007] Heart failure caused by causes such as diabetes, aging, hypertension, ischemic heart disease, coronary heart disease, valvular heart disease, and hereditary and idiopathic cardiomyopathy is the leading cause of morbidity and mortality worldwide. For this increasingly common disease process, available drug therapies are limited. The standard of care for heart failure includes a variety of drug therapies targeting various mechanisms involved in the complex pathophysiology of the disease. Unfortunately, even with guideline-directed therapy, the prognosis for these patients remains poor, with a 5-year mortality rate approaching 50%. In advanced systolic heart failure, patients may often be unable to tolerate commonly used oral drug therapies due to hemodynamic, renal, and arrhythmogenic side effects, or may not be able to achieve adequate relief from such therapies. For these patients, advanced therapies such as intravenous inotropes, mechanical support devices, and heart transplantation are very limited, expensive, and have significant risks.

[0008] Prior to the present disclosure, targeted inhibition of the type II activin receptor (ActRIIA / B) had not been investigated as a prophylactic or therapeutic approach for heart failure or the aforementioned conditions that can lead to heart failure. As disclosed herein, there is now evidence that systemic administration of an ActRIIA / B receptor antagonist, such as CDD866, which is a murinized form of BYM338 (wherein the human Fc region of the antibody has been replaced by the mouse Fc), has a significant beneficial effect on cardiac function in mice subjected to transverse aortic constriction (TAC). TAC is a commonly used experimental model of cardiac hypertrophy and heart failure caused by pressure overload. First validated by Rockman et al., 1991, the murine TAC model has been widely used since 1991 as a valuable tool for modeling human cardiovascular disease and understanding the basic signaling processes involved in the development of cardiac hypertrophic responses and heart failure (deAlmeida et al., 2010). As disclosed herein, CDD866 not only prevents TAC-mediated cardiac dysfunction, but is also able to restore cardiac function within 1-2 weeks of drug administration after heart failure is established. It also enhances skeletal muscle growth (which tends to atrophy in advanced forms of heart failure) and induces minimal cardiac effects in control mice that have not been subjected to pathological stress / injury by TAC.

[0009] Disclosed herein are ActRII receptor antagonists for treating and / or preventing heart failure, including heart failure caused by or associated with conditions such as valvular heart disease, ischemic heart disease, coronary artery disease, hypertension, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic or infectious agents, and hereditary and / or idiopathic dilated cardiomyopathy. Also disclosed are ActRII receptor antagonists for treating structural and / or functional cardiac abnormalities associated with the above conditions. Also provided are methods of using such ActRII antagonists to treat and / or prevent heart failure, and treating structural and / or functional cardiac abnormalities associated with the above conditions. Summary of the invention

[0010] ActRII receptor antagonists for treating and / or preventing heart failure are disclosed herein. Heart failure may be caused by or associated with a variety of conditions, such as valvular disease (such as aortic stenosis), coronary artery disease, hypertension, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic or infectious agents, and hereditary or idiopathic dilated cardiomyopathy. Heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF) are included herein.

[0011] Also disclosed herein are ActRII receptor antagonists for treating structural and / or functional cardiac abnormalities associated with conditions such as valvular heart disease, coronary artery disease, hypertension, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic or infectious agents, and hereditary or idiopathic dilated cardiomyopathy. In some cases, peripartum cardiomyopathy occurs in late pregnancy or during the 6 months postpartum. Stress cardiomyopathy typically occurs in elderly women in the postmenopausal period. An example of valvular heart disease is aortic stenosis, which can be associated with weakness and / or sarcopenia. Stress cardiomyopathy can occur following psychological, pathological, or physical stress.

[0012] Disclosed herein are methods for treating and / or preventing heart failure. The method includes administering a therapeutically effective amount of an ActRII receptor antagonist, such as bimagrumab, to a subject having heart failure or at risk of developing heart failure. Included herein are heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF). Heart failure can be diagnosed in a patient using well-known methods, including, for example, measuring brain natriuretic peptide, followed by cardiac ultrasound if the brain natriuretic peptide is positive, and imaging such as echocardiography.

[0013] Patients are at risk for developing heart failure when they have conditions such as valvular heart disease, coronary artery disease (including previous myocardial infarction), hypertension, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, and hereditary or idiopathic dilated cardiomyopathy.

[0014] Also disclosed herein are methods for treating structural and / or functional cardiac abnormalities associated with conditions such as valvular heart disease, coronary artery disease, hypertension, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic or infectious agents, and hereditary or idiopathic dilated cardiomyopathy. The method comprises administering an effective amount of an ActRII receptor antagonist to a subject having such structural and / or functional cardiac abnormalities associated with such conditions.

[0015] Examples of ActRII receptor antagonists for use in the methods described herein are ActRII receptor binding molecules that block access of ActRII interacting ligands such as myostatin, GDF11, and activin A to ActRII. ActRII receptor binding molecules can bind to ActRIIA and / or to ActRIIB receptors. Examples of ActRII binding molecules include, but are not limited to, antibodies that bind to ActRIIA and / or ActRIIB receptors, such as anti-ActRII receptor antibodies. Preferably, the anti-ActRII receptor antibody is BYM338, also known as bimagrumab.

[0016] Another example of an ActRII receptor antagonist for use in the methods described herein is a soluble form of the extracellular domain of the ActRIIA receptor or the ActRIIB receptor that can bind to an ActRII interacting ligand, such as myostatin, GDF11, and activin A. This "receptor-body" inhibits the function of a cell-bound ActRII receptor by competing away its ligand.

[0017] Disclosed herein are ActRII receptor antagonists for use in or for use in the methods described herein, wherein the ActRII receptor antagonist is an anti-ActRII antibody that binds to an epitope of ActRIIB consisting of amino acids 19-134 of SEQ ID NO: 181 (SEQ ID NO: 182).

[0018] Disclosed herein are ActRII receptor antagonists for use in or in the methods described herein, wherein the anti-ActRII antibody binds to an epitope of ActRIIB comprising or consisting of:

[0019] (a) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN - SEQ ID NO: 188);

[0020] (b) amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC - SEQ ID NO: 186);

[0021] (c) amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 190);

[0022] (d) amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189);

[0023] (e) amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187);

[0024] (f) amino acids 29-41 of SEQ ID NO: 181 (CIYYNANWELERT-SEQ ID NO: 191);

[0025] (g) amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN - SEQ ID NO: 192); or

[0026] (h) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR).

[0027] Other anti-ActRIIB antibodies for use in the methods described herein include, for example

[0028] a) an anti-ActRIIB antibody that binds to an epitope of ActRIIB, the epitope comprising:

[0029] (a) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN - SEQ ID NO: 188);

[0030] (b) amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC - SEQ ID NO: 186);

[0031] (c) amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 190);

[0032] (d) amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189);

[0033] (e) amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187);

[0034] (f) amino acids 29-41 of SEQ ID NO: 181 (CIYYNANWELERT-SEQ ID NO: 191);

[0035] (g) amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN - SEQ ID NO: 192); or

[0036] (h) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR); and

[0037] b) an ActRIIB antagonist antibody that binds to an epitope of ActRIIB comprising amino acids 78-83 of SEQ ID NO: 181 (WLDDFN - SEQ ID NO: 188);

[0038] (b) amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC - SEQ ID NO: 186);

[0039] (c) amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 190);

[0040] (d) amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189);

[0041] (e) amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187);

[0042] (f) amino acids 29-41 of SEQ ID NO: 181 (CIYYNANWELERT-SEQ ID NO: 191);

[0043] (g) amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN - SEQ ID NO: 192); or

[0044] (h) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR), wherein the K D About 2pM.

[0045] In one embodiment, an ActRII receptor antagonist for use in the methods described herein is an antibody that binds to ActRIIB with an affinity that is about 10 times or greater than that of the antibody that binds to ActRIIA.

[0046] The ActRII receptor antagonist used in or for use in the methods described herein can be an antibody comprising: a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14; a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 28; a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29 to SEQ ID NO: 42; a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 43 to SEQ ID NO: 56; a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 70; and a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71 to SEQ ID NO: 84.

[0047] The ActRII receptor antagonist for use in the methods described herein may be an antibody comprising:

[0048] (a) the heavy chain variable region CDR1 shown in SEQ ID NO:1; the heavy chain variable region CDR2 shown in SEQ ID NO:15; the heavy chain variable region CDR3 shown in SEQ ID NO:29; the light chain variable region CDR1 shown in SEQ ID NO:43; the light chain variable region CDR2 shown in SEQ ID NO:57; and the light chain variable region CDR3 shown in SEQ ID NO:71,

[0049] (b) the heavy chain variable region CDR1 shown in SEQ ID NO:2; the heavy chain variable region CDR2 shown in SEQ ID NO:16; the heavy chain variable region CDR3 shown in SEQ ID NO:30; the light chain variable region CDR1 shown in SEQ ID NO:44; the light chain variable region CDR2 shown in SEQ ID NO:58; and the light chain variable region CDR3 shown in SEQ ID NO:72,

[0050] (c) the heavy chain variable region CDR1 shown in SEQ ID NO:3; the heavy chain variable region CDR2 shown in SEQ ID NO:17; the heavy chain variable region CDR3 shown in SEQ ID NO:31; the light chain variable region CDR1 shown in SEQ ID NO:45; the light chain variable region CDR2 shown in SEQ ID NO:59; and the light chain variable region CDR3 shown in SEQ ID NO:73,

[0051] (d) the heavy chain variable region CDR1 shown in SEQ ID NO:4; the heavy chain variable region CDR2 shown in SEQ ID NO:18; the heavy chain variable region CDR3 shown in SEQ ID NO:32; the light chain variable region CDR1 shown in SEQ ID NO:46; the light chain variable region CDR2 shown in SEQ ID NO:60; and the light chain variable region CDR3 shown in SEQ ID NO:74,

[0052] (e) the heavy chain variable region CDR1 shown in SEQ ID NO:5; the heavy chain variable region CDR2 shown in SEQ ID NO:19; the heavy chain variable region CDR3 shown in SEQ ID NO:33; the light chain variable region CDR1 shown in SEQ ID NO:47; the light chain variable region CDR2 shown in SEQ ID NO:61; and the light chain variable region CDR3 shown in SEQ ID NO:75,

[0053] (f) the heavy chain variable region CDR1 shown in SEQ ID NO:6; the heavy chain variable region CDR2 shown in SEQ ID NO:20; the heavy chain variable region CDR3 shown in SEQ ID NO:34; the light chain variable region CDR1 shown in SEQ ID NO:48; the light chain variable region CDR2 shown in SEQ ID NO:62; and the light chain variable region CDR3 shown in SEQ ID NO:76,

[0054] (g) the heavy chain variable region CDR1 shown in SEQ ID NO:7; the heavy chain variable region CDR2 shown in SEQ ID NO:21; the heavy chain variable region CDR3 shown in SEQ ID NO:35; the light chain variable region CDR1 shown in SEQ ID NO:49; the light chain variable region CDR2 shown in SEQ ID NO:63; and the light chain variable region CDR3 shown in SEQ ID NO:77,

[0055] (h) the heavy chain variable region CDR1 shown in SEQ ID NO:8; the heavy chain variable region CDR2 shown in SEQ ID NO:22; the heavy chain variable region CDR3 shown in SEQ ID NO:36; the light chain variable region CDR1 shown in SEQ ID NO:50; the light chain variable region CDR2 shown in SEQ ID NO:64; and the light chain variable region CDR3 shown in SEQ ID NO:78,

[0056] (i) the heavy chain variable region CDR1 shown in SEQ ID NO:9; the heavy chain variable region CDR2 shown in SEQ ID NO:23; the heavy chain variable region CDR3 shown in SEQ ID NO:37; the light chain variable region CDR1 shown in SEQ ID NO:51; the light chain variable region CDR2 shown in SEQ ID NO:65; and the light chain variable region CDR3 shown in SEQ ID NO:79,

[0057] (j) the heavy chain variable region CDR1 shown in SEQ ID NO:10; the heavy chain variable region CDR2 shown in SEQ ID NO:24; the heavy chain variable region CDR3 shown in SEQ ID NO:38; the light chain variable region CDR1 shown in SEQ ID NO:52; the light chain variable region CDR2 shown in SEQ ID NO:66; and the light chain variable region CDR3 shown in SEQ ID NO:80,

[0058] (k) the heavy chain variable region CDR1 shown in SEQ ID NO:11; the heavy chain variable region CDR2 shown in SEQ ID NO:25; the heavy chain variable region CDR3 shown in SEQ ID NO:39; the light chain variable region CDR1 shown in SEQ ID NO:53; the light chain variable region CDR2 shown in SEQ ID NO:67; and the light chain variable region CDR3 shown in SEQ ID NO:81,

[0059] (l) the heavy chain variable region CDR1 shown in SEQ ID NO:12; the heavy chain variable region CDR2 shown in SEQ ID NO:26; the heavy chain variable region CDR3 shown in SEQ ID NO:40; the light chain variable region CDR1 shown in SEQ ID NO:54; the light chain variable region CDR2 shown in SEQ ID NO:68; and the light chain variable region CDR3 shown in SEQ ID NO:82,

[0060] (m) the heavy chain variable region CDR1 shown in SEQ ID NO:13; the heavy chain variable region CDR2 shown in SEQ ID NO:27; the heavy chain variable region CDR3 shown in SEQ ID NO:41; the light chain variable region CDR1 shown in SEQ ID NO:55; the light chain variable region CDR2 shown in SEQ ID NO:69; and the light chain variable region CDR3 shown in SEQ ID NO:83, or

[0061] (n) the heavy chain variable region CDR1 shown in SEQ ID NO:14; the heavy chain variable region CDR2 shown in SEQ ID NO:28; the heavy chain variable region CDR3 shown in SEQ ID NO:42; the light chain variable region CDR1 shown in SEQ ID NO:56; the light chain variable region CDR2 shown in SEQ ID NO:70; and the light chain variable region CDR3 shown in SEQ ID NO:84.

[0062] In another embodiment, the ActRII receptor antagonist for use in the methods described herein can be an antibody comprising a full length heavy chain amino acid sequence having at least 95% sequence identity to at least one sequence selected from the group consisting of SEQ ID NO:146 to SEQ ID NO:150 and SEQ ID NO:156 to SEQ ID NO:160.

[0063] The ActRII receptor antagonist for use in the methods described herein may be an antibody comprising a full length light chain amino acid sequence having at least 95% sequence identity to at least one sequence selected from the group consisting of SEQ ID NO:141 to SEQ ID NO:145 and SEQ ID NO:151 to SEQ ID NO:155.

[0064] The ActRII receptor antagonist for use in the methods described herein may be an antibody comprising:

[0065] (a) the variable heavy chain sequence shown in SEQ ID NO:99 and the variable light chain sequence shown in SEQ ID NO:85;

[0066] (b) the variable heavy chain sequence shown in SEQ ID NO: 100 and the variable light chain sequence shown in SEQ ID NO: 86;

[0067] (c) the variable heavy chain sequence shown in SEQ ID NO: 101 and the variable light chain sequence shown in SEQ ID NO: 87;

[0068] (d) the variable heavy chain sequence shown in SEQ ID NO: 102 and the variable light chain sequence shown in SEQ ID NO: 88;

[0069] (e) the variable heavy chain sequence shown in SEQ ID NO: 103 and the variable light chain sequence shown in SEQ ID NO: 89;

[0070] (f) the variable heavy chain sequence shown in SEQ ID NO: 104 and the variable light chain sequence shown in SEQ ID NO: 90;

[0071] (g) the variable heavy chain sequence shown in SEQ ID NO: 105 and the variable light chain sequence shown in SEQ ID NO: 91;

[0072] (h) the variable heavy chain sequence shown in SEQ ID NO: 106 and the variable light chain sequence shown in SEQ ID NO: 92;

[0073] (i) the variable heavy chain sequence shown in SEQ ID NO: 107 and the variable light chain sequence shown in SEQ ID NO: 93;

[0074] (j) the variable heavy chain sequence shown in SEQ ID NO: 108 and the variable light chain sequence shown in SEQ ID NO: 94;

[0075] (k) the variable heavy chain sequence shown in SEQ ID NO: 109 and the variable light chain sequence shown in SEQ ID NO: 95;

[0076] (l) the variable heavy chain sequence shown in SEQ ID NO: 110 and the variable light chain sequence shown in SEQ ID NO: 96;

[0077] (m) the variable heavy chain sequence shown in SEQ ID NO: 111 and the variable light chain sequence shown in SEQ ID NO: 97; or

[0078] (n) the variable heavy chain sequence shown in SEQ ID NO:112 and the variable light chain sequence shown in SEQ ID NO:98.

[0079] The ActRII receptor antagonist for use in the methods described herein may be an antibody comprising:

[0080] (a) the heavy chain sequence shown in SEQ ID NO: 146 and the light chain sequence shown in SEQ ID NO: 141;

[0081] (b) the heavy chain sequence shown in SEQ ID NO: 147 and the light chain sequence shown in SEQ ID NO: 142;

[0082] (c) the heavy chain sequence shown in SEQ ID NO: 148 and the light chain sequence shown in SEQ ID NO: 143;

[0083] (d) the heavy chain sequence shown in SEQ ID NO: 149 and the light chain sequence shown in SEQ ID NO: 144;

[0084] (e) the heavy chain sequence shown in SEQ ID NO: 150 and the light chain sequence shown in SEQ ID NO: 145;

[0085] (f) the heavy chain sequence shown in SEQ ID NO: 156 and the light chain sequence shown in SEQ ID NO: 151;

[0086] (g) the heavy chain sequence shown in SEQ ID NO: 157 and the light chain sequence shown in SEQ ID NO: 152;

[0087] (h) the heavy chain sequence shown in SEQ ID NO: 158 and the light chain sequence shown in SEQ ID NO: 153;

[0088] (i) the heavy chain sequence shown in SEQ ID NO: 159 and the light chain sequence shown in SEQ ID NO: 154; or

[0089] (j) the heavy chain sequence shown in SEQ ID NO:160 and the light chain sequence shown in SEQ ID NO:155.

[0090] Also disclosed is an ActRII receptor antagonist for use in or for use in the methods described herein, the ActRII receptor antagonist being an anti-ActRII receptor antibody that cross-blocks or is cross-blocked by at least one of the antibodies described above.

[0091] The ActRII receptor antagonist used in or for use in the methods described herein may be an anti-ActRII receptor antibody having altered effector function by mutation of the Fc region.

[0092] Exemplary of an antibody for use in or in the methods described herein is an anti-ActRII antibody encoded by pBW522 (DSM22873) or pBW524 (DSM22874).

[0093] The working examples described herein utilize CDD866, which is a murinized version of BYM338 in which the human Fc region of the antibody has been replaced by a mouse Fc.

[0094] However, a preferred antibody for use in or in the methods described herein is bimagrumab (BYM338), which is a fully human antibody (modified IgG1, 234-235-Ala-Ala, λ2).

[0095] "ActRII binding molecule" refers to any molecule that is capable of binding to a human ActRII receptor (ActRIIA and / or ActRIIB) alone or in association with other molecules. The binding reaction can be shown by standard methods (qualitative assays), including, for example, binding assays with reference to negative control tests, competition assays, or bioassays for determining inhibition of binding of the ActRII receptor to myostatin, or any type of binding assay in which antibodies with irrelevant specificity but ideally the same isotype, such as anti-CD25 antibodies, are used. Non-limiting examples of ActRII receptor binding molecules include small molecules, such as aptamers or other nucleic acid molecules designed to and / or undergo binding to the receptor, ligand decoys, and antibodies to the ActRII receptor produced by B cells, or hybridomas and chimeric CDR grafts or human antibodies or any fragments thereof (e.g., F(ab')2 fragments and Fab fragments), as well as single chain or single domain antibodies. Preferably, the ActRII receptor binding molecule antagonizes (eg, reduces, inhibits, decreases, delays) the binding of a natural ligand to an ActRII receptor. In some embodiments of the disclosed methods, regimens, kits, processes, uses, and compositions, an ActRIIB receptor binding molecule is employed.

[0096] In another embodiment, the composition comprises an anti-ActRII antibody that binds to a binding domain consisting of amino acids 19-134 of SEQ ID NO: 181 (SEQ ID NO: 182), and the epitope comprises or consists of: (a) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN - SEQ ID NO: 188); (b) amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC - SEQ ID NO: 186); (c) amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 190); (d) amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189); (e) amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187); (f) amino acids 77-80 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 191); (g) amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN-SEQ ID NO: 192); or (h) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR).

[0097] In yet another alternative embodiment, the above composition comprises an anti-ActRII antibody that binds to ActRIIB with an affinity that is 10 times or greater than its affinity for ActRIIA.

[0098] In addition, the present disclosure relates to a composition, wherein the anti-ActRIIB antibody comprises: a heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14; a heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 28; a heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29 to SEQ ID NO: 42; a light chain variable region CDR1, wherein the light chain variable region CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 43 to SEQ ID NO: 56; a light chain variable region CDR2, wherein the light chain variable region CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 70; and a light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 71 to SEQ ID NO: 84.

[0099] In certain embodiments, the present disclosure provides a composition, wherein the anti-ActRII antibody comprises: (a) a heavy chain variable region CDR1 as set forth in SEQ ID NO: 1; a heavy chain variable region CDR2 as set forth in SEQ ID NO: 15; a heavy chain variable region CDR3 as set forth in SEQ ID NO: 29; a light chain variable region CDR1 as set forth in SEQ ID NO: 43; a light chain variable region CDR2 as set forth in SEQ ID NO: 57; and a light chain variable region CDR3 as set forth in SEQ ID NO: 71, (b) a heavy chain variable region CDR1 as set forth in SEQ ID NO: 2; a heavy chain variable region CDR2 as set forth in SEQ ID NO: 16; a heavy chain variable region CDR3 as set forth in SEQ ID NO: 30; a light chain variable region CDR1 as set forth in SEQ ID NO: 44; a light chain variable region CDR2 as set forth in SEQ ID NO: 58; and a light chain variable region CDR3 as set forth in SEQ ID NO: 72, (c) a heavy chain variable region CDR1 as set forth in SEQ ID NO: 3; a heavy chain variable region CDR2 as set forth in SEQ ID NO: 17; a light chain variable region CDR2 as set forth in SEQ ID NO: 18 NO:31; the heavy chain variable region CDR3 shown in SEQ ID NO:45; the light chain variable region CDR2 shown in SEQ ID NO:59; and the light chain variable region CDR3 shown in SEQ ID NO:73, (d) the heavy chain variable region CDR1 shown in SEQ ID NO:4; the heavy chain variable region CDR2 shown in SEQ ID NO:18; the heavy chain variable region CDR3 shown in SEQ ID NO:32; the light chain variable region CDR1 shown in SEQ ID NO:46; the light chain variable region CDR2 shown in SEQ ID NO:60; and the light chain variable region CDR3 shown in SEQ ID NO:74, (e) the heavy chain variable region CDR1 shown in SEQ ID NO:5; the heavy chain variable region CDR2 shown in SEQ ID NO:19; the heavy chain variable region CDR3 shown in SEQ ID NO:33; the light chain variable region CDR1 shown in SEQ ID NO:47; the light chain variable region CDR2 shown in SEQ ID NO:61; and the light chain variable region CDR3 shown in SEQ ID NO:74 NO:75, (f) the heavy chain variable region CDR1 shown in SEQ ID NO:6; the heavy chain variable region CDR2 shown in SEQ ID NO:20; the heavy chain variable region CDR3 shown in SEQ ID NO:34; the light chain variable region CDR1 shown in SEQ ID NO:48; the light chain variable region CDR2 shown in SEQ ID NO:62; and the light chain variable region CDR3 shown in SEQ ID NO:76, (g) the heavy chain variable region CDR1 shown in SEQ ID NO:7;The heavy chain variable region CDR2 shown in SEQ ID NO:21; the heavy chain variable region CDR3 shown in SEQ ID NO:35; the light chain variable region CDR1 shown in SEQ ID NO:49; the light chain variable region CDR2 shown in SEQ ID NO:63; and the light chain variable region CDR3 shown in SEQ ID NO:77, (h) the heavy chain variable region CDR1 shown in SEQ ID NO:8; the heavy chain variable region CDR2 shown in SEQ ID NO:22; the heavy chain variable region CDR3 shown in SEQ ID NO:36; the light chain variable region CDR1 shown in SEQ ID NO:50; the light chain variable region CDR2 shown in SEQ ID NO:64; and the light chain variable region CDR3 shown in SEQ ID NO:78, (i) the heavy chain variable region CDR1 shown in SEQ ID NO:9; the heavy chain variable region CDR2 shown in SEQ ID NO:23; the heavy chain variable region CDR3 shown in SEQ ID NO:37; the light chain variable region CDR1 shown in SEQ ID NO:51; SEQ ID NO:65; and the light chain variable region CDR2 shown in SEQ ID NO:79, (j) the heavy chain variable region CDR1 shown in SEQ ID NO:10; the heavy chain variable region CDR2 shown in SEQ ID NO:24; the heavy chain variable region CDR3 shown in SEQ ID NO:38; the light chain variable region CDR1 shown in SEQ ID NO:52; the light chain variable region CDR2 shown in SEQ ID NO:66; and the light chain variable region CDR3 shown in SEQ ID NO:80, (k) the heavy chain variable region CDR1 shown in SEQ ID NO:11; the heavy chain variable region CDR2 shown in SEQ ID NO:25; the heavy chain variable region CDR3 shown in SEQ ID NO:39; the light chain variable region CDR1 shown in SEQ ID NO:53; the light chain variable region CDR2 shown in SEQ ID NO:67; and the light chain variable region CDR3 shown in SEQ ID NO:81, (i) the heavy chain variable region CDR1 shown in SEQ ID NO:12; the heavy chain variable region CDR2 shown in SEQ ID NO:25; the heavy chain variable region CDR3 shown in SEQ ID NO:39; the light chain variable region CDR1 shown in SEQ ID NO:53; the light chain variable region CDR2 shown in SEQ ID NO:67; and the light chain variable region CDR3 shown in SEQ ID NO:81 NO:26; SEQ ID NO:40; SEQ ID NO:54; SEQ ID NO:68; and SEQ ID NO:82; (m) SEQ ID NO:13; SEQ ID NO:27; SEQ ID NO:41; and SEQ ID NO:55;The light chain variable region CDR2 shown in SEQ ID NO:69; and the light chain variable region CDR3 shown in SEQ ID NO:83, or (n) the heavy chain variable region CDR1 shown in SEQ ID NO:14; the heavy chain variable region CDR2 shown in SEQ ID NO:28; the heavy chain variable region CDR3 shown in SEQ ID NO:42; the light chain variable region CDR1 shown in SEQ ID NO:56; the light chain variable region CDR2 shown in SEQ ID NO:70; and the light chain variable region CDR3 shown in SEQ ID NO:84. ;

[0100] In another embodiment, the anti-ActRII antibody comprises (i) a full-length heavy chain amino acid sequence having at least 95% sequence identity to at least one sequence selected from the group consisting of SEQ ID NO: 146 to SEQ ID NO: 150 and SEQ ID NO: 156 to SEQ ID NO: 160, (ii) a full-length light chain amino acid sequence having at least 95% sequence identity to at least one sequence selected from the group consisting of SEQ ID NO: 141 to SEQ ID NO: 145 and SEQ ID NO: 151 to SEQ ID NO: 155, or (iii) (a) the variable heavy chain sequence set forth in SEQ ID NO: 99 and the variable light chain sequence set forth in SEQ ID NO: 85; (b) the variable heavy chain sequence set forth in SEQ ID NO: 100 and the variable light chain sequence set forth in SEQ ID NO: 86; (c) the variable heavy chain sequence set forth in SEQ ID NO: 101 and the variable light chain sequence set forth in SEQ ID NO: 87; (d) the variable heavy chain sequence set forth in SEQ ID NO: 102 and the variable light chain sequence set forth in SEQ ID NO: 88; (e) the variable heavy chain sequence set forth in SEQ ID NO: 103 and the variable light chain sequence set forth in SEQ ID NO: 89. NO: 103 and the variable light chain sequence shown in SEQ ID NO: 89; (f) the variable heavy chain sequence shown in SEQ ID NO: 104 and the variable light chain sequence shown in SEQ ID NO: 90; (g) the variable heavy chain sequence shown in SEQ ID NO: 105 and the variable light chain sequence shown in SEQ ID NO: 91; (h) the variable heavy chain sequence shown in SEQ ID NO: 106 and the variable light chain sequence shown in SEQ ID NO: 92; (i) the variable heavy chain sequence shown in SEQ ID NO: 107 and the variable light chain sequence shown in SEQ ID NO: 93; (j) the variable heavy chain sequence shown in SEQ ID NO: 108 and the variable light chain sequence shown in SEQ ID NO: 94; (k) the variable heavy chain sequence shown in SEQ ID NO: 109 and the variable light chain sequence shown in SEQ ID NO: 95; (l) the variable heavy chain sequence shown in SEQ ID NO: 110 and the variable light chain sequence shown in SEQ ID NO: 96; (m) the variable heavy chain sequence shown in SEQ ID NO: 111 and the variable light chain sequence shown in SEQ ID NO: The variable light chain sequence shown in NO:97; or (n) the variable heavy chain sequence shown in SEQ ID NO:112 and the variable light chain sequence shown in SEQ ID NO:98.

[0101] In certain aspects, the present disclosure relates to the above-mentioned composition, wherein the anti-ActRII antibody comprises (a) a heavy chain sequence as shown in SEQ ID NO: 146 and a light chain sequence as shown in SEQ ID NO: 141; (b) a heavy chain sequence as shown in SEQ ID NO: 147 and a light chain sequence as shown in SEQ ID NO: 142; (c) a heavy chain sequence as shown in SEQ ID NO: 148 and a light chain sequence as shown in SEQ ID NO: 143; (d) a heavy chain sequence as shown in SEQ ID NO: 149 and a light chain sequence as shown in SEQ ID NO: 144; (e) a heavy chain sequence as shown in SEQ ID NO: 150 and a light chain sequence as shown in SEQ ID NO: 145; (f) a heavy chain sequence as shown in SEQ ID NO: 156 and a light chain sequence as shown in SEQ ID NO: 151; (g) a heavy chain sequence as shown in SEQ ID NO: 157 and a light chain sequence as shown in SEQ ID NO: 152; (h) a heavy chain sequence as shown in SEQ ID NO: 158 and a light chain sequence as shown in SEQ ID NO: 153; (i) a heavy chain sequence as shown in SEQ ID NO: 159 and a light chain sequence as shown in SEQ ID NO: 160; The heavy chain sequence shown in SEQ ID NO:159 and the light chain sequence shown in SEQ ID NO:154; or (j) the heavy chain sequence shown in SEQ ID NO:160 and the light chain sequence shown in SEQ ID NO:155.

[0102] Another subject matter of the present disclosure relates to compositions wherein (i) the anti-ActRII antibody cross-blocks or is cross-blocked by one of the above antibodies, (ii) has altered effector function through mutation of the Fc region, and / or (iii) binds to an epitope recognized by one of the above antibodies.

[0103] In yet another alternative embodiment, the above composition comprises an anti-ActRII antibody that binds to ActRIIB with an affinity that is 10 times or greater than its affinity for ActRIIA.

[0104] In another embodiment, the disclosed composition comprises an anti-ActRII antibody encoded by pBW522 (DSM22873) or pBW524 (DSM22874). BRIEF DESCRIPTION OF THE DRAWINGS

[0105] FIG. 1A graphically shows CDD866 plasma levels measured in wild-type C57BL / 6 mice treated with weekly injections of CDD866 or isotype control Ab for 8 weeks.

[0106] Figure 1B graphically shows the heart weight / tibialis muscle length ratio (HW / TL) of control mice with isotype Ab (n=3) (grey bars) and experimental mice with CDD866 Ab (n=3) *p<0.05 (black bars), indicating that CDD866 does not significantly increase the heart mass of adult wild-type C57BL / 6 mice.

[0107] 1C is a bar graph showing % fibrosis in mice of control (isotype Ab, grey bars) and experimental groups of mice (CDD866 Ab [n=3], *p<0.05, black bars)). CDD866 reduced myocardial fibrosis, but the % fibrosis at baseline was significantly lower in adult wild-type C57BL / 6 mice.

[0108] Figure ID shows a representative photomicrograph of PAS-stained myocardium, highlighting the size of cardiomyocytes.

[0109] Figure IE graphically shows the finding that CDD866 does not significantly increase cardiomyocyte size in wild-type animals. Data are presented as mean ± SD. Grey = control group, isotype Ab (n = 3). Black = experimental group, CDD866 Ab (n = 3), *p < 0.05

[0110] Figure 2A graphically shows that contractile function, as measured by % fractional shortening (FS), was expectedly reduced with TAC (horizontal bars), but was preserved in CDD866-treated animals subjected to TAC (diagonal bars). SHAM+isotype Ab (n=7), black bars; SHAM+CDD866 Ab (n=7), gray bars; TAC+isotype Ab (n=10), horizontal bars; TAC+CDD866 Ab (n=10), diagonal bars. #P<0.01.

[0111] Figure 2B shows representative echocardiographic images 11 weeks after SHAM or TAC surgery, demonstrating preserved contractile function in TAC animals treated with CDD866.

[0112] Figure 2C graphically shows the lung weight / tibialis muscle length ratio (LW / TL) of mice in different treatment groups. SHAM+isotype Ab (n=7), black bars; SHAM+CDD866 Ab (n=7), gray bars; TAC+isotype Ab (n=10), horizontal bars; TAC+CDD866 Ab (n=10), diagonal bars. *p<0.01. There was a trend toward decreased lung weight in CDD866-treated animals, indicating less lung congestion (a surrogate for heart failure in the mouse model).

[0113] FIG2D graphically shows the significant reduction in the primary endpoint (survival or %FS < 20%) with CDD866 treatment.

[0114] FIG. 3A graphically shows plasma CDD866 levels for various treatment groups: TAC+isotype; TAC+CDD866; Sham+isotype; and Sham+CDD866.

[0115] Figure 3B is a bar graph showing that cardiac follistatin-like 3 (FSTL3) expression increases with TAC, indicating increased cardiac ActRII-A / B signaling in this cardiac injury model. CDD866 treatment reduces cardiac FSTL3 expression, indicating that it effectively blocks TAC-induced ActRII-A / B signaling in the heart. Black = SHAM + isotype Ab (n = 7). Gray = SHAM + CDD866Ab (n = 7). Horizontal bars = TAC + isotype Ab (n = 10). Diagonal bars = TAC + CDD866Ab (n = 10). *p < 0.05. #p < 0.01.

[0116] FIG3C graphically shows that the relative mRNA expression of pathological cardiac hypertrophy genes is reduced with CDD866 treatment. ANP (atrial natriuretic peptide); BNP (brain natriuretic peptide); aMHC (alpha myosin heavy chain); bMHC (beta myosin heavy chain). Black = SHAM + isotype Ab (n = 7). Gray = SHAM + CDD866 Ab (n = 7). Horizontal bars = TAC + isotype Ab (n = 10). Diagonal bars = TAC + CDD866 Ab (n = 10). *p < 0.05. #p < 0.01.

[0117] FIG3D is a bar graph illustrating that treatment with CDD866 reduces relative mRNA expression of pathological cardiac fibrosis genes in TAC-induced heart failure. COL1 (collagen type 1); CTGF (connective tissue growth factor). Black = SHAM + isotype Ab (n = 7). Gray = SHAM + CDD866 Ab (n = 7). Horizontal bars = TAC + isotype Ab (n = 10). Diagonal bars = TAC + CDD866 Ab (n = 10). *p < 0.05. #p < 0.01.

[0118] FIG. 4A graphically shows CDD866 plasma levels measured in mice that developed contractile function following TAC followed by weekly treatment with CDD866 injections for 8 weeks.

[0119] Figure 4B is a bar graph showing the relative mRNA expression levels of FSTL3 (follistatin-like 3), activin A, MSTN (myostatin), ACVR2A (activin A receptor type 2A), and ACVR2B (activin A receptor type 2B). The figure shows that treatment with CDD866 can reduce cardiac FSTL3 expression, indicating that CDD866 can effectively block TAC-induced ActRII-A / B signaling in the heart.

[0120] Figure 4C is a graph of % fractional shortening plotted against time (in weeks), showing that CDD866 reversed systolic dysfunction in TAC-induced heart failure in a progressively improving manner as early as 1 week after treatment.

[0121] Figure 4D graphically shows that CDD866 also reduced the lung weight to tibialis muscle length ratio, a surrogate marker for heart failure in a murine model. Grey = TAC + isotype Ab. Black = TAC + CDD866 Ab. *p<0.05. #p<0.01. LW / TL (lung weight / tibialis muscle length ratio).

[0122] Figure 5A is a graph plotting wall thickness versus weeks after TAC, showing that wall thickness gradually increases with CDD866 treatment. The arrow indicates the onset of CDD866.

[0123] Figure 5B shows serial echo images of ventricular slices during treatment, demonstrating differences in cardiac growth between isotype and CDD866 treated animals. Cdd866-mediated cardiac growth prevents eccentric remodeling associated with progressive contractile dysfunction.

[0124] Figure 5C is a graph showing the heart weight / tibialis muscle length ratio (HW / TL) of TAC+isotype Ab (grey bars) and TAC+CDD866 Ab (black bars) treated mice, indicating that CDD866 increases heart mass in the TAC model. *p<0.05. #p<0.01.

[0125] Figure 5D shows micrographs of PAS-stained myocardium highlighting the size of cardiomyocytes in TAC+isotype Ab and TAC+CDD866Ab treated mice.

[0126] Figure 5E is a graph showing cross-sectional area of ​​cardiomyocytes from TAC+isotype Ab (grey) and TAC+CDD866Ab (black) treated mice, demonstrating that CDD866 increases cardiomyocyte growth in TAC. *p<0.05. #p<0.01.

[0127] FIG6A is a graph showing that mRNA expression of genes associated with pathological hypertrophy is reduced with CDD866 treatment. ANP (atrial natriuretic peptide); BNP (brain natriuretic peptide); αMHC (α myosin heavy chain); βMHC (β myosin heavy chain). TAC+isotype Ab (grey); TAC+CDD866 Ab (black) are shown. *p<0.05. #p<0.01.

[0128] FIG6B plots fractional shortening, wall thickness, and body weight of mice versus time (in weeks). Arrows = timing of single dose; dashed line = expected trajectory without CDD866 treatment. The figure shows that the effects of CDD866 on cardiac growth and body weight occur rapidly, are transient, and are reversible. The effects of a single dose of CDD866 also occur within a 1-2 week time frame and persist for at least 6 weeks.

[0129] FIG6C shows a photomicrograph of Masson's trichrome stained myocardium (blue = fibrosis; red = muscle), demonstrating reduced cardiac fibrosis in TAC'd mice treated with CDD866.

[0130] Figure 6D is a bar graph showing % fibrosis using TAC + isotype Ab (grey) and TAC + CDD866 Ab (black), demonstrating a trend toward reduced myocardial fibrosis with CDD866 treatment. *p<0.05. #p<0.01.

[0131] Figure 7A is a Western blot of gastrocnemius muscle samples probed with p-SMAD3 and GAPDH antibodies. Samples were collected from T57BL / 6 mice with confirmed cardiac dysfunction after TAC and subsequently treated with CDD866 (TT-2 to TT-10) or isotype control Abs (TT-11 to TT-20) for 8 weeks. The figure generally demonstrates that CDD866 reduces ActRII-A / B signaling in skeletal muscle in a TAC-mediated murine model of heart failure. MSTN stimulation in C2C12 cells was used as a positive control for the assay.

[0132] Figure 7B is a graph where the % change from baseline in mouse weight is measured relative to weeks post-treatment. Diamond data points (red) represent TAC+isotype Ab. Square data points (blue) represent TAC+CDD866 Ab. *p<0.05. #p<0.01. CDD866 resulted in a gradual increase in overall body weight, most likely through increased muscle mass.

[0133] Figure 7C graphically shows the % change in muscle mass relative to control for each of the following skeletal muscle groups: EDL (Extensor digitorum longus), Gastrocnemius, and Tibialis. Red indicates TAC + isotype Ab. Blue indicates TAC + CDD866 Ab. *p<0.05. #p<0.01. CDD866 increased the mass of the three skeletal muscle groups overall.

[0134] Figure 7D is a graph where % fiber distribution is plotted relative to serial tissue sections, demonstrating that CDD866 increases skeletal muscle cell size. Red indicates TAC + isotype Ab treatment. Blue indicates TAC + CDD866 Ab treatment. *p<0.05. #p<0.01.

[0135] Figure 7E shows four graphs in which % fiber distribution is plotted relative to serial tissue sections, indicating that CDD866 induces multiple fiber type switching in skeletal muscle. Red indicates TAC+isotype Ab treatment. Blue indicates TAC+CDD866 Ab treatment.

[0136] FIG8A is a graph showing the change in % fractional shortening (FS) over time in mice with a missense mutation (F764L) in the αMHC gene (a mouse model of dilated cardiomyopathy). Twelve weeks of CDD866 treatment resulted in a modest trend toward increased contractile function. Gray = isotype Ab. Black = CDD866 Ab. *p<0.05. #p<0.01.

[0137] Figure 8B is a bar graph showing the relative mRNA expression levels of various genes associated with the ActRII pathway in cardiac tissue from these mice treated with CDD866 (black) or isotype Ab (grey). There was a trend for CDD866 to reduce cardiac FSTL3 expression, indicating inhibition of ActRII receptor signaling in the heart. *p<0.05. #p<0.01.

[0138] FIG8C is a bar graph showing the relative mRNA expression levels of genes associated with pathological hypertrophy in mice treated with isotype Ab or CDD866 Ab. *p<0.05. #p<0.01. Grey = isotype Ab. Black = CDD866 Ab. *p<0.05. #p<0.01. ANP (atrial natriuretic peptide); BNP (brain natriuretic peptide); αMHC (α myosin heavy chain); βMHC (β myosin heavy chain). There were no significant differences in the pathological hypertrophy gene expression profiles.

[0139] definition

[0140] In order to more readily understand the present disclosure, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0141] The term "comprising" means "including", for example a composition "comprising" X may consist only of X or may include something else, for example X+Y.

[0142] The term "about" in connection with a numerical value x means, for example, x ± 10%.

[0143] The following exemplifies possible preclinical treatment protocols to evaluate the possible effects of treatment with an ActRII binding molecule, more preferably an antagonist antibody to ActRII (eg, bimagrumab).

[0144] Treatment is exemplified using mice subjected to transverse aortic constriction (TAC), a common experimental model of pressure overload-induced cardiac hypertrophy and heart failure. Technicians know how to establish suitable experimental or dosing protocols for other species, particularly humans. For primate studies, anti-ActRII antibodies (e.g., bimagrumab) can be administered to male and female macaques once a week by intravenous injection for up to 3 months. 32 macaques (16 / sex) can be assigned to one of four treatment groups (3 to 5 animals / sex / group), and vehicle or ActRIIB antibodies (e.g., BYM338) can be administered intravenously once a week at 10, 30 or 100 mg / kg for 13 weeks (a total of 14 doses; the dose should be selected based on the symptomology of the heart disease).

[0145] The terms "ActRIIA" and "ActRIIB" refer to activin receptors. Activins signal through a heterodimeric complex of receptor serine kinases, which include at least two type I (I and IB) and two type II (IIA and IIB, also known as ACVR2A and ACVR2B) receptors. These receptors are all transmembrane proteins, consisting of a ligand-binding extracellular domain (which has a cysteine-rich region), a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine specificity. Type I receptors are essential for signal transduction, while type II receptors are required for binding ligands and expression / recruitment of type I receptors. Type I and type II receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors. Activin receptor IIB (ActRIIB) is a receptor for myostatin. Activin receptor IIA (ActRIIA) is also a receptor for myostatin. The term ActRIIB or Act IIB receptor refers to human ActRIIB as defined in SEQ ID NO: 181 (AAC64515.1, GI: 3769443). Research grade polyclonal and monoclonal anti-ActRIIB antibodies are known in the art, such as those provided by R&D Those manufactured in MN, USA. Of course, antibodies can be raised against ActRIIB from other species and used to treat pathological conditions in those species.

[0146] The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules (e.g., antibodies, cytokines, and complement) produced by the above cells or the liver that results in the selective injury, destruction, or elimination from the human body of invading pathogens, cells or tissues infected by pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0147] "Signaling activity" refers to a biochemical cause-effect relationship, usually initiated by a protein-protein interaction (such as the binding of a growth factor to a receptor), resulting in the transmission of a signal from one part of a cell to another part of the cell. Typically, the transmission involves specific phosphorylation of one or more tyrosine residues, serine residues, or threonine residues on one or more proteins in a series of reactions, resulting in signal transduction. The penultimate process usually includes nuclear events, resulting in changes in gene expression.

[0148] The term "antibody" as used herein includes intact antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as V L ) and the light chain constant region. The light chain constant region consists of a domain C L Composition. H and V L The V region can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), and more conserved regions, called framework regions (FRs), interspersed with the CDRs. H and V L It is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0149] As used herein, the term "antigen-binding portion" of an antibody (or simply "antigen portion") refers to the full length of an antibody, or one or more fragments of an antibody (e.g., a portion of ActRIIB) that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: Fab fragments, which are fragments of V L 、V H , C L A monovalent fragment consisting of a CH1 domain and a F(ab)2 fragment, wherein the F(ab)2 fragment is a bivalent fragment comprising two Fab fragments, each Fab fragment binds to the same antigen and is connected by a disulfide bond in the hinge region; an Fd fragment, wherein the Fd fragment consists of a V H and CH1 domains; Fv fragment, the Fv fragment consists of the V L and V H Domain composition; dAb fragment (Ward et al., 1989 Nature 341: 544-546), the dAb fragment consists of V H domain composition; and isolated complementarity determining regions (CDRs).

[0150] In addition, although the two domains V L and V H are encoded by separate genes, but they can be joined using recombinant methods by a synthetic linker that enables them to be made into a single protein chain in which V L and V H The regions are paired to form monovalent molecules (called single-chain Fv (scFv); see, for example, Bird et al., 1988 Science 242: 423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding region" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.

[0151] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds ActRIIB is substantially free of antibodies that specifically bind an antigen other than ActRIIB). However, an isolated antibody that specifically binds ActRIIB may have cross-reactivity with other antigens (such as ActRIIB molecules from other species). In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0152] The term "cross-blocking" refers to the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to ActRIIB, particularly the ligand binding domain, in a standard competitive binding assay.

[0153] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0154] As used herein, the term "human (class) antibody" is intended to include such antibodies, the antibody has a variable region, in which the framework region and CDR region are all derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or mutant forms of human germline sequences, or antibodies containing a consensus framework sequence derived from human framework sequence analysis, such as described in Knappik et al. (2000.J Mol Biol 296, 57-86). Human antibodies of the present disclosure may include amino acid residues not encoded by human sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human (class) antibody" is not intended to include such antibodies, in which the CDR sequences derived from the germline of another mammalian species (such as mice) have been transplanted to human framework sequences.

[0155] The term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has a variable region in which both the framework and CDR regions are derived from human sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal such as a transgenic mouse, the B cell having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0156] As used herein, the term "recombinant human (class) antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express human antibodies (e.g., antibodies from transfectomas), antibodies isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, produced or isolated by any other method involving splicing all or part of the sequence of human immunoglobulin genes to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework regions and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, somatic mutagenesis in the receptor), whereby the V H Area and V L The amino acid sequence of the region is such a sequence that, although the sequence is derived from human germline V H Sequence and V L V H Sequence and V L Sequence related, but may not occur naturally in the human antibody germline repertoire.

[0157] As used herein, "isotype" refers to the antibody class provided by the heavy chain constant region genes (eg, IgM, IgE, IgG, such as IgGl or IgG2).

[0158] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0159] As used herein, an antibody that "specifically binds to an ActRIIB polypeptide" means an antibody that binds to an ActRIIB polypeptide with a K of about 100 nM or less, about 10 nM or less, about 1 nM or less, or about 1 nM or less. D An antibody that binds to a human ActRIIB polypeptide. An antibody that "cross-reacts with an antigen other than ActRIIB" means an antibody that cross-reacts with an antigen other than ActRIIB at about 10×10 -9 M or less, about 5×10 -9 M or less, or about 2×10 -9 M or lower K D An antibody that "does not cross-react with a specific antigen" means an antibody that binds to an antigen at a rate of about 1.5×10 -8 M or higher K D , or about 5-10×10 -8 M or about 1×10 -7 M or higher K DIn certain embodiments, such antibodies that do not cross-react with the antigen exhibit substantially undetectable binding to these proteins in standard binding assays. Biosensor systems such as The system or solution is titrated to determine K D .

[0160] As used herein, the term "antagonist antibody" means an antibody that inhibits ActRIIB-induced signaling activity in the presence of myostatin or other ActRIIB ligands (such as activin or GDF-11), and / or means an antibody that inhibits ActRIIA-induced signaling activity in the presence of myostatin or other ActRIIA ligands (such as activin or GDF-11). Examples of assays for detecting this include inhibition of myostatin-induced signaling (e.g., by Smad-dependent reporter gene assay), inhibition of myostatin-induced Smad phosphorylation (P-Smad ELISA), and inhibition of myostatin-induced inhibition of skeletal muscle cell differentiation (e.g., by creatine kinase assay).

[0161] In some embodiments, the antibody inhibits myostatin-induced signaling as measured in a Smad-dependent reporter gene assay, IC 50 About 10 nM or less, about 1 nM or less, or about 100 pM or less.

[0162] As used herein, an antibody with "no agonistic activity" is intended to refer to an antibody that does not significantly increase ActRIIB-mediated signaling activity in the absence of myostatin in a cell-based assay, such as inhibition of myostatin-induced signaling (e.g., by Smad-dependent reporter gene assay), inhibition of myostatin-induced Smad phosphorylation (P-Smad ELISA), and inhibition of myostatin-induced inhibition of skeletal muscle cell differentiation (e.g., by creatine kinase assay).

[0163] As used herein, the term "K assoc ” or “K a " is intended to represent the association rate of a particular antibody-antigen interaction, while the term "K" as used herein dis ” or “K d " is intended to represent the off-rate of a particular antibody-antigen interaction. As used herein, the term "K D " is intended to represent the dissociation constant, which is the dissociation constant from K d With K a The ratio (K d / K a) and expressed as molar concentration (M). The K of an antibody can be determined using well-established methods in the art. D Used to determine the K value of an antibody D The method is to use surface plasmon resonance, such as Biosensor systems, or solution equilibrium titration (SET) (see Friguet B et al., (1985) J. Immunol Methods; 77(2): 305-319, and Hanel C et al., (2005) Anal Biochem; 339(1): 182-184).

[0164] As used herein, the term "affinity" refers to the strength of the interaction between an antigen and an antibody at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at many sites through weak non-covalent forces; the more interactions there are, the stronger the affinity.

[0165] As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of an antibody-antigen complex. It is controlled by three main factors: antibody epitope affinity; the valence of both the antigen and the antibody; and the structural arrangement of the interacting parts. Ultimately, these factors define the specificity of the antibody, i.e., the likelihood that a particular antibody will bind to a precise antigen epitope.

[0166] As used herein, the term "ADCC" or "antibody-dependent cellular cytotoxicity" activity refers to human B cell depletion activity. ADCC activity can be measured by a human B cell depletion assay known in the art.

[0167] In order to obtain a probe with higher affinity, a dimer conjugate (two antibody protein molecules coupled to a FACS marker) can be constructed to make it easier to detect low-affinity interactions (such as interactions with germline antibodies) by FACS. In addition, another method to increase antigen binding affinity involves producing dimers, trimers or polymers of any construct of the anti-ActRIIB antibodies described herein. Such polymers can be produced by covalent bonding between the individual modules, for example, by simulating natural C to N-terminal bonding or by simulating antibody dimers held together by the constant region of the antibody. The bonds engineered into the Fc / Fc interface can be covalent or non-covalent. In addition, dimerization or multimerization of partners other than Fc can be used in ActRIIB hybrids to create such higher-order structures. For example, a multimerization domain can be used, such as a trimerization domain described in WO2004 / 039841 or a pentamerization domain described in WO98 / 18943.

[0168] As used herein, the term "selectivity" of an antibody refers to the ability of the antibody to bind to a particular target polypeptide but not to closely related polypeptides.

[0169] As used herein, the term "high affinity" of an antibody refers to the K of the antibody for the target antigen. D 1 nM or less. As used herein, the term "subject" includes any human or non-human animal.

[0170] The term "non-human animals" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, mice, horses, cows, chickens, amphibians, reptiles, etc.

[0171] As used herein, the term "(through) optimized" refers to a nucleotide sequence that has been altered to encode an amino acid sequence using codons that are preferred in a production cell or organism, which is typically a eukaryotic cell, such as a cell of Pichia, a cell of Trichoderma, a Chinese hamster ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain the amino acid sequence originally encoded by the starting nucleotide sequence completely or as much as possible, the starting nucleotide sequence is also referred to as the "parent" sequence. The optimized sequences herein have been engineered to have codons that are preferred in CHO mammalian cells, however, the optimized expression of these sequences in other eukaryotic cells is also contemplated herein. The amino acid sequence encoded by the optimized nucleotide sequence is also referred to as optimized.

[0172] Detailed description of the present disclosure

[0173] It has been found that antibodies directed against ActRII receptors, such as bimagrumab, can reduce signaling through these receptors and lead to the prevention and / or treatment of heart disease.

[0174] Therefore, in one aspect, the present disclosure provides a composition comprising an ActRIIA or ActRIIB binding molecule (e.g., bimagrumab), or a functional protein comprising an antigen binding portion of the antibody. The binding molecule may be an ActRIIB binding molecule, such as human ActRIIB. The polypeptide sequence of human ActRIIB is shown in SEQ ID NO: 181 (AAC64515.1, GI: 3769443). In one embodiment, the antibody or functional protein is from a mammal, having an origin such as a human or camelid. Therefore, the antibody contained in the disclosed composition may be a chimeric antibody, a human antibody, or a humanized antibody. In a specific embodiment, the anti-ActRIIB antibody contained in the disclosed composition is characterized by having an antigen binding region that is specific for the target protein ActRIIB and binds to ActRIIB or a fragment of ActRIIB.

[0175] In one embodiment, the antibody contained in the disclosed composition is an ActRII antagonist that has no agonistic activity or has low agonistic activity. In another embodiment, the antibody or functional fragment contained in the disclosed composition binds to the target protein ActRII and reduces the binding of myostatin to ActRII to a basal level. In another aspect of this embodiment, the antibody or functional fragment contained in the disclosed composition completely prevents the binding of myostatin to ActRII. In another embodiment, the antibody or functional fragment contained in the disclosed composition inhibits Smad activation. In another embodiment, the antibody or functional fragment contained in the disclosed composition inhibits type IIB activin receptor-mediated myostatin-induced inhibition of skeletal differentiation through a Smad-dependent pathway.

[0176] Binding can be determined by one or more assays that can be used to measure the antagonistic or agonistic activity of the antibody. Preferably, the assay measures at least one of the effects of the antibody on ActRIIB, including: inhibiting myostatin binding to ActRIIB (by ELISA); inhibiting myostatin-induced signaling (e.g., by Smad-dependent reporter gene assay), inhibiting myostatin-induced Smad phosphorylation (P-Smad ELISA), and inhibiting myostatin-induced inhibition of skeletal muscle cell differentiation (e.g., by creatine kinase assay).

[0177] In one embodiment, the present disclosure provides a composition comprising an antibody that specifically binds to the myostatin binding region (i.e., the ligand binding domain) of ActRIIB. The ligand binding domain consists of amino acids 19-134 of SEQ ID NO: 181 and has been designated herein as SEQ ID NO: 182. The ligand binding domain contains several epitopes described below.

[0178] In one embodiment, the antibodies included in the disclosed compositions have a K of about 100 nM or less, about 10 nM or less, about 1 nM or less, D Binding to ActRIIB. Preferably, the antibodies included in the disclosed compositions bind to ActRIIB with an affinity of 100 pM or less (i.e., about 100 pM, about 50 pM, about 10 pM, about 2 pM, about 1 pM or less). In one embodiment, the antibodies included in the disclosed compositions bind to ActRIIB with an affinity between about 1 pM and about 10 pM.

[0179] In one embodiment, the antibodies included in the disclosed compositions do not cross-react with ActRIIB-related proteins, in particular, do not cross-react with human ActRIIA (NP_001607.1, GI: 4501897). In another embodiment, the antibodies included in the disclosed compositions cross-react with ActRIIA and bind to ActRIIB with equivalent affinity, or bind to ActRIIB with about 1, 2, 3, 4 or 5 times, more preferably about 10 times, still more preferably about 20, 30, 40 or 50 times, still more preferably about 100 times their affinity for binding to ActRIIA.

[0180] In one embodiment, the antibody comprised in the disclosed composition binds to ActRIIA with an affinity of 100 pM or greater (ie, about 250 pM, about 500 pM, about 1 nM, about 5 nM or greater).

[0181] In one embodiment, the antibodies comprised in the disclosed compositions are of the IgG2 isotype.

[0182] In another embodiment, the antibody included in the disclosed composition is of IgG1 isotype. In another embodiment, the antibody included in the disclosed composition is of IgG1 isotype and has an altered effector function by mutation of the Fc region. The altered effector function may be reduced ADCC activity and CDC activity. In one embodiment, the altered effector function is silenced ADCC activity and CDC activity.

[0183] In another related embodiment, the antibody comprised in the disclosed composition is a fully human or humanized IgG1 antibody that has no antibody-dependent cellular cytotoxicity (ADCC) activity or CDC activity and binds to the ActRIIB region consisting of amino acids 19-134 of SEQ ID NO:181.

[0184] In another related embodiment, the antibody comprised in the disclosed composition is a fully human or humanized IgG1 antibody having reduced antibody-dependent cellular cytotoxicity (ADCC) activity or CDC activity and binding to the ActRIIB region consisting of amino acids 19-134 of SEQ ID NO:181.

[0185] The present disclosure also relates to a composition comprising a human anti-ActRIIB antibody or a humanized anti-ActRIIB antibody for use in preventing and / or treating a heart disease as described above.

[0186] In certain embodiments, the antibodies contained in the disclosed compositions are derived from specific heavy and light chain sequences and / or contain specific structural features, such as CDR regions containing specific amino acid sequences. The present disclosure provides isolated ActRIIB antibodies, methods for preparing the antibodies, immunoconjugates and multivalent or multispecific molecules containing the antibodies, and pharmaceutical compositions containing the antibodies, immunoconjugates or bispecific molecules.

[0187] In alternative embodiments, the present disclosure relates to the following aspects:

[0188] 1. An ActRII receptor antagonist for use in treating and / or preventing heart failure, including heart failure associated with or caused by valvular heart disease, hypertension, coronary artery disease, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic or infectious agents, and other forms of inherited or idiopathic cardiomyopathy.

[0189] 2. The ActRII receptor antagonist for use according to aspect 1, wherein the ActRII antagonist is administered to a patient in need thereof at a dose of about 3-10 mg / kg.

[0190] 3. The ActRII receptor antagonist for use according to aspect 2, wherein the myostatin antagonist is administered at a dose of about 3 mg / kg body weight or about 10 mg / kg body weight.

[0191] Alternatively, the ActRII receptor antagonist is administered at a dose of about 3, 4, 5, 6, 7, 8, 9, or about 10 mg / kg body weight.

[0192] 4. The ActRII receptor antagonist for use according to aspects 1-3, wherein the ActRII receptor antagonist is administered intravenously or subcutaneously.

[0193] 5. The ActRII receptor antagonist for use according to any one of aspects 1-4, wherein the ActRII receptor antagonist is administered once every four weeks.

[0194] Alternatively, the ActRII receptor antagonist may be administered once every 8 weeks.

[0195] 6. An ActRII receptor antagonist for use according to any one of aspects 1-5, wherein the ActRII receptor antagonist is administered for at least 3 months.

[0196] 7. An ActRII receptor antagonist for use according to any one of aspects 1-6, wherein the ActRII receptor antagonist is administered for up to 12 months.

[0197] Preferably, the ActRII receptor antagonist is administered for at least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months.

[0198] 8. A method for treating and / or preventing heart failure, the method comprising administering an effective amount of an ActRII receptor antagonist to a subject having heart failure or at risk of developing heart failure.

[0199] In many cases, heart failure can be caused by or associated with conditions such as valvular heart disease, coronary heart disease, hypertension, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, exposure to toxic and infectious agents, and other forms of inherited or idiopathic cardiomyopathy. Patients at risk for developing heart failure may have one or more of these conditions.

[0200] 9. A method for treating structural and / or functional cardiac abnormalities associated with a condition selected from the group consisting of valvular heart disease, hypertension, coronary artery disease, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic and infectious agents, and other forms of inherited or idiopathic cardiomyopathy, the method comprising administering an effective amount of an ActRII receptor antagonist to a subject having the structural and / or functional cardiac abnormalities associated with the condition.

[0201] 10. The method according to aspect 8 or 9, comprising administering the ActRII receptor antagonist to a patient in need thereof at a dose of about 3-10 mg / kg.

[0202] 11. The method according to aspect 8 or 9, comprising administering the ActRII receptor antagonist to a patient in need thereof at a dose of about 3 or about 10 mg / kg body weight.

[0203] 12. The method according to aspect 8 or 9, comprising administering the ActRII receptor antagonist intravenously or subcutaneously.

[0204] 13. The method according to any one of aspects 8 to 10, comprising administering the ActRII receptor antagonist once every four weeks.

[0205] 14. The method according to any one of aspects 8 to 13, comprising administering the ActRII receptor antagonist for at least 3 months.

[0206] 15. The method of aspect 14, comprising administering the ActRII receptor antagonist for up to 12 months.

[0207] 16. An ActRII receptor antagonist for the method or use according to any one of aspects 1-15, wherein the ActRII receptor antagonist is an anti-ActRII receptor antibody.

[0208] 17. An ActRII receptor antagonist for use according to any one of aspects 1-16, wherein the anti-ActRII receptor antibody is bimagrumab.

[0209] 18. An ActRII receptor antagonist for use according to aspect 17, wherein the ActRII receptor antagonist is an anti-ActRII antibody that binds to an epitope of ActRIIB consisting of amino acids 19-134 of SEQ ID NO: 181 (SEQ ID NO: 182).

[0210] 19. The method or ActRII receptor antagonist for use according to any one of aspects 16-18, wherein the anti-ActRII antibody binds to an epitope of ActRIIB comprising or consisting of:

[0211] (a) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN - SEQ ID NO: 188);

[0212] (b) amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC - SEQ ID NO: 186);

[0213] (c) amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 190);

[0214] (d) amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189);

[0215] (e) amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187);

[0216] (f) amino acids 29-41 of SEQ ID NO: 181 (CIYYNANWELERT-SEQ ID NO: 191);

[0217] (g) amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN - SEQ ID NO: 192); or

[0218] (h) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR).

[0219] 20. An ActRII receptor antagonist for use according to any one of aspects 16-19, wherein the anti-ActRIIB antibody is selected from the group consisting of:

[0220] a) an anti-ActRIIB antibody, wherein the anti-ActRIIB antibody binds to an epitope of ActRIIB, wherein the epitope comprises:

[0221] (a) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN - SEQ ID NO: 188);

[0222] (b) amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC - SEQ ID NO: 186);

[0223] (c) amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 190);

[0224] (d) amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189);

[0225] (e) amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187);

[0226] (f) amino acids 29-41 of SEQ ID NO: 181 (CIYYNANWELERT-SEQ ID NO: 191);

[0227] (g) amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN - SEQ ID NO: 192); or

[0228] (h) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR);

[0229] and b) an ActRIIB antagonist antibody that binds to an epitope of ActRIIB comprising amino acids 78-83 of SEQ ID NO: 181 (WLDDFN—SEQ ID NO: 188);

[0230] (b) amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC - SEQ ID NO: 186);

[0231] (c) amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 190);

[0232] (d) amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189);

[0233] (e) amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187);

[0234] (f) amino acids 29-41 of SEQ ID NO: 181 (CIYYNANWELERT-SEQ ID NO: 191);

[0235] (g) amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN - SEQ ID NO: 192); or

[0236] (h) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR), wherein the K D About 2pM.

[0237] 21. An ActRII receptor antagonist for use according to any one of aspects 16-20, wherein the antibody binds to ActRIIB with an affinity that is 10 times or greater than its affinity for binding to ActRIIA.

[0238] 22. An ActRII receptor antagonist for use according to any one of aspects 16-21, wherein the antibody comprises: a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14; a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 28; a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29 to SEQ ID NO: 42; a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 43 to SEQ ID NO: 56; a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 70; and a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71 to SEQ ID NO: The amino acid sequence of the group consisting of NO:84.

[0239] 23. An ActRII receptor antagonist for use according to any one of aspects 16 to 22, wherein the antibody comprises:

[0240] (a) the heavy chain variable region CDR1 shown in SEQ ID NO:1; the heavy chain variable region CDR2 shown in SEQ ID NO:15; the heavy chain variable region CDR3 shown in SEQ ID NO:29; the light chain variable region CDR1 shown in SEQ ID NO:43; the light chain variable region CDR2 shown in SEQ ID NO:57; and the light chain variable region CDR3 shown in SEQ ID NO:71,

[0241] (b) the heavy chain variable region CDR1 shown in SEQ ID NO:2; the heavy chain variable region CDR2 shown in SEQ ID NO:16; the heavy chain variable region CDR3 shown in SEQ ID NO:30; the light chain variable region CDR1 shown in SEQ ID NO:44; the light chain variable region CDR2 shown in SEQ ID NO:58; and the light chain variable region CDR3 shown in SEQ ID NO:72,

[0242] (c) the heavy chain variable region CDR1 shown in SEQ ID NO:3; the heavy chain variable region CDR2 shown in SEQ ID NO:17; the heavy chain variable region CDR3 shown in SEQ ID NO:31; the light chain variable region CDR1 shown in SEQ ID NO:45; the light chain variable region CDR2 shown in SEQ ID NO:59; and the light chain variable region CDR3 shown in SEQ ID NO:73,

[0243] (d) the heavy chain variable region CDR1 shown in SEQ ID NO:4; the heavy chain variable region CDR2 shown in SEQ ID NO:18; the heavy chain variable region CDR3 shown in SEQ ID NO:32; the light chain variable region CDR1 shown in SEQ ID NO:46; the light chain variable region CDR2 shown in SEQ ID NO:60; and the light chain variable region CDR3 shown in SEQ ID NO:74,

[0244] (e) the heavy chain variable region CDR1 shown in SEQ ID NO:5; the heavy chain variable region CDR2 shown in SEQ ID NO:19; the heavy chain variable region CDR3 shown in SEQ ID NO:33; the light chain variable region CDR1 shown in SEQ ID NO:47; the light chain variable region CDR2 shown in SEQ ID NO:61; and the light chain variable region CDR3 shown in SEQ ID NO:75,

[0245] (f) the heavy chain variable region CDR1 shown in SEQ ID NO:6; the heavy chain variable region CDR2 shown in SEQ ID NO:20; the heavy chain variable region CDR3 shown in SEQ ID NO:34; the light chain variable region CDR1 shown in SEQ ID NO:48; the light chain variable region CDR2 shown in SEQ ID NO:62; and the light chain variable region CDR3 shown in SEQ ID NO:76,

[0246] (g) the heavy chain variable region CDR1 shown in SEQ ID NO:7; the heavy chain variable region CDR2 shown in SEQ ID NO:21; the heavy chain variable region CDR3 shown in SEQ ID NO:35; the light chain variable region CDR1 shown in SEQ ID NO:49; the light chain variable region CDR2 shown in SEQ ID NO:63; and the light chain variable region CDR3 shown in SEQ ID NO:77,

[0247] (h) the heavy chain variable region CDR1 shown in SEQ ID NO:8; the heavy chain variable region CDR2 shown in SEQ ID NO:22; the heavy chain variable region CDR3 shown in SEQ ID NO:36; the light chain variable region CDR1 shown in SEQ ID NO:50; the light chain variable region CDR2 shown in SEQ ID NO:64; and the light chain variable region CDR3 shown in SEQ ID NO:78,

[0248] (i) the heavy chain variable region CDR1 shown in SEQ ID NO:9; the heavy chain variable region CDR2 shown in SEQ ID NO:23; the heavy chain variable region CDR3 shown in SEQ ID NO:37; the light chain variable region CDR1 shown in SEQ ID NO:51; the light chain variable region CDR2 shown in SEQ ID NO:65; and the light chain variable region CDR3 shown in SEQ ID NO:79,

[0249] (j) the heavy chain variable region CDR1 shown in SEQ ID NO:10; the heavy chain variable region CDR2 shown in SEQ ID NO:24; the heavy chain variable region CDR3 shown in SEQ ID NO:38; the light chain variable region CDR1 shown in SEQ ID NO:52; the light chain variable region CDR2 shown in SEQ ID NO:66; and the light chain variable region CDR3 shown in SEQ ID NO:80,

[0250] (k) the heavy chain variable region CDR1 shown in SEQ ID NO:11; the heavy chain variable region CDR2 shown in SEQ ID NO:25; the heavy chain variable region CDR3 shown in SEQ ID NO:39; the light chain variable region CDR1 shown in SEQ ID NO:53; the light chain variable region CDR2 shown in SEQ ID NO:67; and the light chain variable region CDR3 shown in SEQ ID NO:81,

[0251] (l) the heavy chain variable region CDR1 shown in SEQ ID NO:12; the heavy chain variable region CDR2 shown in SEQ ID NO:26; the heavy chain variable region CDR3 shown in SEQ ID NO:40; the light chain variable region CDR1 shown in SEQ ID NO:54; the light chain variable region CDR2 shown in SEQ ID NO:68; and the light chain variable region CDR3 shown in SEQ ID NO:82,

[0252] (m) the heavy chain variable region CDR1 shown in SEQ ID NO:13; the heavy chain variable region CDR2 shown in SEQ ID NO:27; the heavy chain variable region CDR3 shown in SEQ ID NO:41; the light chain variable region CDR1 shown in SEQ ID NO:55; the light chain variable region CDR2 shown in SEQ ID NO:69; and the light chain variable region CDR3 shown in SEQ ID NO:83, or

[0253] (n) the heavy chain variable region CDR1 shown in SEQ ID NO:14; the heavy chain variable region CDR2 shown in SEQ ID NO:28; the heavy chain variable region CDR3 shown in SEQ ID NO:42; the light chain variable region CDR1 shown in SEQ ID NO:56; the light chain variable region CDR2 shown in SEQ ID NO:70; and the light chain variable region CDR3 shown in SEQ ID NO:84.

[0254] 24. An ActRII receptor antagonist for use according to any one of aspects 16-23, wherein the antibody comprises a full length heavy chain amino acid sequence having at least 95% sequence identity to at least one sequence selected from the group consisting of SEQ ID NO: 146 to SEQ ID NO: 150 and SEQ ID NO: 156 to SEQ ID NO: 160.

[0255] 25. An ActRII receptor antagonist for use according to any one of aspects 16-24, wherein the antibody comprises a full length light chain amino acid sequence having at least 95% sequence identity to at least one sequence selected from the group consisting of SEQ ID NO: 141 to SEQ ID NO: 145 and SEQ ID NO: 151 to SEQ ID NO: 155.

[0256] 26. An ActRII receptor antagonist for use according to any one of aspects 16 to 25, wherein the antibody comprises:

[0257] (a) the variable heavy chain sequence shown in SEQ ID NO:99 and the variable light chain sequence shown in SEQ ID NO:85;

[0258] (b) the variable heavy chain sequence shown in SEQ ID NO: 100 and the variable light chain sequence shown in SEQ ID NO: 86;

[0259] (c) the variable heavy chain sequence shown in SEQ ID NO: 101 and the variable light chain sequence shown in SEQ ID NO: 87;

[0260] (d) the variable heavy chain sequence shown in SEQ ID NO: 102 and the variable light chain sequence shown in SEQ ID NO: 88;

[0261] (e) the variable heavy chain sequence shown in SEQ ID NO: 103 and the variable light chain sequence shown in SEQ ID NO: 89;

[0262] (f) the variable heavy chain sequence shown in SEQ ID NO: 104 and the variable light chain sequence shown in SEQ ID NO: 90;

[0263] (g) the variable heavy chain sequence shown in SEQ ID NO: 105 and the variable light chain sequence shown in SEQ ID NO: 91;

[0264] (h) the variable heavy chain sequence shown in SEQ ID NO: 106 and the variable light chain sequence shown in SEQ ID NO: 92;

[0265] (i) the variable heavy chain sequence shown in SEQ ID NO: 107 and the variable light chain sequence shown in SEQ ID NO: 93;

[0266] (j) the variable heavy chain sequence shown in SEQ ID NO: 108 and the variable light chain sequence shown in SEQ ID NO: 94;

[0267] (k) the variable heavy chain sequence shown in SEQ ID NO: 109 and the variable light chain sequence shown in SEQ ID NO: 95;

[0268] (l) the variable heavy chain sequence shown in SEQ ID NO: 110 and the variable light chain sequence shown in SEQ ID NO: 96;

[0269] (m) the variable heavy chain sequence shown in SEQ ID NO: 111 and the variable light chain sequence shown in SEQ ID NO: 97; or

[0270] (n) the variable heavy chain sequence shown in SEQ ID NO:112 and the variable light chain sequence shown in SEQ ID NO:98.

[0271] 27. An ActRII receptor antagonist for use according to any one of aspects 16 to 26, wherein the antibody comprises:

[0272] (a) the heavy chain sequence shown in SEQ ID NO: 146 and the light chain sequence shown in SEQ ID NO: 141;

[0273] (b) the heavy chain sequence shown in SEQ ID NO: 147 and the light chain sequence shown in SEQ ID NO: 142;

[0274] (c) the heavy chain sequence shown in SEQ ID NO: 148 and the light chain sequence shown in SEQ ID NO: 143;

[0275] (d) the heavy chain sequence shown in SEQ ID NO: 149 and the light chain sequence shown in SEQ ID NO: 144;

[0276] (e) the heavy chain sequence shown in SEQ ID NO: 150 and the light chain sequence shown in SEQ ID NO: 145;

[0277] (f) the heavy chain sequence shown in SEQ ID NO: 156 and the light chain sequence shown in SEQ ID NO: 151;

[0278] (g) the heavy chain sequence shown in SEQ ID NO: 157 and the light chain sequence shown in SEQ ID NO: 152;

[0279] (h) the heavy chain sequence shown in SEQ ID NO: 158 and the light chain sequence shown in SEQ ID NO: 153;

[0280] (i) the heavy chain sequence shown in SEQ ID NO: 159 and the light chain sequence shown in SEQ ID NO: 154; or

[0281] (j) the heavy chain sequence shown in SEQ ID NO:160 and the light chain sequence shown in SEQ ID NO:155.

[0282] 28. An ActRII receptor antagonist for use according to any one of aspects 16 to 27, wherein the antibody comprised in the composition cross-blocks binding of at least one antibody according to aspect 27 to ActRIIB or is cross-blocked by at least one antibody according to aspect 27 from binding to ActRIIB.

[0283] 29. An ActRII receptor antagonist for use according to any one of aspects 16 to 28, wherein the antibody comprised in the composition has altered effector function by mutation of the Fc region.

[0284] 30. An ActRII receptor antagonist for use according to any one of aspects 16-29, wherein the antibody comprised in the composition binds to an epitope recognized by an antibody listed in aspects 26-27.

[0285] 31. An ActRII receptor antagonist for use according to any one of aspects 16 to 30, wherein the antibody is encoded by pBW522 (DSM22873) or pBW524 (DSM22874).

[0286] 32. Bimagrumab for use in the treatment and / or prevention of heart failure or for the treatment of structural and / or functional cardiac abnormalities associated with a condition selected from the group consisting of valvular heart disease, hypertension, coronary artery disease, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic and infectious agents, and other forms of hereditary or idiopathic cardiomyopathy, wherein bimagrumab is administered intravenously once every 4 weeks at a dose of about 3-10 mg / kg body weight.

[0287] 33. A composition comprising 150 mg / ml of bimagrumab for use in the treatment and / or prevention of heart failure or for the treatment of structural and / or functional cardiac abnormalities associated with a condition selected from the group consisting of valvular heart disease, hypertension, coronary artery disease, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, toxic and infectious agents, and other forms of inherited or idiopathic cardiomyopathy.

[0288] 34. A single dosage form comprising 150 mg / ml of bimagrumab.

[0289] In another embodiment, a single dosage form, i.e. a vial, comprises 100-200 mg / ml of bimagrumab, preferably 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 mg / ml of bimagrumab.

[0290] 35. An infusion bag comprising an appropriate amount of bimagrumab from one or more vials diluted with a solution.

[0291] The solution is preferably a glucose solution.

[0292] In some additional embodiments, the ActRII receptor antagonist or anti-ActRII antibody (such as bimagrumab) is administered at a dose of about 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, 10 mg / kg body weight.

[0293] Disclosed herein are ActRII receptor antagonists for use in the preparation of medicaments for treating and / or preventing heart failure and for treating structural and / or functional cardiac abnormalities associated with conditions such as valvular heart disease, hypertension, coronary artery disease, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, and other forms of inherited or idiopathic dilated cardiomyopathy.

[0294] In further embodiments, all aspects disclosed herein may be used in combination with any other aspects.

[0295] Various aspects of the disclosure are described in more detail in the following subsections. Standard assays for evaluating the binding ability of antibodies to ActRII of various species are known in the art, including, for example, ELISA, Western blot, and RIA. The binding affinity of the antibody can also be assessed by standard assays known in the art, such as by Biacore analysis or solution equilibrium titration. Surface plasmon resonance-based techniques (such as Biacore) can determine binding kinetics, which allows calculation of binding affinity.

[0296] Thus, an antibody that "inhibits" one or more of these ActRII functional properties (e.g., biochemical, immunochemical, cellular, physiological or other biological activities, etc.) as determined according to methods known in the art and described herein should be understood to involve a statistically significant reduction in the specific activity relative to that observed in the absence of the antibody (e.g., or when a control antibody of irrelevant specificity is present). Antibodies that inhibit ActRII activity achieve such a statistically significant reduction of at least 10%, at least 50%, 80% or 90% of the measured parameter, and in certain embodiments, antibodies of the present disclosure may inhibit greater than 95%, 98% or 99% of ActRIIB functional activity.

[0297] The ability or extent to which an antibody or other binding agent is able to interfere with the binding of another antibody or binding molecule to ActRII, and therefore whether it can be said to cross-block according to the present disclosure, can be determined using standard competitive binding assays. One suitable assay involves the use of Biacore technology (e.g., by using a BIAcore instrument (Biacore, Uppsala, Sweden)), which can use surface plasmon resonance technology to measure the extent of interaction. Another assay for measuring cross-blocking uses an ELISA-based method. Another assay uses FACS analysis, in which various antibodies are tested for competition for binding to ActRIIB expressing cells.

[0298] According to the present disclosure, the cross-blocking antibodies or other binding agents according to the present disclosure bind to ActRII in the described BIAcore cross-blocking assay such that the recorded binding of the combination (mixture) of antibodies or binding agents is between 80% and 0.1% (e.g., 80% to 4%) of the maximal theoretical binding of the combination of the two antibodies or binding agents, specifically between 75% and 0.1% (e.g., 75% to 4%) of the maximal theoretical binding of the combination of the two antibodies or binding agents, more specifically between 70% and 0.1% (e.g., 70% to 4%) of the maximal theoretical binding of the combination of the two antibodies or binding agents, more specifically between 65% and 0.1% (e.g., 65% to 4%) of the maximal theoretical binding (as defined above) of the combination of the two antibodies or binding agents.

[0299] If the test antibody is capable of causing a reduction in the binding of the anti-ActRII antibody to ActRIIB by between 60% and 100%, specifically between 70% and 100%, more specifically between 80% and 100%, when compared to a positive control well (i.e., the same anti-ActRIIB antibody and ActRIIB, but without the "test" cross-blocking antibody), the antibody is defined as cross-blocking the anti-ActRIIB antibody of the present disclosure in an ELISA assay. Examples of cross-blocking antibodies cited herein are MOR08159 and MOR08213 (disclosed in WO2010 / 125003). Accordingly, the present disclosure provides a composition comprising an antibody that cross-blocks the binding of MOR08159 or MOR08213 to ActRIIB.

[0300] Recombinant Antibodies

[0301] The antibodies included in the compositions used in the present disclosure, e.g., ActRII antagonist antibodies such as bimagrumab, include isolated and structurally characterized human recombinant antibodies as described herein. H The amino acid sequences are shown in SEQ ID NO: 99 to SEQ ID NO: 112. The V LThe amino acid sequences are shown in SEQ ID NO:85 to SEQ ID NO:98, respectively. Examples of preferred full-length heavy chain amino acid sequences of the antibodies included in the compositions of the present invention are shown in SEQ ID NO:146 to SEQ ID NO:150 and SEQ ID NO:156 to SEQ ID NO:160. Examples of preferred full-length light chain amino acid sequences of the antibodies included in the compositions of the present invention are shown in SEQ ID NO:141 to SEQ ID NO:145 and SEQ ID NO:151 to SEQ ID NO:155, respectively. Other antibodies included in the compositions of the present invention include amino acids that have been mutated by amino acid deletion, insertion or substitution, but have at least 60%, 70%, 80%, 90%, 95%, 97% or 99% identity in the CDR regions to the CDR regions described in the above sequences. In some embodiments, it includes a mutant amino acid sequence in which no more than 1, 2, 3, 4 or 5 amino acids in the CDR region have been mutated by amino acid deletion, insertion or substitution when compared to the CDR region described by the above sequence.

[0302] In addition, variable heavy chain parent nucleotide sequences are shown in SEQ ID NO: 127 to SEQ ID NO: 140. Variable light chain parent nucleotide sequences are shown in SEQ ID NO: 113 to SEQ ID NO: 126. Full-length light chain nucleotide sequences optimized for expression in mammalian cells are shown in SEQ ID NO: 161 to SEQ ID NO: 165 and SEQ ID NO: 171 to SEQ ID NO: 175. Full-length heavy chain nucleotide sequences optimized for expression in mammalian cells are shown in SEQ ID NO: 166 to SEQ ID NO: 170 and SEQ ID NO: 176 to SEQ ID NO: 180. Other antibodies included in the compositions of the present invention include mutated amino acids or are encoded by mutated nucleic acids, but the mutated amino acids or mutated nucleic acids have at least 60 or higher (i.e., 80, 90, 95, 97, 99 or higher) percent identity with the above sequences. In some embodiments, the antibody comprises a mutant amino acid sequence, wherein no more than 1, 2, 3, 4, or 5 amino acids in the variable region have been mutated by amino acid deletion, insertion, or substitution when compared to the variable region depicted in the above sequence.

[0303] Since each of these antibodies binds to the same epitope and is descended from the same parent antibody, V H 、V L, full-length light chain, and full-length heavy chain sequences (nucleotide sequences and amino acid sequences) can be "mixed and matched" to generate other anti-ActRIIB binding molecules of the present disclosure. Such "mixed and matched" antibodies can be tested for ActRIIB binding using the above-mentioned binding assays and well-known methods (e.g., ELISA). When these chains are mixed and matched, the binding activity of the antibodies from a particular V H / V L Paired V H The sequence should be replaced by a structurally similar V H Likewise, a full-length heavy chain sequence from a particular full-length heavy chain / full-length light chain pairing should be replaced with a full-length heavy chain sequence of similar structure. H / V L Paired V L The sequence should be replaced by a structurally similar V L Sequence replacement. Likewise, a full-length chain sequence from a particular full-length heavy chain / full-length light chain pairing should be replaced with a structurally similar full-length chain sequence. Thus, in one aspect, the present disclosure provides a composition comprising a recombinant anti-ActRII antibody or antigen-binding region thereof having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99 to SEQ ID NO: 112; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to SEQ ID NO: 98.

[0304] In another aspect, the present disclosure provides a composition comprising:

[0305] (i) an isolated recombinant anti-ActRII antibody having: a full-length heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99 to SEQ ID NO: 112; and a full-length light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to SEQ ID NO: 98, or

[0306] (ii) a functional protein comprising the antigen-binding portion of the isolated recombinant anti-ActRII antibody.

[0307] In another aspect, the present disclosure provides a composition comprising:

[0308] (i) an isolated recombinant anti-ActRII antibody having: a full-length heavy chain encoded by a nucleotide sequence that has been optimized for expression in a mammalian cell, the nucleotide sequence being selected from the group consisting of SEQ ID NO: 127 to SEQ ID NO: 140; and a full-length light chain encoded by a nucleotide sequence that has been optimized for expression in a mammalian cell, the nucleotide sequence being selected from the group consisting of SEQ ID NO: 113 to SEQ ID NO: 126, or

[0309] (ii) a functional protein comprising the antigen-binding portion of the isolated recombinant anti-ActRII antibody.

[0310] The V of the antibody contained in the composition of the present invention H Examples of amino acid sequences of CDR1 are shown in SEQ ID NO: 1 to SEQ ID NO: 14. H The amino acid sequences of CDR2 are shown in SEQ ID NO: 15 to SEQ ID NO: 28. H The amino acid sequences of CDR3 are shown in SEQ ID NO: 29 to SEQ ID NO: 42. L The amino acid sequences of CDR1 are shown in SEQ ID NO: 43 to SEQ ID NO: 56. L The amino acid sequences of CDR2 are shown in SEQ ID NO: 57 to SEQ ID NO: 70. LThe amino acid sequence of CDR3 is shown in SEQ ID NO:71 to SEQ ID NO:84. The Kabat system is used to describe the CDR region (Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). An alternative method for determining the CDR region uses a method designed by Chothia (Chothia et al., 251989, Nature, 342:877-883). The Chothia definition is based on the position of the structural loop region. However, due to changes in the numbering system used by Chothia (see, for example, http: / / www.biochem.ucl.ac.uk / ~martin / abs / Generallnfo.html and http: / / www.bioinf.org.uk / abs / ), this system is now less commonly used. There are other systems for defining CDRs, and these systems are also mentioned in these two websites.

[0311] Given that each of these antibodies can bind to ActRII and that the antigen-binding specificity is primarily provided by the CDR1, CDR2, and CDR3 regions, V H CDR1, V H CDR2 and V H CDR3 sequence and V L CDR1, V L CDR2 and V L CDR3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, with each antibody containing V H CDR1, V H CDR2 and V H CDR3 and V L CDR1, V L CDR2 and V L CDR3, thereby generating other anti-ActRII binding molecules of the present disclosure. Such "mixed and matched" antibodies can be tested for ActRIIB binding using the binding assays (e.g., ELISA) described above and in the Examples. H When CDR sequences are mixed and matched, H The CDR1, CDR2 and / or CDR3 sequences of the sequence should be replaced by CDR sequences with similar structures. L When CDR sequences are mixed and matched,L The CDR1, CDR2 and / or CDR3 sequences of the sequences should be replaced by structurally similar CDR sequences. It is obvious to the skilled person that by replacing one or more V sequences with structurally similar sequences from the CDR sequences of the monoclonal antibodies shown herein, H and / or V L CDR region sequences can generate new V H Sequence and V L sequence.

[0312] The anti-ActRII antibody or antigen-binding region thereof contained in the disclosed composition has: a heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14; a heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 28; a heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29 to SEQ ID NO: 42; a light chain variable region CDR1, wherein the light chain variable region CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 43 to SEQ ID NO: 56; a light chain variable region CDR2, wherein the light chain variable region CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 70; and a light chain variable region CDR3, wherein the light chain variable region CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 71 to SEQ ID NO: 84.

[0313] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:1; the heavy chain variable region CDR2 shown in SEQ ID NO:15; the heavy chain variable region CDR3 shown in SEQ ID NO:29; the light chain variable region CDR1 shown in SEQ ID NO:43; the light chain variable region CDR2 shown in SEQ ID NO:57; and the light chain variable region CDR3 shown in SEQ ID NO:71.

[0314] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:2; the heavy chain variable region CDR2 shown in SEQ ID NO:16; the heavy chain variable region CDR3 shown in SEQ ID NO:30; the light chain variable region CDR1 shown in SEQ ID NO:44; the light chain variable region CDR2 shown in SEQ ID NO:58; and the light chain variable region CDR3 shown in SEQ ID NO:72.

[0315] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:3; the heavy chain variable region CDR2 shown in SEQ ID NO:17; the heavy chain variable region CDR3 shown in SEQ ID NO:31; the light chain variable region CDR1 shown in SEQ ID NO:45; the light chain variable region CDR2 shown in SEQ ID NO:59; and the light chain variable region CDR3 shown in SEQ ID NO:73.

[0316] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:4; the heavy chain variable region CDR2 shown in SEQ ID NO:18; the heavy chain variable region CDR3 shown in SEQ ID NO:32; the light chain variable region CDR1 shown in SEQ ID NO:46; the light chain variable region CDR2 shown in SEQ ID NO:60; and the light chain variable region CDR3 shown in SEQ ID NO:74.

[0317] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:5; the heavy chain variable region CDR2 shown in SEQ ID NO:19; the heavy chain variable region CDR3 shown in SEQ ID NO:33; the light chain variable region CDR1 shown in SEQ ID NO:47; the light chain variable region CDR2 shown in SEQ ID NO:61; and the light chain variable region CDR3 shown in SEQ ID NO:75.

[0318] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:6; the heavy chain variable region CDR2 shown in SEQ ID NO:20; the heavy chain variable region CDR3 shown in SEQ ID NO:34; the light chain variable region CDR1 shown in SEQ ID NO:48; the light chain variable region CDR2 shown in SEQ ID NO:62; and the light chain variable region CDR3 shown in SEQ ID NO:76.

[0319] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:7; the heavy chain variable region CDR2 shown in SEQ ID NO:21; the heavy chain variable region CDR3 shown in SEQ ID NO:35; the light chain variable region CDR1 shown in SEQ ID NO:49; the light chain variable region CDR2 shown in SEQ ID NO:63; and the light chain variable region CDR3 shown in SEQ ID NO:77.

[0320] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:8; the heavy chain variable region CDR2 shown in SEQ ID NO:22; the heavy chain variable region CDR3 shown in SEQ ID NO:36; the light chain variable region CDR1 shown in SEQ ID NO:50; the light chain variable region CDR2 shown in SEQ ID NO:64; and the light chain variable region CDR3 shown in SEQ ID NO:78.

[0321] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:9; the heavy chain variable region CDR2 shown in SEQ ID NO:23; the heavy chain variable region CDR3 shown in SEQ ID NO:37; the light chain variable region CDR1 shown in SEQ ID NO:51; the light chain variable region CDR2 shown in SEQ ID NO:65; and the light chain variable region CDR3 shown in SEQ ID NO:79.

[0322] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:10; the heavy chain variable region CDR2 shown in SEQ ID NO:24; the heavy chain variable region CDR3 shown in SEQ ID NO:38; the light chain variable region CDR1 shown in SEQ ID NO:52; the light chain variable region CDR2 shown in SEQ ID NO:66; and the light chain variable region CDR3 shown in SEQ ID NO:80.

[0323] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:11; the heavy chain variable region CDR2 shown in SEQ ID NO:25; the heavy chain variable region CDR3 shown in SEQ ID NO:39; the light chain variable region CDR1 shown in SEQ ID NO:53; the light chain variable region CDR2 shown in SEQ ID NO:67; and the light chain variable region CDR3 shown in SEQ ID NO:81.

[0324] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:12; the heavy chain variable region CDR2 shown in SEQ ID NO:26; the heavy chain variable region CDR3 shown in SEQ ID NO:40; the light chain variable region CDR1 shown in SEQ ID NO:54; the light chain variable region CDR2 shown in SEQ ID NO:68; and the light chain variable region CDR3 shown in SEQ ID NO:82.

[0325] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:13; the heavy chain variable region CDR2 shown in SEQ ID NO:27; the heavy chain variable region CDR3 shown in SEQ ID NO:41; the light chain variable region CDR1 shown in SEQ ID NO:55; the light chain variable region CDR2 shown in SEQ ID NO:69; and the light chain variable region CDR3 shown in SEQ ID NO:83.

[0326] In one embodiment, the antibody contained in the composition of the present invention comprises: the heavy chain variable region CDR1 shown in SEQ ID NO:14; the heavy chain variable region CDR2 shown in SEQ ID NO:28; the heavy chain variable region CDR3 shown in SEQ ID NO:42; the light chain variable region CDR1 shown in SEQ ID NO:56; the light chain variable region CDR2 shown in SEQ ID NO:70; and the light chain variable region CDR3 shown in SEQ ID NO:84.

[0327] In one embodiment, the present disclosure provides a composition, the composition comprising an antibody, the antibody comprising: (a) a variable heavy chain sequence shown in SEQ ID NO: 85 and a variable light chain sequence shown in SEQ ID NO: 99; (b) a variable heavy chain sequence shown in SEQ ID NO: 86 and a variable light chain sequence shown in SEQ ID NO: 100; (c) a variable heavy chain sequence shown in SEQ ID NO: 87 and a variable light chain sequence shown in SEQ ID NO: 101; (d) a variable heavy chain sequence shown in SEQ ID NO: 88 and a variable light chain sequence shown in SEQ ID NO: 102; (e) a variable heavy chain sequence shown in SEQ ID NO: 89 and a variable light chain sequence shown in SEQ ID NO: 103; (f) a variable heavy chain sequence shown in SEQ ID NO: 90 and a variable light chain sequence shown in SEQ ID NO: 104; (g) a variable heavy chain sequence shown in SEQ ID NO: 91 and a variable light chain sequence shown in SEQ ID NO: 105; (h) a variable heavy chain sequence shown in SEQ ID NO: 92 and a variable light chain sequence shown in SEQ ID NO: 106; (i) a variable heavy chain sequence shown in SEQ ID NO: 93 and a variable light chain sequence shown in SEQ ID NO: 107; NO:93 and the variable light chain sequence shown in SEQ ID NO:107; (j) the variable heavy chain sequence shown in SEQ ID NO:94 and the variable light chain sequence shown in SEQ ID NO:108; (k) the variable heavy chain sequence shown in SEQ ID NO:95 and the variable light chain sequence shown in SEQ ID NO:109; (l) the variable heavy chain sequence shown in SEQ ID NO:96 and the variable light chain sequence shown in SEQ ID NO:110; (m) the variable heavy chain sequence shown in SEQ ID NO:97 and the variable light chain sequence shown in SEQ ID NO:111; or (n) the variable heavy chain sequence shown in SEQ ID NO:98 and the variable light chain sequence shown in SEQ ID NO:112.

[0328] In one embodiment, the present disclosure provides a composition, the composition comprising an antibody, the antibody comprising: (a) a heavy chain sequence shown in SEQ ID NO: 146 and a light chain sequence shown in SEQ ID NO: 141; (b) a heavy chain sequence shown in SEQ ID NO: 147 and a light chain sequence shown in SEQ ID NO: 142; (c) a heavy chain sequence shown in SEQ ID NO: 148 and a light chain sequence shown in SEQ ID NO: 143; (d) a heavy chain sequence shown in SEQ ID NO: 149 and a light chain sequence shown in SEQ ID NO: 144; (e) a heavy chain sequence shown in SEQ ID NO: 150 and a light chain sequence shown in SEQ ID NO: 145; (f) a heavy chain sequence shown in SEQ ID NO: 156 and a light chain sequence shown in SEQ ID NO: 151; (g) a heavy chain sequence shown in SEQ ID NO: 157 and a light chain sequence shown in SEQ ID NO: 152; (h) a heavy chain sequence shown in SEQ ID NO: 158 and a light chain sequence shown in SEQ ID NO: 153; (i) The heavy chain sequence shown in SEQ ID NO:159 and the light chain sequence shown in SEQ ID NO:154; or (j) the heavy chain sequence shown in SEQ ID NO:160 and the light chain sequence shown in SEQ ID NO:155.

[0329] As used herein, a human antibody comprises a heavy chain variable region or a light chain variable region or a full-length heavy chain or a full-length light chain, and if the variable region or full-length chain of the antibody is obtained from a system using human germline immunoglobulin genes, the heavy chain variable region or light chain variable region or full-length heavy chain or full-length light chain is a "product" or "derived from" a specific germline sequence. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with an antigen of interest, or screening a human immunoglobulin gene library displayed on phage with an antigen of interest. Thus, by comparing the amino acid sequence of a human antibody with the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is closest in sequence to the sequence of the human antibody (i.e., the greatest % identity), it can be identified as a human antibody that is a "product" or "derived from" a human germline immunoglobulin sequence. Human antibodies that are the "product" or "derived from" a specific human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, due to, for example, naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, the amino acid sequence of the selected human antibody is usually at least 90% identical to the amino acid sequence encoded by the human germline immunoglobulin gene, and contains amino acid residues that identify the human antibody as human when compared to the germline immunoglobulin amino acid sequence of other species (e.g., mouse germline sequence). In some cases, the amino acid sequence of the human antibody may be at least 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a specific human germline sequence will show no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, a human antibody may show no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0330] In one embodiment, the antibody comprised in the composition of the present disclosure is the antibody encoded by pBW522 or pBW524 (deposited on August 18, 2009 at DSMZ, Inhoffenstr. 7B, D-38124 Braunschweig, Germany under the deposit numbers DSM22873 and DSM22874, respectively).

[0331] Homologous antibody

[0332] In another embodiment, the antibody contained in the composition of the present invention has a full-length heavy chain amino acid sequence and a full-length light chain amino acid sequence; a full-length heavy chain nucleotide sequence and a full-length light chain nucleotide sequence, a variable region heavy chain amino acid sequence and a variable region light chain amino acid sequence that are homologous to the amino acid sequence and nucleotide sequence of the antibodies described herein, or a variable region heavy chain nucleotide sequence and a variable region light chain nucleotide sequence, and wherein the antibody retains the desired functional properties of the anti-ActRIIB antibodies of the present disclosure.

[0333] For example, the present disclosure provides a composition comprising an isolated recombinant anti-ActRIIB antibody (or a functional protein comprising an antigen-binding portion of an isolated recombinant anti-ActRIIB antibody), wherein the isolated recombinant anti-ActRIIB antibody (or a functional protein comprising an antigen-binding portion of an isolated recombinant anti-ActRIIB antibody) comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence that is at least 80%, or at least 90% (preferably at least 95%, 97% or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 99 to SEQ ID NO: 112; the light chain variable region comprises an amino acid sequence that is at least 80%, or at least 90% (preferably at least 95%, 97% or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to SEQ ID NO:98 is at least 80%, or at least 90% (preferably at least 95%, 97% or 99%) identical to the amino acid sequence of the group consisting of SEQ ID NO:98; or, the composition comprises a recombinant anti-ActRIIB antibody (or a functional protein comprising the antigen-binding portion of a recombinant anti-ActRIIB antibody), wherein the recombinant anti-ActRIIB antibody (or a functional protein comprising the antigen-binding portion of a recombinant anti-ActRIIB antibody) comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises no more than 5 amino acids, or no more than 4 amino acids, or no more than 3 amino acids, or no more than 2 amino acids, or no more than 1 amino acid change compared to the amino acid sequence selected from the group consisting of SEQ ID NO:99 to SEQ ID NO:112; the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:85 to SEQ ID NO: The amino acid sequence of the group consisting of IDNO: 98 has no more than 5 amino acids, or no more than 4 amino acids, or no more than 3 amino acids, or no more than 2 amino acids, or no more than 1 amino acid change compared to the amino acid sequence of the group consisting of IDNO: 98, and the antibody exhibits at least one of the following functional properties: (i) it inhibits myostatin binding in vitro or in vivo, (ii) reduces the inhibition of muscle differentiation through the Smad-dependent pathway, and / or (iii) does not induce hematological changes, especially RBCs. In this article, the term "change / change" refers to insertion, deletion and / or substitution.

[0334] In another example, the present disclosure provides a composition comprising an isolated recombinant anti-ActRII antibody (or a functional protein comprising an antigen-binding portion of an isolated recombinant anti-ActRII antibody), wherein the isolated recombinant anti-ActRII antibody (or a functional protein comprising an antigen-binding portion of an isolated recombinant anti-ActRII antibody) comprises a full-length heavy chain and a full-length light chain, wherein: the full-length heavy chain comprises an amino acid sequence that is at least 80%, or at least 90% (preferably at least 95%, 97% or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 146 to SEQ ID NO: 150 and SEQ ID NO: 156 to SEQ ID NO: 160; the full-length light chain comprises an amino acid sequence that is at least 80%, or at least 90% (preferably at least 95%, 97% or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 141 to SEQ ID NO: 145 and SEQ ID NO: 151 to SEQ ID NO: 155; or, the composition comprises a recombinant anti-ActRII antibody (or a functional protein comprising an antigen-binding portion of a recombinant anti-ActRII antibody), wherein the recombinant anti-ActRII antibody (or a functional protein comprising an antigen-binding portion of a recombinant anti-AcRII antibody) comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises no more than 5 amino acids, or no more than 4 amino acids, or no more than 3 amino acids, or no more than 2 amino acids, or no more than 1 amino acid change compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 146 to SEQ ID NO: 150 and SEQ ID NO: 156 to SEQ ID NO: 160; the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 141 to SEQ ID NO: 145 and SEQ ID NO: 151 to SEQ ID NO: The amino acid sequence of the group consisting of NO: 155 has no more than 5 amino acids, or no more than 4 amino acids, or no more than 3 amino acids, or no more than 2 amino acids, or no more than 1 amino acid change compared to the amino acid sequence of the group consisting of NO: 155, and the antibody exhibits at least one of the following functional properties: (i) it inhibits myostatin binding in vitro or in vivo, (ii) reduces the inhibition of muscle differentiation through the Smad-dependent pathway, and / or (iii) does not induce hematological changes, especially RBCs. Preferably, such antibodies bind to the ligand binding domain of ActRIIB and / or ActRIIA. In this article, the term "change / variation" refers to insertion, deletion and / or substitution.

[0335] In another example, the present disclosure provides a composition comprising an isolated recombinant anti-ActRII antibody (or a functional protein comprising an antigen-binding portion of an isolated recombinant anti-ActRII antibody), wherein the isolated recombinant anti-ActRII antibody (or a functional protein comprising an antigen-binding portion of an isolated recombinant anti-AcRII antibody) comprises a full-length heavy chain and a full-length light chain, wherein: the full-length heavy chain is encoded by a nucleotide sequence that is at least 80%, or at least 90% (preferably at least 95%, 97% or 99%) identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 166 to SEQ ID NO: 170 and SEQ ID NO: 176 to SEQ ID NO: 180; the full-length light chain is encoded by a nucleotide sequence that is at least 80%, or at least 90% (preferably at least 95%, 97% or 99%) identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 161 to SEQ ID NO: 165 and SEQ ID NO: 171 to SEQ ID NO: 175; or, the composition comprises a recombinant anti-ActRIIB antibody (or a functional protein comprising an antigen-binding portion of a recombinant anti-ActRIIB antibody), wherein the recombinant anti-ActRIIB antibody (or a functional protein comprising an antigen-binding portion of a recombinant anti-ActRIIB antibody) comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises no more than 5 amino acids, or no more than 4 amino acids, or no more than 3 amino acids, or no more than 2 amino acids, or no more than 1 amino acid change compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 166 to SEQ ID NO: 170 and SEQ ID NO: 176 to SEQ ID NO: 180; the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 161 to SEQ ID NO: 165 and SEQ ID NO: 171 to SEQ ID NO: The amino acid sequence of the group consisting of NO: 175 has no more than 5 amino acids, or no more than 4 amino acids, or no more than 3 amino acids, or no more than 2 amino acids, or no more than 1 amino acid change compared to the amino acid sequence of NO: 175, and the antibody exhibits at least one of the following functional properties: (i) it inhibits myostatin binding in vitro or in vivo, (ii) reduces the inhibition of muscle differentiation through the Smad-dependent pathway, and / or (iii) does not induce hematological changes, especially RBCs. Preferably, such antibodies bind to the ligand binding domain of ActRIIB. In this article, the term "change / variation" refers to insertion, deletion and / or substitution.

[0336] In various embodiments, the antibodies included in the compositions of the invention may exhibit one or more, two or more, or three of the functional properties discussed above. The antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody. Preferably, the antibody is a fully human IgG1 antibody.

[0337] In other embodiments, V H and / or V L The amino acid sequence may be at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the above sequences. In other embodiments, except for the amino acid substitution at no more than 1, 2, 3, 4 or 5 amino acid positions, V H and / or V L The amino acid sequences may be identical. Having V sequences identical to those of SEQ ID NO: 99 to SEQ ID NO: 112 and SEQ ID NO: 85 to SEQ ID NO: 98, respectively. H and V L The V region has high (i.e., 80% or greater) identity H and V L Antibodies to the regions can be obtained by mutagenizing the nucleic acid molecules SEQ ID NO: 127 to SEQ ID NO: 140 and SEQ ID NO: 113 to SEQ ID NO: 126, respectively (e.g., site-directed mutagenesis or PCR-mediated mutagenesis), and then using the functional assays described herein to test whether the encoded altered antibodies retain function (i.e., the above-mentioned functions).

[0338] In other embodiments, the full-length heavy chain and / or full-length light chain amino acid sequence may be at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the above sequences, or may be identical to the above sequences except for amino acid changes in no more than 1, 2, 3, 4 or 5 amino acid positions. Antibodies having full-length heavy chains and full-length light chains that are highly identical (i.e., at least 80% or more) to the full-length heavy chains shown in any one of SEQ ID NO:146 to SEQ ID NO:150 and SEQ ID NO:156 to SEQ ID NO:160, and the full-length light chains shown in any one of SEQ ID NO:141 to SEQ ID NO:145 and SEQ ID NO:151 to SEQ ID NO:155, respectively, can be obtained by subjecting the nucleic acid molecules SEQ ID NO:166 to SEQ ID NO:170 and SEQ ID NO:176 to SEQ ID NO:180, and SEQ ID NO:161 to SEQ ID NO:165 and SEQ ID NO:171 to SEQ ID NO:175, respectively, to mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis), and then testing whether the encoded altered antibodies retain function (i.e., the above-mentioned functions) using the functional assays described herein.

[0339] In other embodiments, the full-length heavy chain and / or full-length light chain nucleotide sequences may be at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the above sequences.

[0340] In other embodiments, the variable region of the heavy and / or light chain nucleotide sequence may be at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the above sequences, or may be identical to the above sequences except for amino acid changes in no more than 1, 2, 3, 4 or 5 amino acid positions.

[0341] As used herein, the percent identity between two sequences is a function of the number of identical positions that the sequences share (i.e., the number of % identity=identical positions / position total number × 100), taking into account the number of spaces that need to be introduced to achieve the best alignment of the two sequences and the length of each space. As described below, a mathematical algorithm can be used to complete the comparison of sequences and the determination of the percent identity between two sequences.

[0342] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17, 1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48: 444-453, 1970) algorithm, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0343] Antibodies with conservative modifications

[0344] In certain embodiments, the antibodies contained in the compositions of the present invention have a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences have a specified amino acid sequence based on the antibodies described herein, or a variant sequence thereof comprising 1, 2, 3, 4 or 5 amino acid changes or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-ActRIIB antibodies of the present disclosure. Thus, the present disclosure provides a composition comprising an isolated recombinant anti-ActRIIB antibody or a functional protein comprising an antigen-binding portion of an isolated recombinant anti-ActRIIB antibody, wherein the isolated recombinant anti-ActRIIB antibody or a functional protein comprising an antigen-binding portion of an isolated recombinant anti-ActRIIB antibody consists of a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein: the heavy chain variable region CDR1 amino acid sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 14 or a variant sequence thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the heavy chain variable region CDR2 amino acid sequence is selected from SEQ ID NO: 15 to SEQ ID NO: 28 or a variant sequence thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the heavy chain variable region CDR3 amino acid sequence is selected from SEQ ID NO: 29 to SEQ ID NO:42 or a variant sequence thereof containing 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the light chain variable region CDR1 amino acid sequence is selected from SEQ ID NO:43 to SEQ ID NO:56 or a variant sequence thereof containing 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the light chain variable region CDR2 amino acid sequence is selected from SEQ ID NO:57 to SEQ ID NO:70 or a variant sequence thereof containing 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the light chain variable region CDR3 amino acid sequence is selected from SEQ ID NO:71 to SEQ ID NO:84 or a variant sequence thereof containing 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof. Preferably, the antibody exhibits at least one of the following functional properties: (i) it inhibits myostatin binding in vitro or in vivo, (ii) reduces inhibition of muscle differentiation through a Smad-dependent pathway, and / or (iii) does not induce hematological changes, in particular, no changes in RBC.

[0345] In various embodiments, the antibody may exhibit one or both of the functional properties listed above. Such an antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0346] In other embodiments, the antibodies optimized for expression in mammalian cells included in the compositions of the present invention have a full-length heavy chain sequence and a full-length light chain sequence, wherein one or more of these sequences have a specified amino acid sequence based on the antibodies described herein or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-ActRIIB antibodies of the present disclosure. Thus, the present disclosure provides a composition comprising an isolated monoclonal anti-ActRII antibody, the isolated monoclonal anti-ActRII antibody being optimized for expression in mammalian cells, the isolated monoclonal anti-ActRII antibody consisting of a full-length heavy chain and a full-length light chain, wherein: the full-length heavy chain has an amino acid sequence selected from SEQ ID NO: 146 to SEQ ID NO: 150 and SEQ ID NO: 156 to SEQ ID NO: 160 or a variant sequence thereof comprising 1, 2, 3, 4 or 5 amino acid changes and conservative modifications thereof; and the full-length light chain has an amino acid sequence selected from SEQ ID NO: 141 to SEQ ID NO: 145 and SEQ ID NO: 151 to SEQ ID The invention relates to an amino acid sequence of NO:155 or a variant sequence thereof comprising 1, 2, 3, 4 or 5 amino acid changes and conservative modifications thereof; and the antibody exhibits at least one of the following functional properties: (i) it inhibits myostatin binding in vitro or in vivo, (ii) reduces inhibition of muscle differentiation through a Smad-dependent pathway, and / or (iii) does not induce hematological changes, in particular, no changes in RBCs.

[0347] In various embodiments, the antibody may exhibit one or both of the functional properties listed above. Such an antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0348] As used herein, the term "conservative sequence modification" means an amino acid modification that does not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present disclosure by standard techniques known in the art (such as site-directed mutagenesis and PCR-mediated mutagenesis).

[0349] Conservative amino acid substitution is such an amino acid substitution, wherein the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of an antibody of the present disclosure can be replaced with other amino acid residues from the same side chain family, and the functional assay described herein can be used to test whether the changed antibody retains function.

[0350] Antibodies that bind to the same epitope as the anti-ActRII antibodies included in the disclosed compositions

[0351] In another embodiment, the present disclosure provides a composition comprising an antibody that binds to the same epitope as the various specific anti-ActRII antibodies described herein. All antibodies described in the Examples that are capable of blocking myostatin binding to ActRIIA and ActRIIB bind with high affinity to one of the epitopes in ActRIIA and ActRIIB, the epitope being comprised between amino acids 19-134 of SEQ ID NO: 181.

[0352] Thus, additional antibodies can be identified based on their ability to cross-compete with other antibodies of the present disclosure in a standard ActRIIB binding assay (e.g., competitively inhibit the binding of other antibodies of the present disclosure in a statistically significant manner). The ability of the test antibody to inhibit the binding of the antibody contained in the composition of the present invention to human ActRIIB indicates that the test antibody can compete with the antibody for binding to human ActRIIB; according to non-limiting theory, such an antibody can bind to the same or related (e.g., structurally similar or spatially adjacent) epitopes on human ActRIIB as the antibody it competes with. In a certain embodiment, the antibody that binds to the same epitope on human ActRIIA and ActRIIA as the antibody contained in the composition of the present invention is a human recombinant antibody. Such human recombinant antibodies can be prepared and isolated as described in the Examples. Therefore, the present disclosure provides a composition comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence shown in SEQ ID NO: 85 and a variable light chain sequence shown in SEQ ID NO: 99 and / or competes for binding with an antibody having a variable heavy chain sequence shown in SEQ ID NO: 85 and a variable light chain sequence shown in SEQ ID NO: 99.

[0353] Thus, the present disclosure provides a composition comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:86 and a variable light chain sequence as shown in SEQ ID NO:100.

[0354] Therefore, the present disclosure provides a composition comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO: 87 and a variable light chain sequence as shown in SEQ ID NO: 101. Therefore, the present disclosure provides a composition comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO: 88 and a variable light chain sequence as shown in SEQ ID NO: 102. Therefore, the present disclosure provides a composition comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO: 89 and a variable light chain sequence as shown in SEQ ID NO: 103.

[0355] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:90 and a variable light chain sequence as shown in SEQ ID NO:104.

[0356] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:91 and a variable light chain sequence as shown in SEQ ID NO:105.

[0357] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:92 and a variable light chain sequence as shown in SEQ ID NO:106.

[0358] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:93 and a variable light chain sequence as shown in SEQ ID NO:107.

[0359] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:94 and a variable light chain sequence as shown in SEQ ID NO:108.

[0360] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:95 and a variable light chain sequence as shown in SEQ ID NO:109.

[0361] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:96 and a variable light chain sequence as shown in SEQ ID NO:110.

[0362] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:97 and a variable light chain sequence as shown in SEQ ID NO:111.

[0363] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope recognized by an antibody having a variable heavy chain sequence as shown in SEQ ID NO:98 and a variable light chain sequence as shown in SEQ ID NO:112.

[0364] Following more detailed epitope mapping experiments, the binding regions of the preferred antibodies of the compositions of the invention have been more clearly defined.

[0365] Thus, the present disclosure provides compositions comprising an antibody that binds to an epitope comprising amino acids 78-83 (WLDDFN-SEQ ID NO: 188) of SEQ ID NO: 181. The present disclosure also provides compositions comprising an antibody that binds to an epitope comprising amino acids 76-84 (GCWLDDFNC-SEQ ID NO: 186) of SEQ ID NO: 181.

[0366] The present disclosure also provides compositions comprising an antibody that binds to an epitope comprising amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY—SEQ ID NO: 190).

[0367] The present disclosure also provides compositions comprising an antibody that binds to an epitope comprising amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189). The present disclosure also provides compositions comprising an antibody that binds to an epitope comprising amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187).

[0368] The present disclosure also provides compositions comprising an antibody that binds to an epitope comprising or consisting of amino acids 29-41 of SEQ ID NO: 181 (CIYYNANWELERT-SEQ ID NO: 191).

[0369] The present disclosure also provides compositions comprising an antibody that binds to an epitope comprising or consisting of amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN-SEQ ID NO: 192).

[0370] The present disclosure also provides compositions comprising antibodies that bind to epitopes consisting of these sequences or epitopes comprising a combination of these epitope regions.

[0371] Thus, the present disclosure also provides compositions comprising an antibody that binds to an epitope comprising or consisting of amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR).

[0372] Engineered and modified antibodies

[0373] Antibodies included in the compositions of the present invention may also be used with V H and / or V L Antibodies containing one or more of the sequences are prepared as starting materials to engineer modified antibodies that may have altered properties compared to the starting antibody. Antibodies may be engineered by modifying one or two variable regions (i.e., V H and / or V L ), such as one or more residues in one or more CDR regions and / or or one or more residues in one or more framework regions. Additionally or alternatively, the antibody can be engineered by modifying residues in one or more constant regions, such as to alter the effector functions of the antibody.

[0374] One type of variable region engineering that can be performed is CDR transplantation. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). Therefore, the amino acid sequences within the CDRs are more diverse between antibodies than the sequences outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific naturally occurring antibody can be expressed by constructing an expression vector that includes the CDR sequences from a specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. et al., 1989 Proc. Natl. Acad. Sci. USA 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0375] Accordingly, another embodiment of the present disclosure relates to a composition comprising a monoclonal anti-ActRII antibody, or a functional protein comprising an antigen binding portion of a monoclonal anti-ActRII antibody, wherein the monoclonal anti-ActRII antibody or the functional protein comprising an antigen binding portion of a monoclonal anti-ActRII antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: a CDR1 sequence having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14; a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 28; a CDR3 sequence having an amino acid sequence selected from the group consisting of SEQ ID NO: 29 to SEQ ID NO: 42; and the light chain variable region comprises: a CDR1 sequence having an amino acid sequence selected from the group consisting of SEQ ID NO: 43 to SEQ ID NO: 56; a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 70; and a CDR3 sequence having a group consisting of SEQ ID NO: 71 to SEQ ID NO: 84. Therefore, these antibodies contain the V H CDR sequences and V L CDR sequences, but may contain different framework sequences from these antibodies.

[0376] Such framework sequences can be obtained from public references or public DNA databases that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as in Kabat, EA et al., [as described above]; Tomlinson, IM et al., 1992 J. fol. Biol. 227: 776-798; and Cox, JPL et al., 1994 Eur. J Immunol. 24: 827-836. Examples of framework sequences used in antibodies of the present disclosure are those that are structurally similar to the framework sequences used for selected antibodies of the present disclosure (e.g., the consensus sequences and / or framework sequences used for the monoclonal antibodies of the present disclosure). V H CDR1, V H CDR2 and V H CDR3 sequence and V L CDR1, V L CDR2 and V LThe CDR3 sequence is grafted onto a framework region having the same sequence as that found in the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequence can be grafted onto a framework region containing one or more mutations compared to the germline sequence. For example, it has been found that in some cases, mutating residues within the framework region is beneficial for maintaining or enhancing the antigen binding ability of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0377] Another type of variable region modification is to make V H and / or V L The amino acid residue mutation within the CDR1, CDR2 and / or CDR3 region, thereby improving one or more binding properties (e.g., affinity) of the antibody of interest, is referred to as "affinity maturation". Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations, and the impact on antibody binding or other functional properties of interest can be assessed in an in vitro or in vivo assay as described herein and provided in the Examples. Conservative modifications (as described above) can be introduced. The mutation can be an amino acid substitution, addition or deletion. In addition, typically no more than one, two, three, four or five residues are changed in the CDR region.

[0378] Therefore, in another embodiment, the present disclosure provides an isolated anti-ActRII monoclonal antibody, or a functional protein comprising an antigen binding portion of an isolated anti-ActRII monoclonal antibody, wherein the isolated anti-ActRII monoclonal antibody or a functional protein comprising an antigen binding portion of an isolated anti-AcRII monoclonal antibody consists of a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has: V H CDR1 region, the V H The CDR1 region consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 1 to SEQ ID NO: 14; H CDR2 region, the V H The CDR2 region has an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 28, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 15 to SEQ ID NO: 28; H CDR3 region, the V HThe CDR3 region has an amino acid sequence selected from the group consisting of SEQ ID NO: 29 to SEQ ID NO: 42, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 29 to SEQ ID NO: 42; L CDR1 region; the heavy chain variable region has: the V L The CDR1 region has an amino acid sequence selected from the group consisting of SEQ ID NO: 43 to SEQ ID NO: 56, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 43 to SEQ ID NO: 56; L CDR2 region, the V L The CDR2 region has an amino acid sequence of the group consisting of SEQ ID NO: 52 to SEQ ID NO: 70, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 52 to SEQ ID NO: 70; and V L CDR3 region, the V L The CDR3 region has an amino acid sequence of the group consisting of SEQ ID NO:71 to SEQ ID NO:84, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO:71 to SEQ ID NO:84.

[0379] Camelidae Antibodies

[0380] Antibody proteins obtained from camels and members of the dromedary family (Camelus bactrianus and Camelus dromaderius), including New World members such as llama species (Lama paccos, Lama glama and Lamavicugna), have been characterized in terms of size, structural complexity and antigenicity to human subjects. Certain IgG antibodies from this mammal family found in nature lack light chains and are therefore structurally distinct from the typical four-chain quaternary structure of antibodies from other animals, having two heavy chains and two light chains (see WO94 / 04678).

[0381] A region of camelid antibodies identified as V HHSmall single variable domains) can be obtained by genetic engineering to obtain small proteins with high affinity for the target, thereby producing low molecular weight antibody-derived proteins known as "camelid nanobodies" (see US5,759,808; Stijlemans, B. et al., 2004 J Biol Chem 279:1256-1261; Dumoulin, M. et al., 2003 Nature 424:783-788; Pleschberger, M. et al., 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al., 2002 Int J Cancer 89:456-62; and Lauwereys, M. et al., 1998 EMBO J 17:3512-3520). Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, the amino acid sequence of camelid antibodies can be recombinantly altered to obtain a sequence that is more similar to a human sequence, i.e., nanobodies can be "humanized". Thus, the natural low antigenicity of camelid antibodies to humans can be further reduced.

[0382] The molecular weight of camelid nanobodies is about one-tenth that of human IgG molecules, and the physical diameter of the protein is only a few nanometers. One consequence of the small size / dimension is that camelid nanobodies have the ability to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies can be used as reagents for detecting antigens that are hidden when using classical immunological techniques, and as possible therapeutic agents. Therefore, another consequence of the small size / dimension is that camelid nanobodies can inhibit due to binding to specific sites in grooves or narrow clefts of target proteins, and thus can be beneficial in providing the ability to more closely approximate the function of classical low molecular weight drugs than classical antibodies.

[0383] Low molecular weight and compact size further cause camel family nano antibodies to be extremely heat stable, stable to extreme pH and proteolytic digestion, and poor antigenicity. Another result is that camel family nano antibodies are easily moved to tissues from the circulatory system, even across the blood-brain barrier and can treat diseases affecting nervous tissue. Nano antibodies can further promote drug transport across the blood-brain barrier (see US2004 / 0161738). These features and the combination of low antigenicity to humans show huge therapeutic potential. In addition, these molecules can be fully expressed in prokaryotic cells such as E.coli, and are expressed as fusion proteins and are functional with phage.

[0384] Therefore, in one embodiment, the present disclosure relates to a composition comprising a camelid antibody or nanobody with high affinity for ActRIIB. In certain embodiments herein, the camelid antibody or nanobody is naturally produced in camelids, i.e., produced by camelids after immunization with ActRIIB or its peptide fragments using the techniques described herein for other antibodies. Alternatively, the anti-ActRIIB camelid nanobody is engineered, i.e., produced by selecting from a phage library displaying appropriately mutagenized camelid nanobody proteins using a panning procedure targeting ActRIIB, as described in the embodiments herein. The engineered nanobody can be further customized by genetic engineering so that the half-life in the recipient subject is 45 minutes to two weeks. In a specific embodiment, the camelid antibody or nanobody is obtained by transplanting the CDR sequence of the heavy chain or light chain of the human antibody of the present disclosure into a nanobody or a single domain antibody framework sequence, as described, for example, in WO94 / 04678.

[0385] Non-antibody scaffolds

[0386] Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, Adnectin (fibronectin) (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd (Cambridge, MA) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc. (Mountain View, CA)), protein A (Affibody AG, Sweden) and affilin (γ-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany), protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

[0387] (i) Fibronectin scaffold

[0388] The fibronectin scaffold is preferably based on a type III fibronectin domain (e.g., the tenth module of type III fibronectin (10Fn3 domain)). The type III fibronectin domain has 7 or 8 β-strands distributed between two β-sheets, which themselves wrap around each other to form a protein core, and further contain loops (similar to CDRs) that connect the β-strands to each other and are exposed to the solvent. There are at least three such loops at each edge of the β-sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the β-strands (US 6,818,418).

[0389] These fibronectin-based scaffolds are not immunoglobulins, although the overall fold is closely related to the folding of the smallest functional antibody fragment, the heavy chain variable region, which contains the complete antigen recognition unit in camel and llama IgG. Due to this structure, non-immunoglobulin antibodies mimic antigen binding properties similar to antibodies in properties and affinity. These scaffolds can be used for in vitro loop randomization and shuffling strategies, which are similar to the in vivo affinity maturation process of antibodies. These fibronectin-based molecules can be used as scaffolds, where the loop regions of the molecule can be replaced with the CDRs of the present disclosure using standard cloning techniques.

[0390] (ii) Ankyrin-Molecular Partner

[0391] The technology is based on the use of proteins with ankyrin-derived repeat modules as scaffolds for carrying variable regions that can be used to bind to different targets. The ankyrin repeat module is a 33-amino acid polypeptide consisting of two antiparallel α-helices and a β-turn. The binding of the variable regions is mainly optimized by using ribosome display.

[0392] (iii) Maxi-Body / Avimers-Avidia

[0393] Avimers are derived from proteins containing natural A domains, such as LRP-1. These domains are naturally used for protein-protein interactions, and more than 250 proteins in humans are structurally based on A domains. Avimers consist of many different "A-domain" monomers (2-10) connected by amino acid linkers. Avimers capable of binding to target antigens can be produced using methods described in, for example, US2004 / 0175756; US2005 / 0053973; US2005 / 0048512; and US2006 / 0008844.

[0394] (vi) Protein A-Affibody

[0395] The affinity ligand is a simple small protein consisting of a three-helix bundle based on a scaffold of one of the IgG binding domains of protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate a large number of ligand variants. Libraries (see, e.g., US 5,831,012). The molecule mimics antibodies, with a molecular weight of 6 kDa, compared to 150 kDa for antibodies. Despite its small size, The binding site of the molecule is similar to that of an antibody.

[0396] (v)Anticalins-Pieris

[0397] The products are developed by Pieris ProteoLab AG. They are derived from lipocalins, a group of widely distributed small and robust proteins that are often involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins are present in human tissues or body fluids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast to antibodies or their recombinant fragments, lipocalins consist of a single polypeptide chain with 160 to 180 amino acid residues, only slightly larger than a single immunoglobulin domain. A set of four loops that constitute the binding pocket show pronounced structural plasticity and tolerate a variety of side chains. Therefore, the binding site can be reshaped in a proprietary process in order to recognize designated target molecules of different shapes with high affinity and specificity.

[0398] One protein of the lipocalin family, the biletriene binding protein (BBP) of Pieris brassicae, has been used to develop anticalins by mutagenesis of the set of four loops.An example of a patent application describing "anticalins" is WO 1999 / 16873.

[0399] (vi) Affilin-Scil Proteins

[0400] AFFILIN TM The molecules are small non-immunoglobulins designed for specific affinities to proteins and small molecules. New AFFILINs can be selected very quickly from two libraries TM Molecules, each of the libraries is based on a different human scaffold protein.

[0401] AFFILIN TMThe molecule does not show any structural homology to immunoglobulin proteins. Scil Proteins uses two AFFILIN TM Scaffolds, one of which is a gamma-crystalline, human structural eye lens protein and the other is a "ubiquitin" superfamily protein. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturants. This high stability is mainly due to the extended beta sheet structure of the protein. Examples of gamma-crystalline derived proteins are described in WO2001 / 004144, and examples of "ubiquitin-like" proteins are described in WO2004 / 106368.

[0402] (vii) Protein epitope mimics (PEMs)

[0403] PEMs are medium-sized cyclic peptide-like molecules (MW 1-2 kDa) that mimic the β-hairpin secondary structure of proteins, which is the main secondary structure involved in protein-protein interactions.

[0404] Grafting antigen-binding domains into alternative frameworks or scaffolds

[0405] A variety of antibody / immunoglobulin frameworks or scaffolds can be used, as long as the resulting polypeptide comprises at least one binding region that specifically binds to ActRIIB. Such frameworks or scaffolds include the five major idiotypes of human immunoglobulins or fragments thereof (such as those disclosed elsewhere herein), and include immunoglobulins of other animal species, preferably with humanized aspects. In this regard, single heavy chain antibodies, such as those identified in camelids, are of particular interest. Those skilled in the art will continue to explore and develop new frameworks, scaffolds and fragments.

[0406] In one aspect, the compositions of the present disclosure may include non-immunoglobulin-based antibodies using non-immunoglobulin scaffolds, onto which the CDRs of the disclosed antibodies may be grafted. Known or future non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the target protein shown in SEQ ID NO: 181 (preferably, its ligand binding domain as shown in SEQ ID NO: 182). Such compounds are referred to herein as "polypeptides comprising a target-specific binding region". Examples of non-immunoglobulin frameworks are further described in the following sections (Camelidae Antibodies and Non-Antibody Scaffolds).

[0407] Framework or Fc engineering

[0408] The engineered antibodies included in the compositions of the present disclosure include engineered antibodies in which V H and / or VL In some embodiments, the invention relates to a method for modifying the framework residues within an antibody, for example, to improve the properties of the antibody. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one method is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. In order to restore the framework region sequence to its germline configuration, the somatic mutation can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies may also be included in the compositions of the present disclosure.

[0409] Another type of framework modification involves mutating one or more residues within the framework region or even within one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in US2003 / 0153043.

[0410] As a supplement or alternative to modifications made in the framework or CDR regions, the antibodies of the present disclosure may be engineered to include modifications in the Fc region, typically to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular toxicity. In addition, the antibodies included in the compositions of the present disclosure may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or may be modified to change its glycosylation to again change one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0411] In one embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is changed, for example, increased or decreased. This method is further described in US5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is changed, for example, to facilitate the assembly of the light chain and the heavy chain or to increase or decrease the stability of the antibody.

[0412] In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment so that the antibody has impaired staphylococcal protein A (SpA) binding relative to native-Fc hinge domain SpA binding. This method is further described in detail in US 6,165,745.

[0413] In another embodiment, the antibody is modified to extend its biological half-life. Various methods are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in US6,277,375. Alternatively, in order to extend the biological half-life, the antibody can be changed in the CH1 or CL region to include a salvage receptor binding epitope of two loops of the CH2 domain of the Fc region of IgG, as described in US5,869,046 and US6,121,022.

[0414] In other embodiments, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand to which its affinity is changed can be, for example, the C1 component of an Fc receptor or complement. The method is further described in detail in US5,624,821 and US5,648,260 of Winter et al. Specifically, residues 234 and 235 can be mutated. Specifically, these mutations can be to become alanine. Therefore, in one embodiment, the antibody included in the composition of the present disclosure has a mutation in one or both of amino acids 234 and 235 in the Fc region. In another embodiment, one or both of amino acids 234 and 235 can be substituted with alanine. Both amino acids 234 and 235 are substituted with alanine to cause ADCC activity to decrease.

[0415] In another embodiment, one or more amino acids selected from the amino acid residues of the antibody may be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This method is described in further detail in US 6,194,551.

[0416] In another embodiment, one or more amino acid residues of the antibody are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in WO94 / 29351.

[0417] In another embodiment, the Fc region of the antibody is modified by modifying one or more amino acids to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or enhance the affinity of the antibody to Fcγ receptors. This method is further described in WO00 / 42072. In addition, the binding sites of FcγRI, FcγRII, FcγRIII and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields, RL et al., 2001 J. Biol. Chen. 276: 6591-6604).

[0418] In another embodiment, the glycosylation of the antibody contained in the composition of the present disclosure is modified. For example, an aglycoslated antibody (i.e., the antibody lacks glycosylation) can be prepared. Glycosylation can be changed, for example, to increase the affinity of the antibody to the antigen. Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site. Such glycosylation can enhance the affinity of the antibody to the antigen. Such methods are further described in detail in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[0419] Additionally or alternatively, antibodies with altered glycosylation types may be used, such as low-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GsNac structures. Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such carbohydrate modifications may be achieved, for example, by expressing antibodies in host cells with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the disclosed recombinant antibodies therein, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 of Hang et al. describes a cell line with a functionally disrupted FUT8 gene (which encodes a fucosyltransferase) such that antibodies expressed in such cell lines exhibit low fucosylation. Therefore, in one embodiment, the antibodies contained in the compositions of the present disclosure are produced by recombinant expression in a cell line exhibiting a low fucosylation pattern (e.g., a mammalian cell line with defective expression of a FUT8 gene encoding a fucosyltransferase). WO03 / 035835 describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in the host cells (see Shields, RL et al., 2002 J. Biol. Chem. 277: 26733-26740). WO99 / 54342 describes a cell line engineered to express a glycoprotein modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNAc structures, which results in enhanced ADCC activity of the antibody (see Umana et al., 1999 Nat. Biotech. 17: 176-180). Alternatively, the antibodies comprised in the compositions of the present disclosure may be produced in yeast or filamentous fungi engineered to achieve a mammalian-like glycosylation pattern and capable of producing antibodies lacking fucose as a glycosylation pattern (see, e.g., EP1297172B1).

[0420] Another modification of the present disclosure contemplated antibodies herein is pegylation. Antibodies can be pegylated to, for example, extend the biological (e.g., serum) half-life of antibodies. In order to pegylate antibodies, the antibody or its fragment is usually reacted with PEG (such as a reactive ester or aldehyde derivative of PEG) under conditions where one or more polyethylene glycol (PEG) groups become attached to the antibody or antibody fragment. Pegylation can be carried out by alkylation or acylation with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated used is an antibody without glycosylation. Methods for pegylating proteins are known in the art and can be applied to disclosed antibodies (see, for example, EP0154316 and EP0401384).

[0421] Another modification of the antibody contemplated by the present disclosure is a conjugate or protein fusion of at least the antigen binding region of the antibody included in the composition of the present disclosure with a serum protein, such as human serum albumin or a fragment thereof, to extend the half-life of the resulting molecule (see, e.g., EP0322094).

[0422] Another possibility is the fusion of at least the antigen binding region of the antibody comprised in the disclosed composition with a protein capable of binding to serum proteins, such as human serum albumin, in order to extend the half-life of the resulting molecule (see for example EP0486525).

[0423] Methods of engineering altered antibodies

[0424] As described above, the CDR sequences, V H and V L Anti-ActRIIB antibodies containing a sequence or full-length heavy and light chain sequences can be used to generate new anti-ActRIIB antibodies by modifying the CDR sequence, full-length heavy chain and / or light chain sequence, V H and / or V L Sequences, or attached to their constant regions. Therefore, in another aspect of the present disclosure, the structural features of the anti-ActRIIB antibodies included in the compositions of the present disclosure are used to generate structurally related anti-ActRIIB antibodies that retain at least one functional property of the antibodies included in the compositions of the present disclosure (such as binding to human ActRIIB), but also inhibit one or more functional properties of ActRIIB (e.g., inhibiting Smad activation).

[0425] For example, one or more CDR regions or mutations thereof of an antibody included in the compositions of the present disclosure can be recombinantly combined with known framework regions and / or other CDRs to produce additional, recombinantly engineered anti-ActRIIB antibodies included in the compositions of the present disclosure, as described above. Other types of modifications include those described in the previous section. The starting material for the engineering method is a V provided herein. H and / or V L In order to produce an engineered antibody, it is not necessary to actually prepare (i.e., express as a protein) a V sequence as provided herein. H and / or V L Instead, the information contained in the sequence is used as starting material to create a "second generation" sequence derived from the original sequence, which is then prepared and expressed as a protein.

[0426] The antibody sequence of change can also be prepared by screening antibody library, and described antibody library has the fixed CDR3 sequence selected from the group consisting of SEQID NO:29 to SEQ ID NO:42 and SEQ ID NO:71 to SEQ ID NO:84 or the diversity on the minimum necessary binding determinant cluster and CDR1 and CDR2 sequence as described in US2005 / 0255552.Screening can be performed according to any screening technology (such as phage display technology) suitable for screening antibodies from antibody library.

[0427] Standard molecular biology techniques can be used to prepare and express the altered antibody sequences.Antibodies encoded by the altered antibody sequences are antibodies that retain one, some or all of the functional properties of the anti-ActRIIB antibodies described herein, including but not limited to specific binding to human ActRIIB and inhibition of Smad activation.

[0428] The altered antibody may exhibit one or more, two or more, or three or more of the above functional properties.

[0429] The functional properties of the altered antibodies can be assessed using standard assays available in the art and / or described herein, such as those described in the Examples (eg, ELISA).

[0430] Mutations can be selectively or randomly introduced along all or part of the anti-ActRIIB antibody coding sequence, and the resulting modified anti-ActRIIB antibodies can be screened for binding activity and / or other functional properties as described herein. Mutation methods have been described in the art. For example, WO02 / 092780 describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, WO03 / 074679 describes methods for optimizing the physiochemical properties of antibodies using computational screening methods.

[0431] Nucleic acid molecules encoding antibodies included in the compositions of the present disclosure

[0432] Examples of full-length light chain nucleotide sequences optimized for expression in mammalian cells are shown in SEQ ID NO: 161 to SEQ ID NO: 165 and SEQ ID NO: 171 to SEQ ID NO: 175. Examples of full-length heavy chain nucleotide sequences optimized for expression in mammalian cells are shown in SEQ ID NO: 166 to SEQ ID NO: 170 and SEQ ID NO: 176 to SEQ ID NO: 180.

[0433] Nucleic acid can be present in intact cells, in cell lysates, or can be partially purified or substantially pure nucleic acid. When nucleic acid is purified from other cellular components or other contaminants (such as other cellular nucleic acids or proteins) by standard techniques (including alkaline / SDS treatment, CsCI band analysis, column chromatography, agarose gel electrophoresis and other techniques well known in the art), the nucleic acid is "separated" or "made substantially pure". See F.Ausubel et al., ed., 1987Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Standard molecular biology techniques can be used to obtain nucleic acid. For antibodies expressed by hybridomas (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes, as further described below), cDNA encoding the light chain and heavy chain of antibodies produced by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from various phage clones as library members.

[0434] Once the code V is obtained H and V LThe DNA fragments of the segments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region gene into a full-length antibody chain gene, into a Fab fragment gene, or into a scFv gene. L Or code V H A DNA fragment is operably linked to another DNA molecule or a fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" means that two DNA fragments are joined in a functional manner, such as to keep the amino acid sequence encoded by the two DNA fragments in-frame, or to express a protein under the control of a desired promoter.

[0435] By encoding V H The DNA encoding the heavy chain constant region (CH1, CH2 and CH3) can be operably linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). H The DNA of the heavy chain constant region is converted to a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA et al., [as described above]), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. The heavy chain constant region can be selected from the IgG1 isotype. For the Fab fragment heavy chain gene, the encoding V H The DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0436] By encoding V L The DNA encoding the light chain constant region CL can be operably linked to another DNA molecule encoding the light chain constant region CL. L The DNA of the light chain constant region is converted to a full-length light chain gene (as well as to a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA et al., [as described above]), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0437] To create a scFv gene, the V H and code V L The DNA fragment encoding the flexible linker (eg, encoding the amino acid sequence (Gly4-Ser)3) is operably linked to another fragment such that V H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V HThe regions are joined by flexible linkers (see, e.g., Bird et al., 1988 Science 242:423-426; Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature 348:552-554).

[0438] Generation of monoclonal antibodies

[0439] Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein (1975 Nature 256: 495). Many techniques for producing monoclonal antibodies can be used, such as viral or oncogenic transformation of B lymphocytes.

[0440] The animal system used to prepare hybridomas is the murine system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immune spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0441] The chimeric antibody or humanized antibody included in the compositions of the present disclosure can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. The DNA encoding heavy chain and light chain immunoglobulin can be obtained from the mouse hybridoma of interest, and standard molecular biology techniques are used to engineer to include non-mouse (e.g., human) immunoglobulin sequences. For example, in order to produce chimeric antibodies, methods known in the art can be used to connect mouse variable regions to human constant regions (see, e.g., US 4,816,567). In order to produce humanized antibodies, methods known in the art can be used to insert mouse CDR regions into human frameworks (see, e.g., U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0442] In certain embodiments, the antibodies included in the disclosed compositions are human monoclonal antibodies. Such human monoclonal antibodies against ActRIIB can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice".

[0443] HuMAb (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg et al., 1994 Nature 368(6474): 856-859). As a result, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgG KMonoclonal (Lonberg, N. et al., 1994, [supra]; reviewed in Lonberg, N., 1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D., 1995 Intern. Rev. Immunol. 13:65-93, and Harding, F. and Lonberg, N., 1995 Ann. NY Acad. Sci. 764:536-546). The preparation and use of HuMAb mice, and the genomic modifications carried by such mice, are further described in Taylor, L. et al., 1992 Nucleic Acids Research 20:6287-6295; Chen, J. et al., 1993 International Immunology 5:647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724; Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBO J. 12:821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor, L. et al., 1994 International Immunology 579-591; and Fishwild, D. et al., 1996 Nature Biotechnology 14:845-851, the entire contents of which are incorporated herein by reference. See further U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and 5,545,807; and WO92 / 103918, WO93 / 12227, WO94 / 25585, WO97 / 113852, WO98 / 24884; WO99 / 45962; and WO01 / 14424.

[0444] In another embodiment, the human antibodies contained in the compositions of the present disclosure can be produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes (such as mice carrying human heavy chain transgenes and human light chain transchromosomes). Such mice, referred to herein as "KM mice", are described in detail in WO02 / 43478.

[0445] In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to generate anti-ActRIIB antibodies of the present disclosure. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used. Such mice are described in, for example, U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963.

[0446] In addition, alternative transchromosome animal systems expressing human immunoglobulin genes are available in the art and can be used to produce anti-ActRIIB antibodies of the present disclosure. For example, mice carrying human heavy chain transchromosomes and human light chain transchromosomes can be used, and the mice are referred to as "TC mice"; such mice are described in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA 97: 722-727. In addition, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al., 2002 Nature Biotechnology 20: 889-894), which can be used to produce anti-ActRIIB antibodies.

[0447] The human recombinant antibodies included in the disclosed compositions can also be prepared using phage display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art or described in the following examples. See, for example, U.S. Patent Nos. 5,223,409; 5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

[0448] Human monoclonal antibodies included in the compositions of the present disclosure can also be prepared using SCID mice into which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Pat. Nos. 5,476,996 and 5,698,767.

[0449] Generation of hybridomas producing human monoclonal antibodies

[0450] In order to generate hybridomas for producing human monoclonal antibodies included in the disclosed compositions, spleen cells and / or lymph node cells from immunized mice can be isolated and fused to suitable immortalized cell lines, such as mouse myeloma cell lines. The resulting hybridomas can be screened to produce antigen-specific antibodies. For example, a single cell suspension of spleen lymphocytes from immunized mice can be fused with one-sixth the number of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. The cells are seeded at about 2×145 on a flat-bottom microtiter plate and then incubated for two weeks in a selective medium containing 20% ​​fetal clone serum, 18% "653" conditioned medium, 5% origen (IGEN), 4mM L-glutamine, 1mM sodium pyruvate, 5mM HEPES, 0:055mM 2-mercaptoethanol, 50 units / ml penicillin, 50mg / ml streptomycin, 50mg / ml gentamicin and 1XHAT (Sigma; HAT is added 24 hours after fusion). After about two weeks, cells can be cultured in a medium in which HAT is replaced with HT. Individual wells can then be screened for human monoclonal IgM and IgG antibodies by ELISA. Once extensive hybridoma growth occurs, the culture medium can usually be observed after 10-14 days. The antibody-secreting hybridoma can be re-plated, screened again, and if still positive for human IgG, the monoclonal antibody can be subcloned at least twice by limiting dilution. Stable subclones can then be cultured in vitro to produce small amounts of antibodies in tissue culture medium for characterization. In order to purify human monoclonal antibodies, the selected hybridoma can be grown in a 2-liter spinner bottle for monoclonal antibody purification. Before affinity chromatography with protein A-agarose (Pharmacia), the supernatant can be filtered and concentrated. The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be replaced with PBS, and an extinction coefficient of 1.43 can be used to measure the IgG by OD 280 The monoclonal antibody can be aliquoted and stored at -80°C.

[0451] Generation of monoclonal antibody-producing transfectomas

[0452] Antibodies included in the compositions of the present disclosure can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (eg, Morrison, S. (1985) Science 229: 1202).

[0453] For example, in order to express an antibody or its antibody fragment, DNA encoding part or full-length light chain and heavy chain can be obtained by standard molecular biology techniques (e.g., cDNA cloning or PCR amplification using a hybridoma expressing an antibody of interest), and the DNA can be inserted into an expression vector so that the gene is operably connected to transcription and translation control sequences. In this article, the term "operably connected" means that the antibody gene is connected to a vector so that the transcription and translation control sequences in the vector play the expected function of the transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. Antibody light chain genes and antibody heavy chain genes can be inserted into different vectors, or more generally, two genes are inserted into the same expression vector. Antibody genes are inserted into expression vectors by standard methods (e.g., connecting complementary restriction sites on antibody gene fragments and vectors, or flat-end connection if there is no restriction site). The light chain and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting the light chain and heavy chain variable regions into expression vectors that already encode the heavy chain constant region and light chain constant region of the desired isotype, such that V H The segment is operably connected to the CH segment within the carrier, and V LThe segment is operably connected to the CL segment in the vector. In addition or alternatively, the recombinant expression vector can encode a signal peptide that promotes secretion of antibody chains from host cells. The antibody chain gene can be cloned into the vector so that the signal peptide is connected to the amino terminus of the antibody chain gene in frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin). In addition to the antibody chain gene, the recombinant expression vector of the present disclosure also carries a regulatory sequence for controlling the expression of the antibody chain gene in the host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene. Such regulatory sequences are described in, for example, Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA 1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of the host cell to be transformed, the expression level of the desired protein, and the like. Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma virus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the P-globin promoter may be used. Further, the regulatory elements are composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al., 1988 Mol. Cell. Biol. 8: 466-472).

[0454] In addition to antibody chain genes and regulatory sequences, recombinant expression vectors may also carry additional sequences, such as sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in host cells. Selectable marker genes contribute to the selection of host cells introduced into the vector (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, conventional selectable marker genes confer resistance to drugs (e.g., G418, hygromycin, or methotrexate) to host cells introduced into the vector. Selectable marker genes include dihydrofolate reductase (DHFR) genes (for dhfr host cells with methotrexate selection / amplification) and neo genes (for G418 selection).

[0455] In order to express light chain and heavy chain, the expression vector encoding heavy chain and light chain is transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to cover the various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. The antibody of the present disclosure can be expressed in a prokaryotic or eukaryotic host cell in theory. The expression of antibodies in eukaryotic cells, particularly mammalian host cells, has been discussed, because such eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete antibodies that are correctly folded and immunologically active than prokaryotic cells. It is reported that the prokaryotic expression of antibody genes is invalid for producing high-yield active antibodies (Boss, MA and Wood, CR, 1985 Immunology Today 6: 12-13).

[0456] Mammalian host cells for expressing the recombinant antibodies contained in the compositions of the present disclosure include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA 77: 4216-4220, which are used with the DHFR selection marker described, for example, in RJ Kaufman and PA Sharp, 1982 Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. In one embodiment, the host cell is a CHO K1PD cell. Specifically, for use with NSO myeloma cells, another expression system is the GS gene expression system shown in WO87 / 04462, WO89 / 01036, and EP 338,841. Mammalian host cells for expressing the recombinant antibodies contained in the compositions of the present disclosure include mammalian cell lines deficient in FUT8 gene expression, for example as described in US6,946,292B2. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or secreted into the culture medium in which the host cell grows. Standard protein purification methods can be used to recover the antibody from the culture medium.

[0457] Immunoconjugates

[0458] In another aspect, the invention features a composition comprising an anti-ActRIIB antibody or fragment thereof conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Such conjugates are referred to herein as "immunoconjugates." Immunoconjugates comprising one or more cytotoxins are referred to as "immunotoxins." Cytotoxins or cytotoxic agents include any agent that is harmful to cells (e.g., kills cells).

[0459] Cytotoxins can be conjugated to antibodies of the present disclosure using linker technology available in the art. Examples of linker types that have been used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Linkers can be selected, for example, to be easily cleaved by low pH within the lysosomal compartment or to be easily cleaved by proteases, such as proteases preferentially expressed in tumor tissues, such as cathepsins (e.g., cathepsins B, C, D).

[0460] For further discussion of linkers, types of cytotoxins and methods of conjugating therapeutic agents to antibodies, see Saito, G. et al., 2003 Adv. Drug Deliv. Rev. 55: 199-215; Trail, PA et al., 2003 Cancer Immunol. Immunother. 52: 328-337; Payne, G. 2003 Cancer Cell 3: 207-212; Allen, TM, 2002 Nat. Rev. Cancer 2: 750-763; Pastan, I. and Kreitman, RJ, 2002 Curr. Opin. Investig. Drugs 3: 1089-1091; Senter, PD and Springer, CJ, 2001 Adv. Drug Deliv. Rev. 53: 247-264.

[0461] The antibodies included in the compositions of the present disclosure may also be conjugated to radioactive isotopes to generate cytotoxic radiopharmaceuticals, also known as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine. 131 ,indium 111 ,yttrium 90 and Lutetium 177 Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available and include Zevalin TM (DEC Pharmaceuticals) and Bexxar TM(Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the disclosure.

[0462] The antibody conjugates included in the compositions of the present disclosure can be used to modify a given biological response, and the drug moiety should not be construed as being limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having a desired biological activity. These proteins can include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

[0463] Techniques for conjugating such therapeutic moieties to antibodies are well known, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," In Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," In Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," In Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Inmunol. Rev., 62: 119-58 (1982).

[0464] Bispecific molecules

[0465] In another aspect, the present disclosure features a composition comprising a bispecific or multispecific molecule comprising an anti-ActRIIB antibody or fragment thereof of the present disclosure. The antibody or antigen binding region thereof contained in the composition of the present disclosure may be derivatized or connected to another functional molecule, such as another peptide or protein (e.g., another ligand or antibody of a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. In fact, the antibodies of the present disclosure may be derivatized or connected to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be covered by the term "bispecific molecule" as used herein. In order to produce the bispecific molecules of the present disclosure, the antibodies of the present disclosure may be functionally connected (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, so that a bispecific molecule is produced.

[0466] Thus, the present disclosure includes compositions comprising a bispecific molecule comprising at least one first binding specificity for ActRIIB and a second binding specificity for a second target epitope. For example, the second target epitope can be another epitope of ActRIIB that is different from the first target epitope.

[0467] Additionally, for compositions in which the bispecific molecule is multispecific, the molecule can include a third binding specificity in addition to the first and second target epitopes.

[0468] In one embodiment, the bispecific molecule of the disclosed composition comprises at least one antibody or antibody fragment thereof (including, for example, Fab, Fab', F(ab')2, Fv, or single-chain Fv) as a binding specificity. The antibody can also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as a single chain construct or Fv described in Ladner et al., US 4,946,778, the contents of which are expressly incorporated herein by reference.

[0469] Other antibodies that can be used in bispecific molecules are murine, chimeric and humanized monoclonal antibodies.

[0470] The bispecific molecules included in the compositions of the present disclosure can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificity is a protein or peptide, a variety of coupling agents or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229: 81-83) and Glennie et al., 1987 J. Immunol. 139: 2367-2375). The conjugating agents were SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0471] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains. In a particular embodiment, the hinge region is modified to contain an odd number (eg, one) of sulfhydryl residues prior to conjugation.

[0472] Alternatively, the two binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb×mAb, mAb×Fab, Fab×F(ab')2 or ligand×Fab fusion protein. The bispecific molecule contained in the composition of the present disclosure can be a single-chain molecule comprising a single-chain antibody and a binding determinant, or a single-chain bispecific molecule comprising two binding determinants. The bispecific molecule may comprise at least two single-chain molecules. Methods for preparing bispecific molecules are described in, for example, U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

[0473] Binding of the bispecific molecule to its specific target can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot detection. Each of these assays typically detects the presence or absence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0474] Multivalent Antibodies

[0475] In another aspect, the disclosure relates to a composition comprising a multivalent antibody comprising at least two identical or different antigen binding portions of a disclosed antibody that bind to ActRIIB. In one embodiment, the multivalent antibody provides at least two, three, or four antigen binding portions of an antibody. The antigen binding portions can be linked together by protein fusion or covalent or non-covalent attachment. Alternatively, methods of attachment for bispecific molecules have been described. In various embodiments, the composition can be monovalent, bivalent or multivalent (e.g., capable of binding to one, two or several antigens), and / or monospecific, bispecific or multispecific (e.g., having binding regions that can bind to one, two or several different antigens), and the composition can be any combination of these, for example, monovalent and monospecific (having one binding region that binds to one antigen or epitope); or bivalent and bispecific (having two binding regions, each binding region binds to a different epitope or antigen); or bivalent and monospecific (having two binding regions, each binding region binds to the same epitope or antigen); or multivalent and monospecific (having several binding regions, all binding regions bind to the same antigen or epitope); or multivalent and multispecific (having several binding regions that bind to several different antigens or epitopes).

[0476] Pharmaceutical composition

[0477] In another aspect, the present invention provides a composition, such as a pharmaceutical composition, comprising one or a combination of the following items formulated with a pharmaceutically acceptable carrier: the above-mentioned antibody / monoclonal antibody or its antigen-binding portion. Such a composition may include one or a combination (e.g., two or more different) of the following items: the antibody, or an immunoconjugate or a bispecific molecule. For example, a pharmaceutical composition of the present disclosure may include a combination of antibodies that bind to different epitopes on a target antigen or have complementary activities.

[0478] The pharmaceutical compositions of the present disclosure may also be administered in combination therapy, i.e., in combination with other agents. For example, a combination therapy may include an anti-ActRII antibody of the present disclosure in combination with at least one other muscle mass / strength increasing agent, such as IGF-1, IGF-2, or a variant of IGF-1 or IGF-2, an anti-myostatin antibody, a myostatin propeptide, a myostatin decoy that binds to ActRIIB but does not activate it, a β2 agonist, a ghrelin agonist, a SARM, a GH agonist / mimetics, or follistatin. Examples of therapeutic agents that may be used in combination therapy are described in more detail below in the section on uses of the antibodies of the present disclosure.

[0479] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion), preferably suitable for intravenous injection or infusion. Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate or bispecific molecule, can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0480] The pharmaceutical composition of the present disclosure may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects (see, for example, Berge, SM et al., 1977 J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc.; and those from nontoxic organic acids, such as aliphatic monocarboxylic acids and aliphatic dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic sulfonic acids, and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like; and from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0481] The pharmaceutical composition of the present disclosure may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0482] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof; vegetable oils, such as olive oil; and injectable organic esters, such as ethyl oleate. For example, proper fluidity can be maintained by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0483] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. By the sterilization procedures described above and by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.), it is possible to ensure that microorganisms are prevented from existing. It may also be desirable to include isotonic agents, such as sugar, sodium chloride, etc., in the composition. In addition, the absorption of injectable drug forms can be extended by including agents that delay absorption (such as aluminum monostearate and gelatin). Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agents are incompatible with the active compound, it is considered to be used in the pharmaceutical composition of the present disclosure. The active compound supplemented may also be incorporated into the composition.

[0484] The therapeutic composition must generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated into solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants, appropriate fluidity can be maintained. In many cases, isotonic agents such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride can be included in the composition. By including an agent that delays absorption in the composition, such as monostearate and gelatin, extended absorption of injectable compositions can be achieved.

[0485] Sterile injectable solutions can be prepared by incorporating the desired amount of active compound into an appropriate solvent with one or a combination of the agents listed above, if necessary, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other desired agents from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying (lyophilization), thereby producing an active agent powder plus any additional desired agent from its previous sterile filtered solution.

[0486] The amount of active agent that can be combined with a carrier material to produce a single dosage form varies depending on the subject being treated and the specific mode of administration. The amount of active agent that can be combined with a carrier material to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect. Generally speaking, in one hundred percent, the amount will range from about 0.01% to about 99% of the active agent, from about 0.1% to about 70%, or from about 1% to about 30% of the active agent in combination with a pharmaceutically acceptable carrier.

[0487] The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be appropriately reduced or increased depending on the urgency of the treatment situation. It is particularly advantageous to prepare parenteral compositions in dosage unit form for ease of administration and dosage uniformity. As used herein, dosage unit form refers to physically separated units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of active compound and a desired pharmaceutical carrier calculated to produce a desired therapeutic effect. The specifications of the dosage unit form disclosed herein are determined by and directly depend on the following items: the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the art of mixing such active compounds according to individual sensitivity to treatment.

[0488] For the administration of the composition comprising the antibody, the antibody dosage range is from about 0.0001 to about 100 mg / kg host body weight, more generally from about 0.01 to about 30 mg / kg host body weight. For example, the dosage is about 1 mg / kg body weight, about 3 mg / kg body weight, about 5 mg / kg body weight or about 10 mg / kg body weight within the range of about 1-10 mg / kg, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg body weight. The dosage is repeated as needed, and can be within the range of about once a week to about once every 10 weeks, for example, once every 4 to 8 weeks. However, depending on the disease, pulse therapy can be utilized, wherein, for example, in the emergency room, for example, an ActII receptor antagonist injection is given to a patient with acute deterioration of heart disease.

[0489] Administration is preferably performed intravenously. Dosage regimens for the anti-ActRII antibodies (eg, bimagrumab) of the present disclosure include about 1 mg / kg body weight, or about 3 mg / kg body weight, or about 10 mg / kg body weight, once every four weeks by intravenous administration.

[0490] In some methods, two or more monoclonal antibodies with different binding specificities are included in the compositions of the present disclosure and are therefore administered simultaneously, in which case the dose of each antibody administered falls within the ranges shown. The antibodies are typically administered multiple times. Intervals between single doses can be, for example, weekly, monthly, every three months, every six months, or annually. Intervals can also be irregular as indicated by measuring blood levels of antibodies to the target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-about 1000 μg / ml, and in some methods about 25-about 300 μg / ml. For example, an ActRII antibody of the present disclosure can be co-administered with an anti-myostatin antibody.

[0491] Dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals during a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer relatively high dosages at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete improvement of disease symptoms. Thereafter, a preventive regimen may be administered to the patient.

[0492] Administration of a "therapeutically effective dose" of an anti-ActRII antibody comprised in the compositions of the present disclosure can result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of damage or disability resulting from disease affliction, i.e., an increase in cardiac function.

[0493] The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0494] Therapeutic compositions can be administered using medical devices known in the art.

[0495] Uses and methods of the present disclosure

[0496] The disclosed compositions and disclosed antibodies have therapeutic uses in that they have an effect on the treatment of heart disease or on the improvement of the condition of a patient affected by heart disease or on the alleviation of symptoms associated with heart disease.

[0497] The term "subject" or "individual" as used herein is intended to include humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, mice, cows, horses, chickens, amphibians and reptiles.

[0498] Therefore, the present disclosure also relates to a method of treatment, in which the composition of the present disclosure or the disclosed ActRII receptor antagonist, such as an ActRII binding molecule, more preferably an ActRII antibody, such as bimagrumab or BYM338, inhibits (i.e., antagonizes) the function of ActRII, thereby leading to the improvement of various types of heart diseases. The present disclosure provides a method for preventing and or treating heart disease, the method comprising administering to a patient a therapeutically effective amount of an ActRII receptor antagonist (e.g., preferably an ActRIIB binding molecule, more preferably an ActRIIB antagonist antibody (e.g., bimagrumab or BYM338)) or the disclosed composition.

[0499] Examples of ActRII receptor antagonists (e.g., ActRII binding molecules, preferably ActRIIB antagonist antibodies (e.g., bimagrumab or BYM338)) that can be used in the disclosed treatment methods are those disclosed or described in detail above. In certain embodiments, an ActRII antibody (e.g., bimagrumab or BYM338) is included in the compositions of the invention disclosed herein.

[0500] The present disclosure also relates to the use of an ActRII receptor antagonist (eg, an ActRIIA or ActRIIB receptor binding molecule, preferably an ActRII antagonist antibody, such as BYM338) in the manufacture of a medicament for treating various forms of heart disease as described above.

[0501] ActRII binding molecules, preferably ActRII antagonist antibodies, such as bimagrumab or BYM338, can be administered as the sole active agent or in combination with other drugs, such as IGF-1, IGF-2 or variants of IGF-1 or IGF-2, anti-myostatin antibodies, myostatin propeptides, myostatin decoys that bind to ActRIIB but do not activate it, β2 agonists, Ghrelin agonists, SARMs, GH agonists / mimetics, or follistatin. For example, the antagonists of the present disclosure can be used in combination with the IGF-1 mimetics disclosed in WO2007 / 146689.

[0502] In accordance with the foregoing, in yet another aspect, the present disclosure provides a method or use as defined above, comprising co-administering, such as simultaneously or sequentially administering, a therapeutically effective amount of an ActRII receptor antagonist (preferably an ActRII binding molecule, more preferably an ActRII antagonist antibody, such as bimagrumab or BYM338) and at least one second drug, the second drug being IGF-1, IGF-2 or a variant of IGF-1 or IGF-2, an anti-myostatin antibody, a myostatin propeptide, a myostatin decoy that binds to ActRII but does not activate it, a β2 agonist, a Ghrelin agonist, a SARM, a GH agonist / mimetics or follistatin.

[0503] Reagent test kit

[0504] The present invention also includes kits that may include an ActRII receptor antagonist, such as an ActRII receptor binding molecule (e.g., an ActRII receptor antibody or an antigen-binding fragment thereof, such as bimagrumab or BYM338) or an ActRII receptor (i.e., ActRIIB receptor) binding molecule (e.g., an anti-ActRIIB antibody or an antigen-binding fragment thereof) (e.g., in liquid or lyophilized form), or a pharmaceutical composition comprising an ActRII receptor antagonist (as described above). In addition, such kits may include a device for administering the ActRII antagonist (e.g., a syringe and vial, a prefilled syringe, a prefilled pen) and instructions for use. These kits may contain additional therapeutic agents (as described above), for example, for delivery in combination with a blocked ActRII antagonist (e.g., BYM338).

[0505] The phrase "device for administration" is used to indicate any available means for administering a drug systemically to a patient, including but not limited to prefilled syringes, vials and syringes, injection pens, autoinjectors, intravenous drip bags, pumps, etc. With such items, a patient can self-administer the drug (i.e., the administrator administers the drug to himself) or a physician can administer the drug. Each component of the kit is typically packaged in a separate container, and all of the various containers are within a single package together with instructions for use.

[0506] sequence

[0507] Table 1: Sequence Listing

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527] Embodiments of the disclosed methods, treatments, regimens, uses, and kits employ an ActRII receptor antagonist, such as an ActRIIB binding molecule. In another embodiment, the ActRIIB binding molecule is an ActRIIB antagonist antibody.

[0528] In some embodiments of the disclosed methods, treatments, regimens, uses, and kits, the anti-ActRIIB antibody is selected from the group consisting of: a) an anti-ActRIIB antibody that binds to an epitope of ActRIIB comprising amino acids 78-83 of SEQ ID NO: 181 (WLDDFN - SEQ ID NO: 188);

[0529] (b) amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC - SEQ ID NO: 186);

[0530] (c) amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 190);

[0531] (d) amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189);

[0532] (e) amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187);

[0533] (f) amino acids 29-41 of SEQ ID NO: 181 (CIYYNANWELERT-SEQ ID NO: 191);

[0534] (g) amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN - SEQ ID NO: 192); or

[0535] (h) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR); and

[0536] b) an ActRIIB antagonist antibody that binds to an epitope of ActRIIB comprising amino acids 78-83 of SEQ ID NO: 181 (WLDDFN - SEQ ID NO: 188);

[0537] (b) amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC - SEQ ID NO: 186);

[0538] (c) amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY - SEQ ID NO: 190);

[0539] (d) amino acids 52-56 of SEQ ID NO: 181 (EQDKR - SEQ ID NO: 189);

[0540] (e) amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW - SEQ ID NO: 187);

[0541] (f) amino acids 29-41 of SEQ ID NO: 181 (CIYYNANWELERT-SEQ ID NO: 191);

[0542] (g) amino acids 100-110 of SEQ ID NO: 181 (YFCCCEGNFCN - SEQ ID NO: 192); or

[0543] (h) amino acids 78-83 of SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181 (EQDKR), wherein the K D About 2pM.

[0544] In some embodiments of the disclosed methods, treatments, regimens, uses, and kits, the ActRIIB antagonist antibody is a human antibody.

[0545] In some embodiments of the disclosed methods, treatments, regimens, uses, and kits, the antibody is bimagrumab or BYM338.

[0546] The details of one or more embodiments of the present disclosure are set forth in the accompanying description above. Any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present disclosure. Other features, objects and advantages of the present disclosure are apparent from the specification and claims. In the specification and the appended claims, unless the context clearly provides otherwise, the singular form includes plural indicators. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. All patents and publications cited in this specification are incorporated by reference. The following examples are intended to more fully illustrate the present disclosure, but are not meant to limit the scope of the present disclosure in any way. Example

[0547] General Methodology

[0548] ActRIIB antibodies, their characterization and methods related thereto, such as (i) functional assays, (ii) reporter gene assays (RGA), (iii) culture of HEK293T / 17 cell lines, (iv) myostatin-induced luciferase reporter gene assays, (v) specific ELISAs, (vi) ActRIIB / Fc-myostatin binding interaction ELISAs, (vii) FACS titration of hActRIIB-expressing cells and hActRIIA-expressing cells, (viii) binding to primary human skeletal muscle cells, (ix) using surface plasmon resonance (Biacore) to characterize selected anti-human ActRIIB antibodies, have been disclosed in WO 2010 / 125003. Affinity determination of Fab, (x) CK assay, (xi) animal models, (xii) treatment protocol, (xiii) statistical analysis, (xiiii) panning, (xv) antibody identification and characterization, (xvi) optimization of antibodies derived from first affinity maturation, (xvii) IgG2 conversion of affinity matured Fab (first maturation), (xviiii) second affinity maturation, (xx) IgG2 conversion and IgG2 characterization (secondary maturation), (xxi) characterization of anti-ActRIIB antibodies in in vivo murine studies, (xxii) confirmation of affinity by SET, (xxiii) cross-blocking studies, and (xxiv) epitope mapping details and techniques.

[0549] The TAC (transverse aortic constriction) experimental model in mice is a commonly used experimental model of pressure overload-induced cardiac hypertrophy and heart failure and is described, for example, in Rockman et al. (1991) and deAlmeida et al. (2010), which are incorporated herein by reference as if fully set forth.

[0550] Example 1: TAC prevention study

[0551] Materials and methods:

[0552] The study tested whether CDD866 prevented the development of cardiac dysfunction in the established transverse aortic constriction (TAC) mouse model of heart failure.

[0553] The following four groups of 16-week-old male C57BL / 6 mice (n=7-10 / group) were part of this study:

[0554] 1.SHAM+isotype Ab

[0555] 2.SHAM+CDD866 Ab

[0556] 3.TAC+isotype Ab

[0557] 4.TAC+CDD866 Ab

[0558] Antibodies were administered subcutaneously (SQ) at 20 mg / kg once a week, with the last dose given <24 h before sacrifice.

[0559] Echocardiography was performed every two weeks.

[0560] Primary endpoint: Prespecified endpoint at 11 weeks after TAC or % fractional shortening (FS) < 20%

[0561] result:

[0562] As shown in Figures 1A to 1E, CDD866 Ab treatment has minimal cardiac effects in wild-type C57BL / 6 mice. Specifically, as shown in Figure 1A, the measured CDD866 plasma levels confirm that the drug is appropriately administered. CDD866 does not significantly increase heart mass (Figure 1B). CDD866 reduces myocardial fibrosis (Figure 1C), but the fibrosis % at baseline is significantly low in healthy wild-type controls. Representative micrographs of myocardium stained with PAS (Figure 1D) highlight the size of myocardial cells. Figure 1E illustrates the finding that CDD866 does not significantly increase the size of myocardial cells in wild-type animals. Data are expressed as mean ± standard deviation. Gray = control group, isotype Ab (n = 3). Black = experimental group, CDD866 Ab (n = 3), * p < 0.05.

[0563] As shown in Figures 2A to 2D, CDD866 treatment prevents TAC-induced heart failure in mice. Figure 2A graphically shows that contractile function (measured by %FS) is expected to decrease with TAC (horizontal bars), but is still retained in CDD866-treated animals subjected to TAC (diagonal bars). Figure 2B is a representative echocardiographic image 11 weeks after SHAM or TAC surgery, which demonstrates that TAC animals treated with CDD866 maintain contractile function. There is a trend of reduced lung weight in CDD866-treated animals, indicating that pulmonary congestion (a surrogate for heart failure in the mouse model) is less (Figure 2C). There is a significant reduction in the primary endpoint when CDD866 is used for treatment (survival or %FS <20%) (Figure 2D). Data are expressed as mean ± standard deviation. Black = SHAM + isotype Ab (n = 7). Gray = SHAM + CDD866 Ab (n = 7). Horizontal bar = TAC + isotype Ab (n = 10). Diagonal bars = TAC + CDD866 Ab (n = 10). *p < 0.05. #p < 0.01 (black indicates comparison with SHAM + isotype group, red indicates comparison with TAC + isotype group).

[0564] As shown in Figures 3A to 3D, CDD866 Ab effectively blocks cardiac ActRII-A / B signaling in the TAC model of heart failure. In Figure 3 (A), the measured CDD866 plasma levels indicate that the drug is appropriately administered. Cardiac follistatin-like 3 (FSTL3) expression increases with TAC, indicating that cardiac ActRII-A / B signaling increases in this cardiac injury model. CDD866 treatment reduces cardiac FSTL3 expression, indicating that it effectively blocks TAC-induced ActRII-A / B signaling in the heart (Figure 3B). The expression of pathological cardiac hypertrophy genes decreases with CDD866 treatment (Figure 3C). As shown in Figure 3D, treatment with CDD866 reduces the pathological cardiac fibrosis spectrum in TAC-induced heart failure. Data are expressed as mean ± standard deviation. Black = SHAM + isotype Ab (n = 7). Gray = SHAM + CDD866 Ab (n = 7). Horizontal bars = TAC + isotype Ab (n = 10). Diagonal bars = TAC + CDD866 Ab (n = 10). *p < 0.05. #p < 0.01 (black indicates comparison with SHAM + isotype group, red indicates comparison with TAC + isotype group).

[0565] Overall, (i) CDD866 had minimal effects on cardiac growth / function in wild-type controls; (ii) CDD866 effectively prevention development of TAC-induced heart failure, and (iii) CDD866 improves overall survival in the TAC model of heart failure.

[0566] Example 2: TAC treatment study

[0567] Materials and methods:

[0568] The study tested whether CDD866 could reverse Established cardiac dysfunction in animals subjected to TAC rescued the animals from heart failure.

[0569] The following two groups of 16-week-old male C57BL / 6 mice (n=10 / group) were studied:

[0570] TAC+isotype Ab

[0571] TAC+CDD866 Ab

[0572] Only if % shortening fraction decreased > 4 standard deviations after Antibody treatment was started.

[0573] Antibody was administered SQ, 20 mg / kg / week x 8 weeks (last dose <24 h before sacrifice)

[0574] Echocardiography was performed every two weeks.

[0575] Primary endpoint: Pre-specified endpoint at 8 weeks of treatment or %FS < 25%

[0576] result:

[0577] As shown in Figures 4A to 4D, CDD866 treatment restores cardiac function and rescues animals from TAC-induced heart failure. The measured CDD866 plasma levels indicate that the drug is appropriately administered in terms of contractile function (Figure 4A). Figure 4B graphically shows the finding that cardiac FSTL3 expression decreases with CDD866 treatment, indicating that CDD866 effectively blocks TAC-induced ActRII-A / B signaling in the heart. CDD866 reverses contractile dysfunction in TAC-induced heart failure, and gradual improvement is seen as early as 1 week after treatment (Figure 4C). CDD866 also reduces lung weight, which is a surrogate marker for heart failure in a mouse model (Figure 4D). Data are expressed as mean ± standard deviation. Gray = TAC + isotype Ab. Black = TAC + CDD866 Ab. * p < 0.05. # P < 0.01.

[0578] CDD866 treatment induces cardiac growth in the TAC model. As shown in Figure 5A, with CDD866 treatment, wall thickness gradually increases (black arrow indicates Rx start). Figure 5B shows continuous echo images of ventricular slices during treatment, indicating the difference in cardiac growth between isotype and CDD866 treated animals. CDD866 increases cardiac mass in the TAC model, as shown in Figure 5C. Figure 5D shows a micrograph of the myocardium stained with PAS, highlighting the size of myocardial cells. CDD866 also increases myocardial cell growth in TAC (Figure 5E). Data are expressed as mean ± standard deviation. Gray = TAC + isotype Ab. Black = TAC + CDD866 Ab. *p<0.05. #P<0.01.

[0579] The results also show that CDD866 induces physiological cardiac growth, which has a protective effect in heart failure. Figure 6A graphically shows that the expression of genes associated with pathological hypertrophy is reduced with CDD866 treatment. The effects of CDD866 on cardiac growth and body weight are transient and reversible, as shown in Figure 6B. The improvement in cardiac function induced by a single dose of CDD866 lasts for at least 6 weeks. (Figure 6B, arrow = timing of single dose; dotted line = expected trajectory without CDD866 treatment. Figure 6C shows a micrograph of the myocardium stained with Masson's trichrome (blue = fibrosis; red = muscle). As shown in Figure 6D, there is a trend towards reduced myocardial fibrosis using CDD866 treatment. Data are expressed as mean ± standard deviation. Gray = TAC + isotype Ab. Black = TAC + CDD866 Ab. *p<0.05. #P<0.01.

[0580] CDD866 induces skeletal muscle growth in TAC-mediated heart failure, as shown in Figures 7A to 7E. CDD866 reduces p-SMAD3 expression in skeletal muscle, indicating that CDD866 effectively blocks ActRII-A / B signaling in skeletal muscle in this heart failure model (Figure 7A). CDD866 gradually increases overall body weight; it is likely to be achieved by increasing muscle mass (Figure 7B). Figure 7C shows that CDD866 increases the total mass of each skeletal muscle group (EDL, gas, TC). CDD866 also increases skeletal muscle cell size (Figure 7D). CDD866 also induces fiber type switching in skeletal muscle (Figure 7E). Red = TAC + isotype Ab. Blue = TAC + CDD866Ab. *p<0.05. #p<0.01.

[0581] In conclusion, CDD866 effectively reverseCDD866 enhanced cardiac growth and reduced myocardial fibrosis, data indicating physiological cardiac hypertrophy with protective effects in heart failure. CDD866 enhanced skeletal muscle growth in the TAC model of heart failure, suggesting the use of CDD866 in improving cardiac cachexia in advanced heart failure.

[0582] Example 3 :MHCF764L Time Point Study

[0583] Materials and methods:

[0584] This study tested whether CDD866 could improve cardiac function in a genetic model of dilated cardiomyopathy (MHCF764L).

[0585] Two groups of 14-24 week old male MHC F764L+ / - mutant mice were studied:

[0586] Isotype Ab (n=3)

[0587] CDD866 Ab (n=3)

[0588] Antibody was administered SQ, 20 mg / kg / week x 12 weeks (last dose <24 h before sacrifice)

[0589] Echocardiography was performed every two weeks (q2wk).

[0590] Primary endpoint: pre-specified endpoint of 12-week treatment or %FS < 20%.

[0591] result:

[0592] CDD866 had minimal cardiac effects in a genetic model of dilated cardiomyopathy (but only a modest cardiac phenotype at baseline). As illustrated in Figure 8A, CDD866 induced a trend toward a slight increase in contractile function in MHCF74L mice. Figure 8B shows a trend toward reduced cardiac FSTL3 expression with CDD866 treatment, indicating that CDD866 effectively blocks ActRII-A / B signaling in the heart. As shown in Figure 8C, no significant differences in pathological hypertrophy gene expression profiles were observed. Data are expressed as mean ± standard deviation. Gray = isotype Ab. Black = CDD866 Ab. *p<0.05. #p<0.01.

[0593] Overall, a slight increase in contractile function was observed with CDD866 treatment. No significant differences in the gene expression profiles of heart failure were detected.

[0594] References:

[0595] Akpan I,Goncalves MD,Dihr R,Yin X,Pistilli E,Bogdanovich S,Khurana,T,Ucran,J,Lachey,J,Ahima,RS.The effects of a soluble activin type IIB receptoron obesity and insulin sensitivity.Int J Obes(Lond)2009Nov;33(11):1265-1273.

[0596] deAlmeida AC,van Oort RJ,Wehrens XH.Transverse aortic constriction inmice.J Vis Exp 2010Apr 21;(38). http: / / www.jove.com / details.php?id=1729 ,doi:10.3791 / 1729.Lee SJ,McPherron AC.Regulation of myostatin activity and musclegrowth.Proc Natl Acad Sci U S A.2001Jul 31;98(16):9306-11.

[0597] Lee SJ,Reed LA,Davies MV,Girgenrath S,Goad ME,Tomkinson KN,Wright JF,Barker C,Ehrmantraut G,Holmstrom J,Trowell B,Gertz B,Jiang MS,Sebald SM,Matzuk M,Li E,Liang LF,Quattlebaum E,Stotish RL,Wolfman NM.Regulation ofmuscle growth by multiple ligands signaling through activin type IIreceptors.Proc Natl Acad Sci U S A.2005Dec 13;102(50):18117-22.

[0598] Rebbapragada A,Benchabane H,Wrana JL,Celeste AJ,Attisano L.Myostatinsignals through a transforming growth factor beta-like signaling pathway toblock adipogenesis.Mol Cell Biol.2003;23:7230-7242.

[0599] Rockman HA,Ross RS,Harris AN,Knowlton KU,Steinhelper ME,Field LJ,RossJr.J,Chien KR.Segregation of atrial-specific and inducible expression of anatrial natriuretic factor transgene in an in vivo murine model of cardiachypertrophy.Proc Natl Acad Sci USA.1991September;88:8277-8281.

[0600] Whittemore LA,Song K,Li X,Aghajanian J,Davies M,Girgenrath S,Hill JJ,Jalenak M,Kelley P,Knight A,Maylor R,O'Hara D,Pearson A,Quazi A,Ryerson S,TanXY,Tomkinson KN,Veldman GM,Widom A,Wright JF,Wudyka S,Zhao L,WolfmanNM.Inhibition of myostatin in adult mice increases skeletal muscle mass andstrength.Biochem Biophys Res Commun.2003Jan 24;300(4):965-71

Claims

1. An ActRII receptor antagonist for use in treating and / or preventing heart failure.

2. An ActRII receptor antagonist for use in treating structural and / or functional cardiac abnormalities associated with a condition selected from the group consisting of valvular heart disease, coronary artery disease, hypertension, diabetes, aging, arrhythmias, peripartum cardiomyopathy, stress cardiomyopathy, hereditary cardiomyopathy, and idiopathic dilated cardiomyopathy.

3. An ActRII receptor antagonist for use in the treatment and / or prevention of heart failure according to claim 1, wherein the heart failure is caused by or is associated with at least one of the following: valvular heart disease, coronary artery disease, hypertension, diabetes, aging, arrhythmia, peripartum cardiomyopathy, stress cardiomyopathy, toxic or infectious agents, hereditary cardiomyopathy or idiopathic dilated cardiomyopathy.

4. The ActRII receptor antagonist for use in treating and / or preventing heart failure according to claim 1, wherein the heart failure is heart failure with reduced ejection fraction.

5. The ActRII receptor antagonist for use in treating and / or preventing heart failure according to claim 1, wherein the heart failure is heart failure with preserved ejection fraction.

6. The ActRII receptor antagonist for use in treating and / or preventing heart failure according to claim 3, wherein the valvular heart disease is aortic valve stenosis.

7. An ActRII receptor antagonist for use in treating structural and / or functional cardiac abnormalities associated with the disorder according to claim 2, wherein the valvular heart disease is aortic stenosis.

8. The ActRII receptor antagonist for use in treating and / or preventing heart failure according to claim 6, wherein the aortic valve stenosis is accompanied by frailty and / or sarcopenia.

9. An ActRII receptor antagonist for use in treating structural and / or functional cardiac abnormalities associated with the disorder according to claim 7, wherein the aortic valve stenosis is accompanied by frailty and / or sarcopenia.

10. The ActRII receptor antagonist for use in treating and / or preventing heart failure according to claim 3, wherein the peripartum cardiomyopathy occurs during late pregnancy or within 6 months after delivery.

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