Compositions and methods for increasing body weight and lean muscle mass using antagonists to leptin receptor, gdf8, and activin a

Abstract

The present invention provides a combination comprising a leptin receptor antagonist, a GDF8 antagonist, and an activin A antagonist, and methods of use thereof. Such compositions are effective, for example, in causing an increase in lean body mass, at least in part, at the expense of fat mass. Methods for treating malnutrition, cachexia, and other conditions characterized by nutritional deficiencies and weight loss are also provided.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 781,226, filed December 18, 2018, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention provides, in part, compositions comprising inhibitors of LEPR, GDF8, and activin A, and methods of treatment for increasing body weight and lean muscle mass.

[0003] Sequence Listing An official copy of the Sequence Listing is being submitted electronically via EFS-Web contemporaneously herewith as an ASCII formatted Sequence Listing with the filename "10547WOseqlist_ST25.txt", a creation date of December 17, 2019, and an approximate size of 26KB. The Sequence Listing contained in this ASCII format document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] 2. Background of the Invention Growth and differentiation factor-8 (GDF8, also known as myostatin) and activin A are two key regulators of the development and maintenance of skeletal muscle and muscle mass.

[0005] GDF8 is a secreted ligand belonging to the transforming growth factor-β (TGF-β) superfamily of growth factors that acts as a negative regulator of muscle mass. GDF8 antagonists have been used in adult mice with significant positive effects on skeletal muscle mass. The receptor that GDF8 binds to and negatively regulates muscle mass is activin receptor type IIB (ACVR2B). GDF8 is not the only negative regulator of muscle mass that acts through ACVR2B. Activin A (ActA) also negatively regulates muscle mass through this receptor (Latres et al., Activin A more prominently regulates muscle mass in primates than does GDF8, Nature Comm. 8:15153 (2017) (Non-Patent Document 1)). ActRIIB binds to ligands including activin A, B, C and E, GDF11, bone morphogenetic protein 9 (BMP9), and BMP10. Data suggest that activin A acts in concert with GDF8 to regulate skeletal muscle mass, and that combined inhibition of GDF8 and activin A may be effective in treating muscle atrophy in patients with muscle wasting disorders (Latres et al. (2017)).

[0006] Leptin is a polypeptide hormone that is expressed primarily by adipose tissue and skeletal muscle and is involved in the regulation of metabolism, energy balance, and food intake. Leptin activity is mediated by interaction with and signaling through the leptin receptor. The leptin receptor (also known as "LEPR", "WSX", "OB receptor", "OB-R", and "CD295") is a single transmembrane receptor of the class I cytokine receptor family with a large extracellular domain. LEPR regulates body weight through JAK-STAT3 signaling. Altered signaling from leptin or LEPR or both may contribute to multiple disorders, including but not limited to anorexia or other psychiatric eating disorders, cachexia, autoimmune disorders, cardiovascular diseases, and neurodegenerative disorders. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Latres et al.,Activin A more prominently regulates muscle mass in primates than does GDF8,Nature Comm.8:15153(2017) Summary of the Invention

[0008] The present invention relates to compositions that are useful not only for increasing overall body mass, but also for increasing body mass in a manner that results in a more desirable overall body composition.As described above, administration of antagonist anti-LEPR, anti-GDF8, and anti-ActA antibodies (triple combination) resulted in an increase in total body weight greater than that observed with anti-LEPR alone, or anti-ActA and anti-GDF8.An additional beneficial effect was observed with respect to the lean mass of subjects receiving the triple combination.In these subjects, the overall lean mass increased, and the ratio of fat mass to lean mass decreased (compared to anti-LEPR alone, or anti-ActA and anti-GDF8).This increase in lean mass was confirmed, for example, insofar as subjects receiving the triple combination showed greater muscle mass and muscle fiber size (area) in some specific muscle structures assayed.

[0009] The present invention provides a combination comprising a leptin receptor (e.g., human leptin receptor) antagonist associated with a GDF8 (e.g., human GDF8) antagonist associated with an activin A (e.g., human activin A) antagonist (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) and, optionally, a pharmaceutically acceptable carrier. For example, such a combination may comprise a co-formulation comprising at least two antagonists selected from a leptin receptor antagonist, a GDF8 antagonist, and an activin A antagonist, or the antagonists may be in separate compositions. In one embodiment of the present invention, the leptin receptor antagonist, the GDF8 antagonist, and / or the activin A antagonist are antibodies or antigen-binding fragments thereof that specifically bind to leptin receptor, GDF8, and / or activin A, respectively. In one embodiment of the invention, the leptin receptor antagonist is an antibody or antigen-binding fragment that specifically binds to the receptor and does not compete with leptin for binding to the receptor. In one embodiment of the invention, the LEPR antagonist is an antibody or antigen-binding fragment that specifically binds to the receptor and does not compete with leptin for binding to the receptor. In one embodiment of the invention, the LEPR antagonist is an antibody or antigen-binding fragment that specifically binds to the receptor and does not compete with leptin for binding to the receptor. In one embodiment of the invention, the LEPR antagonist is an antibody or antigen-binding fragment that specifically binds to the receptor and does not compete with leptin for binding to the receptor. In one embodiment of the invention, the LEPR antagonist is an antibody or antigen-binding fragment that specifically binds to the receptor and does not compete with leptin for binding to the receptor. and CDR-L1, CDR-L2, CDR-L3 of a light chain variable region selected from H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2, or an antibody or antigen-binding fragment thereof that specifically binds to LEPR and binds to the same epitope on LEPR as the aforementioned antibody or fragment and / or competes with the aforementioned antibody or fragment for binding to LEPR.In one embodiment of the invention, the GDF8 antagonist comprises a heavy chain variable region comprising CDR-H1, CDR-H2, CDR-H3 of a heavy chain variable region selected from 21-E5;21-B9;21-E9;21-A2;22-D3;22-E6;22-G10;1A2;20B12;58C8;19F2;8D12-1;4E3-7;9B11-12;4B9;1H4-5;9B4-3;3E2-1;4G3-25;4B6-6;H4H1657N2 or H4H1669P and H4H18508P2, and a heavy chain variable region comprising CDR-H1, CDR-H2, CDR-H3 of a heavy chain variable region selected from 21-E5;21-B9;21-E9;21-A2;22-D3;22-E6;22-G10; The antibody or antigen-binding fragment thereof specifically binds to GDF8, and comprises CDR-L1, CDR-L2 and CDR-L3 of a light chain variable region selected from 1A2; 20B12; 58C8; 19F2; 8D12-1; 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P and H4H18508P2, or an antibody or antigen-binding fragment thereof which binds to the same epitope on GDF8 as the aforementioned antibody or fragment and / or competes with the aforementioned antibody or fragment for binding to GDF8. In one embodiment of the invention, activin A is H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P. 2 , H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N; and a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P 2, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N, and CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region selected from H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N, or an antibody or antigen-binding fragment thereof that binds to the same epitope on activin A as the aforementioned antibody or fragment and / or competes with the aforementioned antibody or fragment for binding to activin A. In one embodiment of the invention, the LEPR antagonist comprises a heavy chain variable region selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2, and a heavy chain variable region selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2. and a light chain variable region selected from H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2, or an antibody or antigen-binding fragment thereof that binds to the same epitope on LEPR as the aforementioned antibody or fragment and / or competes with the aforementioned antibody or fragment for binding to LEPR.In one embodiment of the invention, the GDF8 antagonist comprises a heavy chain variable region selected from 21-E5;21-B9;21-E9;21-A2;22-D3;22-E6;22-G10;1A2;20B12;58C8;19F2;8D12-1;4E3-7;9B11-12;4B9;1H4-5;9B4-3;3E2-1;4G3-25;4B6-6;H4H1657N2 or H4H1669P and H4H18508P2, and a heavy chain variable region selected from 21-E5;21-B9;21-E9;21-A2;22-D3;22-E6;22-G1 and a light chain variable region selected from among 0; 1A2; 20B12; 58C8; 19F2; 8D12-1; 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P and H4H18508P2, or an antibody or antigen-binding fragment thereof that binds to the same epitope on GDF8 as the aforementioned antibody or fragment and / or competes with the aforementioned antibody or fragment for binding to GDF8. In one embodiment of the invention, activin A is H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P. 2 , H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N, and a heavy chain variable region selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P 2, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N, or which binds to the same epitope on activin A as the aforementioned antibodies or fragments and / or competes with the aforementioned antibodies or fragments for binding to activin A. Such combinations optionally include one or more additional therapeutic agents (e.g., appetite stimulants, cannabinoids, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, smooth muscle relaxants, nitrates, diuretics, iron, bronchodilators, anticholinergics, corticosteroids, antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), immunosuppressants, HMG-CoA reductase inhibitors, antidepressants, anticancer therapies, and / or topical agents).

[0010] The invention provides an injection device (eg, a hypodermic needle and syringe, an autoinjector, or a pre-filled syringe) or container (eg, a vial) comprising a combination of the invention (eg, H4H18457P2 / H4H1657N2 / H4H10446P2). The invention further provides a method for administering the combination of the invention to a subject (e.g., a human), the method comprising the step of introducing the components of the combination into the subject's body, for example parenterally, for example by injection using an injection device according to the invention. In one embodiment of the invention, the subject suffers from malnutrition, poor growth, inadequate food intake, eating disorders, cachexia, muscle atrophy or wasting, and muscle damage, and / or is undergoing physical therapy.

[0011] The present invention also provides a method for inhibiting LEPR, GDF8, and activin A in a subject (e.g., human), comprising administering to the subject a therapeutically effective amount of a combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2), for example parenterally, for example by injection using an injection device according to the present invention. In one embodiment of the present invention, the subject suffers from malnutrition, poor growth, inadequate food intake, eating disorders, cachexia, muscle atrophy or wasting, and muscle damage, and / or is undergoing physical therapy.

[0012] The present invention also provides a method for increasing food intake, fat deposition, body weight, muscle strength, muscle fiber size, and / or lean mass (e.g., at the expense of fat mass) in a subject in need thereof, the method comprising administering to the subject (e.g., a human) a therapeutically effective amount of a combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). In one embodiment of the present invention, the subject suffers from malnutrition, stunted growth, inadequate food intake, eating disorders, cachexia, muscle atrophy or wasting, and muscle damage, and / or is undergoing physical therapy.

[0013] The present invention further provides a method for improving athletic performance in a subject (e.g., a human) in need thereof, comprising administering to the subject a therapeutically effective amount of a combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). In one embodiment of the present invention, the subject suffers from malnutrition, stunted growth, inadequate food intake, eating disorders, cachexia, muscle atrophy or wasting, and muscle damage, and / or is undergoing physical therapy (e.g., stroke rehabilitation).

[0014] The present invention provides a method for reducing an increase in hepatic triglyceride content in a subject administered a leptin receptor antagonist, the method comprising administering to the subject a GDF8 antagonist associated with an activin A antagonist (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) in association with the leptin receptor antagonist.

[0015] The present invention also provides a method for treating or preventing malnutrition, cachexia, failure to thrive, eating disorders characterized by insufficient caloric intake, muscle atrophy, age-related sarcopenia, or muscle damage in a subject (e.g., a human) in need thereof, the method comprising administering to the subject a therapeutically effective amount of a combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). For example, in one embodiment of the present invention, (i) the subject suffering from malnutrition is suffering from hospital malnutrition, childhood stunting, anorexia, and / or bulimia, an eating disorder characterized by insufficient caloric intake, (ii) the subject suffering from cachexia is suffering from anorexia, bulimia, an eating disorder, pulmonary disease, chronic obstructive pulmonary disorder (COPD), chronic kidney disease, infectious disease, HIV infection, acquired immune deficiency syndrome (AIDS), congestive heart failure, radiation therapy, cancer, hepatocellular carcinoma, melanoma, breast cancer, an autoimmune disorder, inflammatory bowel disease, lupus erythematosus, , multiple sclerosis, rheumatoid arthritis, Crohn's disease, psoriasis, cystic fibrosis, cardiovascular disease, hypertension, depression, and / or a neurodegenerative disorder, and / or (iii) the subject suffering from muscle atrophy or muscle wasting has or is undergoing sepsis, AIDS, renal failure, heart failure, glucocorticoid excess, Cushing's syndrome, trauma, muscle disuse, immobilization, bed rest, injury, hip fracture, hip replacement, knee replacement, and / or mechanical ventilation.

[0016] Methods for making the combinations of the invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) comprising co-forming a LEPR antagonist, a GDF8 antagonist, and an activin A antagonist, and a pharma- ceutically acceptable carrier, are also part of the invention.

[0017] The invention also provides a method of making a device or container containing a combination of the invention (eg, H4H18457P2 / H4H1657N2 / H4H10446P2), the method comprising introducing the components of the combination into the container or device. [The present invention 1001] Related to activin A antagonists Related to GDF8 antagonists A leptin receptor antagonist, Optionally, with a pharma- ceutically acceptable carrier or diluent. Including, combinations. [The present invention 1002] The combination of claim 1001, wherein at least two of said antagonists selected from a leptin receptor antagonist, a GDF8 antagonist, and an activin A antagonist are co-formulated. [The present invention 1003] The combination of 1001 or 1002 according to the present invention, wherein said antagonist is in a separate composition. [The present invention 1004] Any of the combinations of claims 1001 to 1003, wherein the leptin receptor antagonist, the GDF8 antagonist, and / or the activin A antagonist are antibodies or antigen-binding fragments thereof that specifically bind to the leptin receptor, GDF8, and / or activin A, respectively. [The present invention 1005] The leptin receptor antagonist is an antibody or antigen-binding fragment that specifically binds to the receptor and does not compete with leptin for binding to the receptor Any combination of 1001 to 1004 of the present invention. [The present invention 1006] (i) the leptin receptor antagonist is selected from the group consisting of: a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of a heavy chain variable region selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2; a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2; An antibody or antigen-binding fragment thereof that specifically binds to a leptin receptor, comprising and / or an antibody or antigen-binding fragment thereof that binds to the same epitope on the leptin receptor as said antibody or fragment and / or competes with said antibody or fragment for binding to the leptin receptor; That is, (ii) the GDF8 antagonist is selected from the group consisting of: a heavy chain variable region comprising CDR-H1, CDR-H2, CDR-H3 of a heavy chain variable region selected from 21-E5; 21-B9; 21-E9; 21-A2; 22-D3; 22-E6; 22-G10; 1A2; 20B12; 58C8; 19F2; 8D12-1; 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P and H4H18508P2; a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of a light chain variable region selected from 21-E5;21-B9;21-E9;21-A2;22-D3;22-E6;22-G10;1A2;20B12;58C8;19F2;8D12-1;4E3-7;9B11-12;4B9;1H4-5;9B4-3;3E2-1;4G3-25;4B6-6;H4H1657N2 or H4H1669P and H4H18508P2; An antibody or antigen-binding fragment thereof that specifically binds to GDF8, comprising: and / or an antibody or antigen-binding fragment thereof that binds to the same epitope on GDF8 as said antibody or fragment and / or competes with said antibody or fragment for binding to GDF8. and / or (iii) the activin A antagonist is selected from the group consisting of: a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N; a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N; An antibody or antigen-binding fragment thereof that specifically binds to activin A, comprising: and / or an antibody or antigen-binding fragment thereof that binds to the same epitope on activin A as said antibody or fragment and / or competes with said antibody or fragment for binding to activin A. That is, Any combination of 1001 to 1005 of the present invention. [The present invention 1007] (i) the LEPR antagonist is a heavy chain variable region of an antibody selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2; a light chain variable region of an antibody selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2; an antibody or antigen-binding fragment thereof comprising: or an antibody or antigen-binding fragment thereof that binds to the same epitope on LEPR as said antibody or fragment and / or competes with said antibody or fragment for binding to LEPR That is, (ii) the GDF8 antagonist is selected from the group consisting of: a heavy chain variable region of an antibody selected from 21-E5; 21-B9; 21-E9; 21-A2; 22-D3; 22-E6; 22-G10; 1A2; 20B12; 58C8; 19F2; 8D12-1; 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P and H4H18508P2; a light chain variable region of an antibody selected from 21-E5;21-B9;21-E9;21-A2;22-D3;22-E6;22-G10;1A2;20B12;58C8;19F2;8D12-1;4E3-7;9B11-12;4B9;1H4-5;9B4-3;3E2-1;4G3-25;4B6-6;H4H1657N2 or H4H1669P and H4H18508P2; an antibody or antigen-binding fragment thereof comprising: or an antibody or antigen-binding fragment thereof that binds to the same epitope on GDF8 as said antibody or fragment and / or competes with said antibody or fragment for binding to GDF8. and / or (iii) the activin A antagonist is selected from the group consisting of: a heavy chain variable region of an antibody selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N; a light chain variable region of an antibody selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N; an antibody or antigen-binding fragment thereof comprising: or an antibody or antigen-binding fragment thereof that binds to the same epitope on activin A as said antibody or fragment and / or competes with said antibody or fragment for binding to activin A. That is, Any combination of 1001 to 1006 of the present invention. [The present invention 1008] Any of the combinations of 1001 to 1007 of the present invention, comprising the antibody H4H18457P2, the antibody REGN2477, and the antibody REGN1033. [The present invention 1009] Any of the combinations of 1001 to 1008, comprising an additional therapeutic agent. [The present invention 1010] The combination of claim 1009, wherein said additional therapeutic agent is one or more selected from appetite stimulants, cannabinoids, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, smooth muscle relaxants, nitrates, diuretics, iron, bronchodilators, anticholinergics, corticosteroids, antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), immunosuppressants, HMG-CoA reductase inhibitors, antidepressants, anticancer therapies, or topical agents. [The present invention 1011] An injection device or container comprising any combination of the present inventions 1001 to 1010. [The present invention 1012] The device or container of the present invention 1011, wherein the container is a vial. [The present invention 1013] The device or container of the present invention 1011, which is an injection device that is a hypodermic needle and syringe, an auto-injector, or a pre-filled syringe. [The present invention 1014] A method for administering to a subject a combination comprising a leptin receptor antagonist associated with a GDF8 antagonist associated with an activin A antagonist, and optionally a pharma- ceutically acceptable carrier or diluent, comprising introducing components of the combination into the body of the subject. [The present invention 1015] The method of claim 1014, wherein said component is administered parenterally. [The present invention 1016] A method for inhibiting LEPR, GDF8, and activin A in a subject's body, comprising administering to the subject a therapeutically effective amount of a combination comprising a leptin receptor antagonist associated with a GDF8 antagonist associated with an activin A antagonist. [The present invention 1017] A method for increasing food intake, fat accumulation, body weight, muscle strength, muscle fiber size, and / or lean mass in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a combination comprising a leptin receptor antagonist in association with a GDF8 antagonist in association with an activin A antagonist. [The present invention 1018] said increase being at the expense of fat mass; The method of the present invention 1017 for increasing lean mass. [The present invention 1019] A method for improving athletic performance in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a combination comprising a leptin receptor antagonist in association with a GDF8 antagonist in association with an activin A antagonist. [The present invention 1020] The method of claim 1019, wherein the subject is undergoing physical therapy. [The present invention 1021] The object is suffers from one or more of the following: malnutrition, failure to thrive, inadequate food intake, eating disorders, cachexia, muscle atrophy or wasting, and muscle damage; and / or undergoing stroke rehabilitation, Any of the methods according to the present invention 1014 to 1020. [The present invention 1022] A method for treating or preventing malnutrition, cachexia, failure to thrive, eating disorders characterized by insufficient caloric intake, muscle atrophy, age-related sarcopenia, or muscle damage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a combination comprising a leptin receptor antagonist in association with a GDF8 antagonist in association with an activin A antagonist. [The present invention 1023] A method for reducing an increase in hepatic triglyceride content in a subject administered a leptin receptor antagonist, comprising administering to the subject a GDF8 antagonist in association with an activin A antagonist in association with the leptin receptor antagonist. [The present invention 1024] (i) the LEPR antagonist is a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of a heavy chain variable region selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2; a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2; An antibody or antigen-binding fragment thereof that specifically binds to LEPR, comprising: and / or an antibody or antigen-binding fragment thereof that binds to the same epitope on LEPR as said antibody or fragment and / or competes with said antibody or fragment for binding to LEPR That is, (ii) the GDF8 antagonist is selected from the group consisting of: a heavy chain variable region comprising CDR-H1, CDR-H2, CDR-H3 of a heavy chain variable region selected from 21-E5; 21-B9; 21-E9; 21-A2; 22-D3; 22-E6; 22-G10; 1A2; 20B12; 58C8; 19F2; 8D12-1; 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P and H4H18508P2; a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of a light chain variable region selected from 21-E5;21-B9;21-E9;21-A2;22-D3;22-E6;22-G10;1A2;20B12;58C8;19F2;8D12-1;4E3-7;9B11-12;4B9;1H4-5;9B4-3;3E2-1;4G3-25;4B6-6;H4H1657N2 or H4H1669P and H4H18508P2; An antibody or antigen-binding fragment thereof that specifically binds to GDF8, comprising: and / or an antibody or antigen-binding fragment thereof that binds to the same epitope on GDF8 as said antibody or fragment and / or competes with said antibody or fragment for binding to GDF8. and / or (iii) the activin A antagonist is selected from the group consisting of: a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N; a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N; An antibody or antigen-binding fragment thereof that specifically binds to activin A, comprising: and / or an antibody or antigen-binding fragment thereof that binds to the same epitope on activin A as said antibody or fragment and / or competes with said antibody or fragment for binding to activin A. That is, Any of the methods of 1014 to 1023 of the present invention. [The present invention 1025] A method for making any of the combinations of inventions 1001 to 1010, comprising co-forming the LEPR antagonist, the GDF8 antagonist, the activin A antagonist, and, optionally, a pharma- ceutically acceptable carrier. [The present invention 1026] A combination that is the product of the method of the present invention 1025. [The present invention 1027] A method for making the device or container of any of inventions 1011 to 1013, comprising introducing the components of the combination into the container or device. [The present invention 1028] A device or container which is the product of the method of the present invention 1027. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention The "LEPR / GDF8 / ActA" combinations of the present invention include compositions or kits, such as pharmaceutical compositions, that include a pharma- ceutically acceptable carrier and one or more LEPR antagonists, one or more GDF8 antagonists, and one or more activin A antagonists, in association with each other and, optionally, with one or more additional therapeutic agents. In one embodiment of the present invention, the LEPR / GDF8 / ActA combination includes the anti-LEPR antibody H4H18457P2 or an antigen-binding fragment thereof (or a variant thereof), the anti-GDF8 antibody H4H1657N2 or an antigen-binding fragment thereof (or a variant thereof), and the anti-ActA antibody H4H10446P2 or an antigen-binding fragment thereof (or a variant thereof) (H4H18457P2 / H4H1657N2 / H4H10446P2).

[0019] The term "associated with" indicates that the components of the LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) are linked together. For example, the LEPR antagonist, GDF8 antagonist, and activin A antagonist may be formulated into a single composition, e.g., for simultaneous delivery, or may be formulated separately into two or more compositions (e.g., and included in a kit). The combination may be, for example, a first composition having a LEPR antagonist co-formulated with a GDF8 antagonist, linked to a second composition having a separately formulated activin A antagonist. Alternatively, the combination may be three separately formulated compositions: a first LEPR antagonist composition, a second GDF8 antagonist composition, and a third activin A antagonist composition. Each component of the combination, when formulated separately, may be administered to a subject at a time that is different from that at which the other components are administered, e.g., each administration may be given non-concurrently (e.g., separately or sequentially) spaced apart over a given period of the treatment regimen. Further, the separate components may be administered to a subject by the same route or by different routes.

[0020] The anti-LEPR, anti-GDF8, and / or anti-ActA antibodies, or antigen-binding fragments thereof, whose sequences appear in the preceding publications are referred to herein. In one embodiment of the present invention, such antibodies or fragments have at least one heavy chain variable domain (VL) whose sequences are referred to herein, except that each may independently have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations and / or point deletions. H ) and / or at least one light chain variable region (V L The anti-LEPR, anti-GDF8, and / or anti-ActA antibodies or fragments may each independently have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations and / or point deletions, and may be any of the V, ... H and / or the anti-LEPR, anti-GDF8, and / or anti-ActA antibodies or fragments may each independently have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations and / or point deletions, and / or the heavy chain CDRs (HCDRs) of V, ... L The polypeptide (e.g., V) may comprise a light chain CDR (HCDR) of the V. Antibodies or fragments containing such mutations may be referred to herein as "variants." L , V H A "variant" of a nucleotide sequence (e.g., HCDR, or LCDR) can include a sequence having at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identity or similarity to a reference sequence when, for example, the comparison is made by a BLAST algorithm, where the parameters of the algorithm are selected to obtain maximum matches between the respective sequences over the entire length of the respective reference sequences (e.g., expectation threshold: 10; word size: 3; maximum match in query range: 0; BLOSUM 62 matrix; gap cost: presence 11, extension 1; conditional composition score matrix adjustment).

[0021] Sequence identity refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids that share similar properties and may be interchangeable are discussed above.

[0022] Leptin receptor (LEPR, OB receptor, OB-R, CD295, or WSX), in one embodiment of the invention, is a human leptin receptor comprising the amino acid sequence set forth in UniProtKB / Swiss-Prot Accession No. P48357.

[0023] GDF8 (growth and differentiation factor-8, MSTN, or myostatin) comprises a protein having the amino acid sequence set forth in UniProtKB / Swiss-Prot Accession No. O14793.

[0024] Activins are homodimeric and heterodimeric molecules that contain beta subunits, namely inhibin βA, inhibin βB, inhibin βθ, and / or inhibin βE. Activin A is a homodimer of two βA subunits, activin B is a homodimer of two βB subunits, activin AB is a heterodimer of one βA subunit and one βB subunit, and activin AC is a heterodimer of one βA subunit and one βθ subunit. In one embodiment of the present invention, the inhibin beta A chain amino acid sequence is described under UniProtKB / Swiss-Prot accession number P08476.

[0025] Lean mass, in one embodiment of the present invention, is as determined using micro-computed tomography (μCT) or dual energy x-ray absorptiometry (DXA).

[0026] General method The standard method in molecular biology is the method described by Sambrook, Fritsch and Maniatis (1982 & 1989 2 nd Edition, 2001 3 rd (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.). Standard methods are also found in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0027] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization have been described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391). The production, purification, and fragmentation of polyclonal and monoclonal antibodies have been described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques are available for characterizing ligand / receptor interactions (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0028] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1999) J. Immunol. 274:27371-27378). (see, e.g., Chothia et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

[0029] An alternative method of humanization is to use human antibody libraries displayed on phage or in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol. 17:397-399). Single chain antibodies and diabodies have been described (see, e.g., Malecki et al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218, Conrath et al. (2001) J. Biol. Chem. 276:7346-7350, Desmyter et al. (2001) J. Biol. Chem. 276:26285-26290, Hudson and Kortt (1999) J. Immunol. Methods 231:177-189, and U.S. Patent No. 4,946,778).Bifunctional antibodies have been provided (e.g., Mack, et al. (1995) Proc. Natl. Acad. Sci. USA 92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel, et al. (2001) Protein Engineering 14:815-823; Segal, et al. (2001) J. Immunol. Methods 248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al. (1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207; Traunecker, et al. (1991) EMBO (See, e.g., J. 10:3655-3659, and U.S. Patent Nos. 5,932,448, 5,532,210, and 6,129,914.) Fully human antibodies can also be developed in genetically engineered mice, such as the VelociMouse. See, e.g., DeChiara et al., Producing fully ES cell-derived mice from eight-cell stage embryo injections, Methods Enzymol, 476:285-94 (2010); Dechiara et al., VelociMouse: fully ES cell-derived F0-generation mice obtained from the injection of ES cells into eight-cell-stage embryos. Methods Mol Biol, 530:311-24 (2009); U.S. Patent Nos. 7,576,259, 7,659,442, or 7,294,754, and US2008 / 0078000A1.

[0030] Purification of antigens is typically not required for the production of antibodies. Animals can be immunized with cells bearing the antigen of interest. Spleen cells can then be isolated from the immunized animals, and the spleen cells can be fused with myeloma cell lines to produce hybridomas (see, for example, Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithana et al. (1999) J. Immunol. 163:5157-5164).

[0031] Antibodies can be conjugated to, for example, small drug molecules, enzymes, liposomes, polyethylene glycol (PEG).Antibodies are useful for treatment, diagnosis, kits, or other purposes, including, for example, antibodies conjugated to dyes, radioisotopes, enzymes, or metals, such as gold colloids (see, for example, Le Doussal et al. (1991) J.Immunol.146:169-175, Gibellini et al. (1998) J.Immunol.160:3891-3898, Hsing and Bishop (1999) J.Immunol.162:2804-2811, Everts et al. (2002) J.Immunol.168:883-889).

[0032] Methods for flow cytometry, including fluorescence-activated cell sorting (FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2.sup.nd ed., Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). For example, fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies for use as diagnostic reagents are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

[0033] Standard methods for immune system histology have been described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0034] For example, software packages and databases for determining antigen fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments are available (e.g., GenBank, Vector NTI.RTM. Suite (Informax, Inc., Bethesda, Md.), GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.), DeCypher.RTM. (TimeLogic Corp., Crystal Bay, Nev.), Menne, et al. (2000) Bioinformatics 16:741-742, Menne, et al. (2000) Bioinformatics Applications Note 16:741-742, Wren, et al. (2002) Comput. Methods Programs Biomed. 68:177-181, von See Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0035] Leptin receptor antagonists The present invention includes LEPR / GDF8 / ActA combinations (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) that include one or more LEPR antagonists. In one embodiment of the invention, the LEPR antagonist is an anti-LEPR antibody or antigen-binding fragment thereof that does not compete with leptin for binding to LEPR.

[0036] LEPR antagonists include antibodies and antigen-binding fragments thereof, as well as other substances (e.g., peptides and small molecules) that specifically bind to LEPR and antagonize one or more biological activities of LEPR. Molecules that specifically bind to LEPR may be referred to as "anti-LEPR". In one embodiment of the invention, LEPR antagonists inhibit LEPR signaling, for example, by binding to human LEPR and antagonizing the activation of LEPR-dependent intracellular signaling cascades. Antagonism of LEPR may be achieved, in one embodiment of the invention, by blocking LEPR / leptin binding, for example, by the formation of a complex between the antagonist and leptin or LEPR or both. LEPR signaling antagonism includes, for example, the reduction of LEPR-dependent transcriptional activation of STAT3. See, for example, Villanueva & Myers, Int. J. Obes. 32 (Suppl 7): S8-12 (2008), and Park & ​​Ahima, F1000 Prime Reports 6: 73 (2014).

[0037] In one embodiment of the invention, the LEPR antagonist is a mutant form of leptin, such as a PEGylated mutant leptin. For example, in one embodiment of the invention, the LEPR antagonist is a mammalian (e.g., human) leptin polypeptide, in which the LDFI hydrophobic binding site is modified by replacing 2-4 amino acid residues of said hydrophobic binding site with different amino acid residues to make said site less hydrophobic, said modified mammalian leptin polypeptide being a leptin antagonist, or a fragment of said modified mammalian leptin polypeptide comprising said altered hydrophobic binding site, said fragment being itself a leptin antagonist. For example, when two or more amino acids of the LDFI motif are replaced with alanine, arginine, aspartic acid, glutamic acid, glycine, lysine, or serine, e.g., having the mutations L39A / D40A / F41A / I42A. Such leptin variants may be PEGylated, for example with one or more 4000-6000 dalton PEG molecules, see US Patent No. 7,307,142.

[0038] In one embodiment of the invention, the LEPR antagonist is an antibody or antigen-binding fragment thereof selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2, or any other antibody or antigen-binding fragment described in WO2018 / 089532.

[0039] In one embodiment of the invention, the anti-LEPR antibody is H4H18457P2.

[0040] In one embodiment of the invention, the anti-LEPR antibody or fragment of the invention comprises: (i) the HCDRs (HCDR1, HCDR2, and HCDR3) (or having one or more variants of the HCDRs) of the heavy chain variable region, and / or the LCDRs (LCDR1, LCDR2, and LCDR3) (or having one or more variants of the LCDRs) of the light chain variable region of an antibody selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2; and / or (ii) a heavy chain variable region (or a variant thereof) and / or a light chain variable region (or a variant thereof) of an antibody selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2; and / or (iii) competes with H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and / or H4H18508P2 for binding to LEPR (e.g., LEPR with a C-terminal myc-myc-His6 tag); and / or (iv) binds to LEPR at the same epitope as H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and / or H4H18508P2. For example, the epitope is the extracellular domain of LEPR, the LEPR CRH (cytokine receptor homology) domain 2, the CRH2 domain, the FNIII (fibronectin type III) domain, or the Ig(D3) domain.

[0041] See International Patent Application Publication No. WO2018 / 89532.

[0042] In one embodiment of the invention, such an antibody or fragment comprises a heavy chain constant domain selected from IgG1, IgG2, IgG3, and IgG4, and / or a light chain constant domain selected from kappa and lambda.

[0043] In one embodiment of the invention, the LEPR antagonist is the antibody H4H18457P2 or an antigen-binding fragment thereof.

[0044] In one embodiment of the invention, the LEPR antagonist is an antibody or antigen-binding fragment thereof, including: (1) the amino acid sequence: V, including TIFF0007660507000001.tif10150 (SEQ ID NO: 1, or a variant thereof) H and / or The amino acid sequence: TIFF0007660507000002.tif10150 (SEQ ID NO: 2, or a variant thereof), L , and / or (2) Its CDR-L, e.g. CDR-L1, comprising the amino acid sequence: Gln Ser Ile Ser Ser Tyr (SEQ ID NO: 3, or a variant thereof); A CDR-L2 comprising the amino acid sequence: Ala Ala Ser (SEQ ID NO: 4, or a variant thereof); and CDR-L3 comprising the amino acid sequence: Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr (SEQ ID NO: 5, or a variant thereof). Including, V L and / or The CDR-H, for example, CDR-H1, comprising the amino acid sequence: Gly Phe Thr Phe Asp Asp Tyr Gly (SEQ ID NO: 6, or a variant thereof); A CDR-H2 comprising the amino acid sequence: Ile Ser Trp Asn Gly Gly Ile Thr (SEQ ID NO: 7, or a variant thereof); and CDR-H3 comprising the amino acid sequence: Ala Arg Ala Arg Tyr Gly Gly Ala Asp Tyr (SEQ ID NO: 8, or a variant thereof). Including, V H , and / or (3) the amino acid sequence: A heavy chain immunoglobulin comprising TIFF0007660507000003.tif34163 (SEQ ID NO: 25) (or a variant thereof), and The amino acid sequence: A light chain immunoglobulin comprising TIFF0007660507000004.tif15163 (SEQ ID NO: 26) (or a variant thereof).

[0045] In one embodiment of the present invention, such a V L is linked to a human kappa or lambda light chain immunoglobulin constant domain and / or H is linked to a human IgG (eg, IgG1, IgG2, IgG3, or IgG4 (eg, S228P mutated IgG4)) heavy chain immunoglobulin constant domain.

[0046] GDF8 antagonists The present invention includes LEPR / GDF8 / ActA combinations (eg, H4H18457P2 / H4H1657N2 / H4H10446P2) that include one or more GDF8 antagonists.

[0047] GDF8 antagonists include antibodies and their antigen-binding fragments, as well as other substances (e.g., peptides and small molecules) that specifically bind to GDF8 and antagonize one or more biological activities of GDF8. Molecules that specifically bind to GDF8 can be referred to as "anti-GDF8". For example, in one embodiment of the present invention, the antagonist blocks the interaction between GDF8 and activin RIIB (or its Fc fusion), or inhibits the SMAD-dependent activation of A204 cells that stably express Smad-dependent (CAGA12) luciferase.

[0048] In one embodiment of the invention, the GDF8 antagonist is a small molecule such as dorsomorphin (Millipore Sigma; St. Louis, Mo.) or LDN-193189 (Millipore Sigma; St. Louis, Mo.).

[0049] In one embodiment of the present invention, the GDF8 antagonist specifically binds to GDF8 but does not bind to other ActRIIB ligands, such as, for example, GDF3, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, activin A, activin B, activin AB, and / or Nodal.

[0050] In one embodiment of the present invention, GDF8 antagonist is anti-GDF8 antibody or its antigen-binding fragment.Anti-GDF8 antibody is mentioned in, for example, US Patent No. 6096506, US Patent No. 7320789, US Patent No. 7261893, US Patent No. 7807159, US Patent No. 7888486, US Patent No. 7635760, US Patent No. 7632499, US Patent Application Publication No. 2007 / 0178095, US Patent No. 2010 / 0166764 and US Patent No. 2009 / 0148436, and International Patent Application Publication No. WO2010 / 070094.

[0051] Anti-GDF8 antibodies are also described in U.S. Patent Application No. 13 / 115,170 (filed May 25, 2011, published as US2011 / 0293630, now U.S. Patent No. 8,840,894), or International Patent Application No. PCT / US2012 / 064911 (filed November 14, 2012 (WO2013 / 074557)), which are incorporated herein by reference in their entirety. The antibodies designated as 1-E9; 21-A2; 22-D3; 22-E6; 22-G10; 1A2; 20B12; 58C8; 19F2; 8D12-1 (or 8D12); 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P, or antigen-binding fragments thereof.

[0052] In one embodiment of the invention, the GDF8 antagonist is an anti-GDF8 antibody or antigen-binding fragment selected from 21-E5; 21-B9; 21-E9; 21-A2; 22-D3; 22-E6; 22-G10; 1A2; 20B12; 58C8; 19F2; 8D12-1 (or 8D12); 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P, or any anti-GDF8 antibody or antigen-binding fragment described in US2011 / 0293630.

[0053] In one embodiment of the invention, the anti-GDF8 antibody is H4H1657N2.

[0054] In one embodiment of the invention, the anti-GDF8 antibody or fragment of the invention comprises: (i) the HCDRs (HCDR1, HCDR2, and HCDR3) (or having one or more variants of the HCDRs) of the heavy chain variable region, and / or the LCDRs (LCDR1, LCDR2, and LCDR3) (or having one or more variants of the LCDRs) of the light chain variable region of an antibody selected from 21-E5; 21-B9; 21-E9; 21-A2; 22-D3; 22-E6; 22-G10; 1A2; 20B12; 58C8; 19F2; 8D12-1 (or 8D12); 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P, and / or (ii) a heavy chain variable region (or a variant thereof) and / or a light chain variable region (or a variant thereof) of an antibody selected from 21-E5; 21-B9; 21-E9; 21-A2; 22-D3; 22-E6; 22-G10; 1A2; 20B12; 58C8; 19F2; 8D12-1 (or 8D12); 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P. and / or (iii) competes with 21-E5; 21-B9; 21-E9; 21-A2; 22-D3; 22-E6; 22-G10; 1A2; 20B12; 58C8; 19F2; 8D12-1 (or 8D12); 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2 or H4H1669P for binding to GDF8; and / or (iv) 21-E5; 21-B9; 21-E9; 21-A2; 22-D3; 22-E6; 22-G10; 1A2; 20B12; 58C8; 19F2; 8D12-1 (or 8D12); 4E3-7; 9B11-12; 4B9; 1H4-5; 9B4-3; 3E2-1; 4G3-25; 4B6-6; H4H1657N2, or H4H1669P bind to GDF8 at the same epitope. For example, the epitope is GDF8 amino acids 1-14, 48-65, 48-69, 48-72, 52-65, 52-72, 56-65, 56-72, and / or 73-90, or the epitope consists of such amino acids (e.g., including a C-terminal tag such as biotin).

[0055] See published U.S. patent application US2011 / 0293630. In one embodiment of the invention, such an antibody or fragment comprises a heavy chain constant domain selected from IgG1, IgG2, IgG3, and IgG4, and / or a light chain constant domain selected from kappa and lambda.

[0056] The antibody H4H1657N2 may be referred to as REGN1033 or trevoglumab.

[0057] In one embodiment of the invention, the GDF8 antagonist is an antibody or antigen-binding fragment thereof, including: (1) the amino acid sequence: TIFF0007660507000005.tif10150 (SEQ ID NO: 9, or a variant thereof), H , and The amino acid sequence: V, including TIFF0007660507000006.tif10150 (SEQ ID NO: 10, or a variant thereof) L and / or (2) Its CDR-L, e.g. CDR-L1, comprising the amino acid sequence: Gln Asp Ile Ser Asp Tyr (SEQ ID NO: 11, or a variant thereof); CDR-L2, comprising the amino acid sequence: Thr Thr Ser (SEQ ID NO: 12, or a variant thereof); CDR-L3 comprising the amino acid sequence: Gln Lys Tyr Asp Ser Ala Pro Leu Thr (SEQ ID NO: 13, or a variant thereof). Including, VL , and / or The CDR-H, for example, CDR-H1, comprising the amino acid sequence: Gly Phe Thr Phe Ser Ala Tyr Ala (SEQ ID NO: 14, or a variant thereof); CDR-H2, comprising the amino acid sequence: Ile Ser Gly Ser Gly Gly Ser Ala (SEQ ID NO: 15, or a variant thereof); CDR-H3 comprising the following amino acid sequence: Ala Lys Asp Gly Ala Trp Lys Met Ser Gly Leu Asp Val (SEQ ID NO: 16, or a variant thereof). Including, V H , and / or (3) the amino acid sequence: A heavy chain immunoglobulin comprising TIFF0007660507000007.tif34163 (SEQ ID NO: 27) (or a variant thereof), and The amino acid sequence: A light chain immunoglobulin comprising TIFF0007660507000008.tif16163 (SEQ ID NO: 28) (or a variant thereof).

[0058] In one embodiment of the present invention, such a V L is linked to a human kappa or lambda light chain immunoglobulin constant domain and / or H is linked to a human IgG (eg, IgG1, IgG2, IgG3, or IgG4 (eg, S228P mutated IgG4)) heavy chain immunoglobulin constant domain.

[0059] Activin A antagonists The present invention includes LEPR / GDF8 / ActA combinations (eg, H4H18457P2 / H4H1657N2 / H4H10446P2) that include one or more activin A antagonists.

[0060] Activin A antagonists include antibodies and antigen-binding fragments thereof, as well as other substances (e.g., peptides and small molecules) that specifically bind to activin A and antagonize one or more biological activities of activin A. Molecules that specifically bind to activin A may be referred to as "anti-activin A" or "anti-ActA." In one embodiment of the present invention, such ActA antagonists are: (i) disrupting the interaction between activin A and an activin A receptor (e.g., activin type IIA receptor, activin type IIB receptor, activin type I receptor, etc.); (ii) preventing the formation of an activin-activin receptor complex; and / or (iii) resulting in inhibition of at least one biological function of activin A, such as phosphorylation and activation of type I activin receptors and phosphorylation of SMAD2 and 3 proteins.

[0061] Activin A antagonists, such as antibodies and antigen-binding fragments thereof, as well as other substances (e.g., peptides), specifically bind to activin A or its βA subunit. Antigen-specific binding proteins that specifically bind to the βA subunit can recognize both activin A (βA / βA homodimer) and activin AB (βA / βB heterodimer). In one embodiment of the present invention, activin A-specific binding proteins bind to both activin A and activin AB (but not activin B). Anti-activin A antibodies and antigen-binding fragments are described, for example, in US2009 / 0234106. A particular anti-activin A antibody is designated "MAB3381" and is commercially available from R&D Systems, Inc, Minneapolis, MN. MAB3381 specifically binds to activin A (homodimer) as well as activin AB (heterodimer).

[0062] In one embodiment of the invention, the activin A antagonist is H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P 2 , H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N, or any other antibody or antigen-binding fragment described in WO2015 / 017576.

[0063] In one embodiment of the invention, the anti-activin A antibody is garetosumab.

[0064] In one embodiment of the invention, the anti-activin A antibody or fragment of the invention comprises: (i) H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P 2 , H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N; and / or the HCDRs (HCDR1, HCDR2, and HCDR3) (or having one or more variants of the HCDRs) of the heavy chain variable region, and / or the LCDRs (LCDR1, LCDR2, and LCDR3) (or having one or more variants of the LCDRs) of the light chain variable region of an antibody selected from (ii) H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2 , H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P 2 , H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, or H2aM10965N; and / or (iii) For binding to activin A, H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, and H4H10446P 2 , H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and / or H2aM10965N, and / or (iv)H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2 , H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P 2 , H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and / or H2aM10965N bind to activin A at the same epitope. It can be characterized as:

[0065] See International Patent Application Publication No. WO2015 / 017576. In one embodiment of the invention, such an antibody or fragment comprises a heavy chain constant domain selected from IgG1, IgG2, IgG3, and IgG4, and / or a light chain constant domain selected from kappa and lambda.

[0066] The antibody H4H10446P2 may be referred to as REGN2477 or garetosumab.

[0067] In one embodiment of the invention, the activin A antagonist is an antibody or antigen-binding fragment thereof, including: (1) the amino acid sequence: V, including TIFF0007660507000009.tif10151 (SEQ ID NO: 17, or a variant thereof) H , and The amino acid sequence: V, including TIFF0007660507000010.tif10150 (SEQ ID NO: 18, or a variant thereof) L and / or (2) Its CDR-L, e.g. CDR-L1, comprising the amino acid sequence: Gln Ser Val Ser Ser Ser Tyr (SEQ ID NO: 19, or a variant thereof); A CDR-L2 comprising the amino acid sequence: Gly Ala Ser (SEQ ID NO: 20, or a variant thereof); and CDR-L3 comprising the amino acid sequence: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 21, or a variant thereof). Including, V L , and The CDR-H, for example, CDR-H1, comprising the amino acid sequence: Gly Gly Ser Phe Ser Ser His Phe (SEQ ID NO: 22, or a variant thereof); A CDR-H2 comprising the amino acid sequence: Ile Leu Tyr Thr Gly Gly Thr (SEQ ID NO: 23, or a variant thereof); and CDR-H3 comprising the amino acid sequence: Ala Arg Ala Arg Ser Gly Ile Thr Phe Thr Gly Ile Ile Val Pro Gly Ser Phe Asp Ile (SEQ ID NO: 24, or a variant thereof). Including, V H , and / or (3) the amino acid sequence: A heavy chain immunoglobulin comprising TIFF0007660507000011.tif34163 (SEQ ID NO: 29) (or a variant thereof), and The amino acid sequence: A light chain immunoglobulin comprising TIFF0007660507000012.tif16163 (SEQ ID NO: 30) (or a variant thereof).

[0068] In one embodiment of the present invention, such a V L is linked to a human kappa or lambda light chain immunoglobulin constant domain and / or H is linked to a human IgG (eg, IgG1, IgG2, IgG3, or IgG4 (eg, S228P mutated IgG4)) heavy chain immunoglobulin constant domain.

[0069] Pharmaceutical Compositions The invention includes the LEPR / GDF8 / ActA combinations of the invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) (e.g., comprising one or more (e.g., three) of its components, mixed with a pharma- ceutically acceptable carrier or excipient). See, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984). Methods for making such pharmaceutical formulations, including mixing a pharma- ceutically acceptable carrier or excipient with the component(s), form part of the invention, as do the pharmaceutical compositions produced by such methods.

[0070] The scope of the present invention includes a dried, e.g., lyophilized, LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2), or one or more components thereof, or pharmaceutical composition thereof, comprising a pharma- ceutically acceptable carrier but substantially free of water. In one embodiment of the present invention, the pharmaceutical formulation is aqueous (containing water). In one embodiment of the present invention, the pharmaceutical formulation is sterile.

[0071] Pharmaceutical formulations of therapeutic agents can be prepared, for example, by mixing with acceptable carriers, excipients, or stabilizers in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension (see, e.g., Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse (See, for example, Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0072] The method of administration of the LEPR / GDF8 / ActA combination can vary, including oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal, or intra-arterial.

[0073] The present invention provides a method for administering a pharmaceutical formulation comprising a combination of LEPR / GDF8 / ActA (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) to a subject (e.g., a human), the method comprising introducing the formulation into the subject's body, e.g., into a vein, subcutaneous tissue, or muscle tissue of the subject. For example, the method comprises puncturing the subject's body with a syringe needle and injecting the formulation into the subject's body. Such a method comprises introducing into the subject's body a formulation comprising all three components of the combination that are co-formulated, or, for example, introducing into the subject's body three separately formulated components of the combination.

[0074] The invention provides one or more containers (e.g., a plastic or glass vial, e.g., with a cap, or a chromatography column, hollow needle, or syringe cylinder) containing the LEPR / GDF8 / ActA combination of the invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) or a pharmaceutical composition thereof, including a pharma- ceutically acceptable carrier. The invention includes a method for preparing one or more containers containing the combination, the method comprising introducing the components of the combination into one or more containers, e.g., a single container containing a combination of components that are co-formulated. In one embodiment of the invention, the container(s) are then introduced into a kit.

[0075] The present invention also provides a device, such as an injection device, comprising the LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) of the present invention or a pharmaceutical composition thereof, and a method of using the same. An injection device is a device that introduces a substance into a subject's body via a parenteral route, such as intramuscular, subcutaneous, or intravenous. For example, an injection device can be a syringe (e.g., an autoinjector, which is pre-filled with a pharmaceutical composition or is filled at the time of use, e.g., by a user or clinician), which includes, for example, a cylinder or barrel for holding the fluid to be injected (e.g., comprising an antibody or fragment or a pharmaceutical composition thereof), a needle for puncturing the skin and / or blood vessel for injecting the fluid, and a plunger for pushing the fluid from the cylinder through the needle hole.

[0076] The pharmaceutical compositions disclosed herein may also be administered with a needleless hypodermic injection device, such as those disclosed in U.S. Patent Nos. 6,620,135, 6,096,002, 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Such needleless devices containing the pharmaceutical compositions and methods of use thereof are also part of the present invention.

[0077] The invention includes methods for preparing one or more injection devices (e.g., pre-filled syringes or auto-injectors) containing a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2), which methods include introducing the components of the combination into one or more of such devices, e.g., a single device that contains the components of the combination that are co-formulated. In one embodiment of the invention, the injection device(s) are then introduced into a kit.

[0078] The present invention also includes kits that include the LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). In one embodiment of the present invention, the kit includes each antagonist in a separate container or injection device (e.g., a pre-filled syringe or auto-injector), or all three antagonists co-formulated in a single container or injection device. The kit may include a package insert that includes information about the pharmaceutical compositions and dosage forms in the kit. Generally, such information will assist patients and physicians in effectively and safely using the enclosed pharmaceutical composition. For example, any of the following information about the combination of the present invention may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, cautions, side effects, overdosage, appropriate dosage and administration, method of supply, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0079] Treatment and Administration The LEPR / GDF8 / ActA combination demonstrated a very good ability to produce an increase in lean mass in subjects administered the combination. Such an increase in lean mass may be at the expense of an increase in fat mass. The LEPR / GDF8 / ActA combination produced a greater increase in lean mass versus blockade of GDF8 and ActA alone, with a smaller increase in fat mass compared to that observed with the LEPR antagonist alone. This may be due to calories from fat now being used to build additional muscle.

[0080] The method for administering the LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) to a subject includes introducing the components of the combination into the subject's body in association with each other. Such introduction may be by any acceptable route, for example, parenterally. Such components may be co-formulated in a single composition or may be formulated in separate compositions. The administration of one or more of the components in separate compositions may be separated in time if such administration is part of a regimen. In one embodiment of the invention, the method involves injecting all three components into the subject at once, in another embodiment of the invention, the GDF8 antagonist and the ActA antagonist are formulated together and injected at once with the LEPR antagonist in a separate injection, and in another embodiment of the invention, all three antagonists are injected separately in three individual injections (e.g., subcutaneously, intravenously, or intramuscularly, or a combination of two or three of such routes).

[0081] The scope of the present invention provides a method for increasing food intake, fat accumulation, body weight (e.g., at the expense of fat mass), muscle strength, muscle fiber size, and / or lean mass (e.g., at the expense of fat mass) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0082] Furthermore, the LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) - treating or preventing a disease or condition that is cured or ameliorated, to any extent, by antagonism of (i) LEPR (e.g., hyperleptinemia or increased expression of the OB-R leptin receptor resulting in excessive LEPR signaling), (ii) GDF8 (e.g., muscle atrophy / wasting), and / or (iii) ActA; and / or - treating or preventing a disease or condition that is cured or ameliorated to any extent by causing an increase or a reversal decrease in food intake, adiposity, body weight (e.g., at the expense of fat mass), muscle fiber size, muscle strength, or lean mass (e.g., at the expense of fat mass), It can be used for the treatment of the disease or condition by administering to the subject a therapeutically effective amount of LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).Such diseases or conditions that can be treated or prevented include, for example, malnutrition, stunted growth, inadequate food / calorie intake, eating disorder, cachexia, muscle atrophy / wasting, age-related sarcopenia, and muscle damage.Such conditions and diseases are discussed in detail herein.

[0083] Malnutrition is an example of a condition in a subject that will benefit from LEPR / GDF8 / ActA combination therapy.Malnutrition is a term used to describe any nutritional imbalance, from overnutrition to undernutrition, as found, for example, in hospitals and residential care facilities.For the purpose of the present invention, malnutrition refers to undernutrition (unless otherwise stated).Therefore, the present invention provides a method for treating or preventing malnutrition in a subject, for example, in a hospital or residential care facility, by administering a therapeutically effective amount of LEPR / GDF8 / ActA combination to the subject in need thereof.

[0084] Malnutrition can result from: -Inadequate dietary intake, - increased requirements associated with a pathology that are not adequately compensated for by increased dietary intake (e.g., HIV-infected subjects and those suffering from AIDS (acquired immune deficiency syndrome) or cystic fibrosis may require increased dietary intake to maintain normal body weight); and / or -Complications of an underlying disease that cannot be adequately compensated for by increased dietary intake (e.g., patients with cirrhosis, chronic pancreatitis, lactase deficiency, pancreatic cancer, amyloidosis, celiac disease, Crohn's disease, radiation enteritis, and Addison's disease may have malabsorption due to insufficient digestive agents; patients with cancer or infections (or other diseases) may develop cachexia secondary to the underlying disease (see below)).

[0085] Thus, the present invention includes a method for reversing or halting malnutrition in a subject that is the result of inadequate dietary intake, increased requirements associated with a pathology (e.g., HIV or AIDS), and / or complications of an underlying disease, the method comprising administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0086] Malnutrition is associated with negative outcomes for patients, including higher infection and complication rates, increased muscle weakness, impaired wound healing, longer hospital stay, and increased morbidity and mortality.Therefore, the present invention includes a method for reducing the negative outcomes for subjects associated with malnutrition (e.g., for reducing the likelihood of infection or for preventing impaired wound healing), which comprises administering to subjects a therapeutically effective amount of LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0087] Numerous nutrition screening and assessment tools exist to identify risk of malnutrition and diagnose malnutrition.For example, the Malnutrition Screening Tool (MST) is a simple three-question tool that assesses recent weight and appetite loss that has been validated for use in general medical, surgical, and oncology patients (Anthony, Nutrition screening tools for hospitalized patients. Nutr. Clin. Pract. 2008; 23: 373-382; Ferguson et al., Validation of a malnutrition screening tool for patients receiving radiotherapy. Australas. Radiol. 1999; 43: 325-327). The Mini Nutritional Assessment (MNA) was developed specifically for use among elderly patients (age 65 and older) in hospitals, nursing homes, and the community and is therefore limited to this demographic (Anthony, Nutrition screening tools for hospitalized patients, Nutr. Clin. Pract. 2008;23:373-382; Gibson, Principles of Nutritional Assessment. 2008;23:373-382). nded. Oxford University Press, Inc; New York, NY, USA: 2005). The Nutrition Risk Screening (NRS-2002) uses recent weight loss, decreased BMI, and reduced dietary intake in combination with a subjective assessment of disease severity (based on increased nutritional requirements and / or metabolic stress) to generate a nutritional risk score (Anthony, Nutrition screening tools for hospitalized patients, Nutr. Clin. Pract. 2008;23:373-382). The 4-item Short Nutritional Assessment Questionnaire (SNAQ) has been developed to diagnose malnutrition in hospitalized patients, provide indications for nutritional referral, and outline nutritional treatment plans (Anthony, Nutrition screening tools for hospitalized patients, Nutr. Clin. Pract. 2008;23:373-382; Kruizenga et al., Development and validation of a hospital screening tool for malnutrition: the short nutritional assessment questionnaire (SNAQ) Clin. Nutr. 2005;24:75-82). It has been validated for inpatient and outpatient use, as well as for home-based patients, and does not require calculation of BMI (Kruizenga et al. The SNAQ(RC), an easy traffic light system as a first step in the recognition of undernutrition in residential care. J. Nutr. Health Aging. 2010;14:83-89; Neelemaat et al., Screening malnutrition in hospital outpatients. Can the SNAQ malnutrition screening tool also be applied to this population? Clin. Nutr. 2008;27:439-446).The subjective global assessment (SGA) is one of the most commonly used nutritional assessment tools and assesses nutritional status by completing a questionnaire that includes data on weight change, change in food intake, gastrointestinal symptoms, changes in functional capacity related to malnutrition, as well as assessment of fat and muscle stores, and the presence of edema and ascites (Detsky et al., What is subjective global assessment of nutritional status? J Parenter Enteral Nutr. 1987;11:8-13).

[0088] Hospital malnutrition is a condition that hospitalized subjects experience. Malnutrition can be pre-existing or can occur during the subject's hospitalization. The present invention provides a method for treating or preventing hospital malnutrition in a subject, comprising administering to the subject a therapeutically effective amount of a combination of LEPR / GDF8 / ActA (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0089] Malnutrition is also a common condition that subjects with cystic fibrosis suffer from. A variety of complex factors, both related and unrelated, can cause energy imbalance in patients with cystic fibrosis. The net effect on growth potential varies greatly from patient to patient, depending on the difference in disease expression and with the progression of the disease. The present invention provides a method for treating or preventing malnutrition in subjects with cystic fibrosis, comprising administering to the subject a therapeutically effective amount of LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0090] Failure to thrive in early childhood is a state of undernutrition due to inadequate caloric intake, inadequate caloric absorption, or excess caloric consumption. In newborns, failure to thrive may be associated with common underlying conditions such as short gut, volvulus, and intestinal resection after necrotizing enterocolitis, congenital malabsorption and structural defects of the small intestine, and inadequate food intake. In infants (ages 2-8 months), failure to thrive may be associated with common underlying conditions such as inadequate food intake, neglect, intestinal allergy to cow's milk proteins, esophagitis with gastroesophageal reflux, cystic fibrosis, feeding disorders and / or increased energy requirements in cases of underlying cardiac, neurological, neoplastic, or renal disease, celiac disease, chronic diarrhea in cases of immune system deficiencies, autoimmune enteropathy, postenteritis and malabsorption syndromes, and Munchausen syndrome by proxy. In young children (ages 9-36 months), failure to thrive may be related to common underlying conditions such as inadequate food intake, neglect, celiac disease, cystic fibrosis, feeding disorders and / or increased energy requirements in cases of underlying cardiac, neurological, neoplastic, or renal disease, chronic diarrhea in cases of immune system deficiencies, and Munchausen syndrome by proxy. In children (ages 3-16 years), failure to thrive may be related to common underlying conditions such as inadequate food intake, neglect, psychiatric conditions such as anorexia nervosa, chronic inflammatory bowel disorders, celiac disease, cystic fibrosis, feeding disorders and / or increased energy requirements in cases of underlying cardiac, neurological, neoplastic, or renal disease, chronic diarrhea in cases of immune system deficiencies, and Giardiasis, as well as other chronic enteric infections. The present invention provides a method for treating or preventing early childhood deficiency in a subject (e.g., a newborn, infant, toddler, or child), e.g., characterized by any one or more of the diseases or conditions discussed herein, the method comprising administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0091] Inadequate food intake may be associated with the following conditions: loss of appetite, chronic vomiting, swallowing and chewing disorders, esophageal motility disorders, and shortness of breath, for example, associated with cardiac and pulmonary disorders. The present invention provides a method for treating or preventing inadequate food intake in a subject, for example, associated with the conditions discussed herein, comprising administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0092] Eating disorders characterized by inadequate caloric intake include anorexia nervosa and / or bulimia.The present invention provides a method for treating or preventing anorexia nervosa and / or bulimia in a subject, comprising administering to the subject a therapeutically effective amount of a combination of LEPR / GDF8 / ActA (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).In one embodiment of the present invention, the anorexia is anorexia nervosa, age-related anorexia, anorexia in patients undergoing hemodialysis.

[0093] Cachexia is a complex metabolic syndrome that is often associated with an underlying disease and is characterized by muscle weakness with or without loss of fat mass. The hallmark clinical feature of cachexia is weight loss in adults (corrected for fluid retention) or poor growth in children (excluding endocrine disorders). Anorexia, inflammation, insulin resistance, and increased muscle protein degradation are symptoms that are often associated with cachexia. The present invention provides a method for treating or preventing cachexia (or any symptoms of cachexia) in a subject, for example, at any stage described herein (e.g., refractory cachexia) and / or as defined by any of the standards described herein or in the art, comprising administering to the subject a therapeutically effective amount of a combination of LEPR / GDF8 / ActA (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0094] Cachexia secondary to other diseases, conditions, or treatments includes cachexia secondary to anorexia or other psychiatric eating disorders, pulmonary disease (e.g., chronic obstructive pulmonary disorder (COPD)), chronic kidney disease, infectious disease (e.g., HIV infection or acquired immune deficiency syndrome (AIDS)), congestive heart failure, radiation therapy, cancer (e.g., hepatocellular carcinoma, melanoma, and / or breast cancer), chronic heart failure, autoimmune disorders (e.g., inflammatory bowel disease, lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Crohn's disease, or psoriasis), cystic fibrosis, cardiovascular disease, hypertension, depression, and / or neurodegenerative disorders. The present invention provides a method for treating or preventing cachexia in a subject secondary to any disease or condition, such as any of the diseases or conditions described herein (e.g., cancer), the method comprising administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0095] The International Consensus Statement on the Definition and Classification of Cancer Cachexia, published in May 2011 in Lancet Oncology, established these criteria for the diagnosis of cachexia in cancer patients: (i) greater than 5 percent weight loss over the past 6 months, or (ii) a BMI of less than 20 and any degree of weight loss of more than 2 percent; or (iii) an appendicular skeletal muscle index consistent with sarcopenia (another wasting syndrome) and weight loss of more than 2 percent. Fearon et al., Lancet Oncol. 2011 May;12(5):489-95.

[0096] The stages of cancer cachexia agreed upon by the panel are as follows: Pre-cachexia: Less than 5 percent weight loss and other symptoms such as glucose intolerance or anorexia, Cachexia: weight loss of more than 5 percent or other symptoms and conditions consistent with the diagnostic criteria for cachexia, and Refractory cachexia: Patients experiencing cachexia that no longer responds to cancer treatment, have poor performance scores, and have a life expectancy of less than 3 months.

[0097] Muscle atrophy or wasting occurs in muscles with denervation or inactivity, but is also a systemic response to fasting and various diseases and conditions. Muscle atrophy can be in the form of myopenia, which is a decrease in muscle mass, and / or dynapenia, which is a decrease in muscle strength. These diseases and conditions include sepsis, AIDS, renal and cardiac failure, excess glucocorticoids (e.g., Cushing's syndrome), and trauma, and muscle atrophy also occurs in 80% of cancer patients. In addition, muscle atrophy or wasting can be caused by disuse, immobility, bed rest, injury (e.g., hip fracture), neurodegenerative diseases associated with motor neuron loss, medical or surgical procedures (e.g., hip replacement, knee replacement, etc.), or the need for mechanical ventilation. The present invention provides a method for treating or preventing muscle atrophy or muscle wasting in a subject (e.g., secondary to a disease or condition, such as AIDS or cancer), the method comprising administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). In one embodiment of the present invention, the muscle atrophy is sarcopenia, such as age-related sarcopenia, or sarcopenia in subjects undergoing hemodialysis and / or suffering from chronic kidney disease cachexia. Age-related sarcopenia is the degenerative loss of skeletal muscle mass (e.g., about 0.5-1% decline per year after age 50), quality, and strength associated with aging. Thus, the present invention includes a method for treating or preventing muscle atrophy due to age-related sarcopenia.

[0098] Muscle damage can be caused by strain from excessive effort or sudden twisting, such as muscle contusion or torn muscle. Torn muscle can cause swelling, pain, and severe bleeding that can lead to blood clots. Severe torn muscle may require surgery. The muscle stimulating properties of the LEPR / GDF8 / ActA combination make it useful for treating or preventing muscle damage. The present invention provides a method for treating or preventing muscle damage in a subject, the method includes administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0099] Other conditions that may be treated or prevented using the LEPR / GDF8 / ActA combination of the present invention include amyotrophic lateral sclerosis, arthritis, autoimmune disorders, benign and malignant pheochromocytoma, breast cancer, cancer, cardiovascular disease, chronic heart failure, chronic obstructive pulmonary disease, depression, diabetes, hypertension, glucocorticoid-induced myopathy, hepatocellular carcinoma, inflammatory bowel disease, keloids and hypertrophic scars, lupus erythematosus, melanoma, metabolic syndrome, and the like. These include: Rome, multiple sclerosis, muscular dystrophies (e.g., myotonic, Duchenne, Becker, limb-girdle, facioscapulohumeral (FSHD, also known as Landauzy-Dejerine disease), congenital, oculopharyngeal, distal, Emery-Dreifuss, etc.), neurodegenerative disorders, organ atrophy, osteoarthritis, osteopenia, osteoporosis, Parkinson's disease, preeclampsia, psoriasis, pulmonary arterial hypertension, sepsis, and uterine fibroids / leiomyomas.

[0100] The LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) is effective in increasing lean muscle mass and muscle strength, and is therefore effective in enhancing athletic performance. Thus, the present invention provides a method for enhancing athletic performance in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). Athletic performance includes walking speed, sprinting speed, sitting and standing capacity per unit of time (e.g., over 30 seconds), walking distance per unit of time (e.g., 6 minutes), biceps curl weight, chest press weight, stair climbing speed (e.g., time to climb 4 steps), and / or grip strength (e.g., measured using manual dynamometry). In one embodiment of the present invention, the subject is undergoing stroke rehabilitation (for example, stroke hemiplegia rehabilitation) or physical therapy.Therefore, the LEPR / GDF8 / ActA combination of the present invention can be used as an adjunct to any therapeutic procedure where it is desired to improve motor skills, for example, stroke rehabilitation, or for surgery (for example, knee surgery or knee replacement to repair tendon or ligament damage) or recovery from physical injury, for example, physical therapy.

[0101] A subject (e.g., a human) administered a leptin receptor antagonist (e.g., an anti-LEPR antibody) may experience an increase in liver triglyceride levels or serum triglyceride levels.One method of alleviating this increase is to administer an activin A antagonist (e.g., an anti-ActA antibody or an antigen-binding fragment thereof) and a GDF8 antagonist (e.g., an anti-GDF8 antibody or an antigen-binding fragment thereof) in conjunction with the LEPR antagonist.For example, such a method may include administering a therapeutically effective amount of a combination of LEPR / GDF8 / ActA (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0102] A "subject" is a mammal, such as, for example, a human, dog, cat, horse, cow, mouse, rat, monkey (eg, a cynomolgous monkey, such as Macaca fascicularis or Macaca mulatta), or rabbit.

[0103] An effective dose or therapeutically effective dose of the LEPR / GDF8 / ActA combination of the invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) is about 0.1 to about 200 mg / kg (of all three antibodies or antigen-binding fragments), for example, for the treatment or prevention of any of the diseases or conditions discussed herein (e.g., cachexia).

[0104] In certain embodiments, the LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) may be used alone or in association with any other additional therapeutic agents and / or therapeutic procedures that are useful, for example, to increase food intake, fat accumulation, body weight, lean mass (e.g., muscle mass), and / or muscle strength in a subject, and / or to treat or prevent any of the diseases or conditions discussed herein, e.g., cachexia.

[0105] In one embodiment of the present invention, the therapeutic procedure is nasogastric tube feeding.

[0106] In one embodiment of the invention, the additional therapeutic agent is any one or more of an appetite stimulant, a cannabinoid, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, a smooth muscle relaxant, a nitrate, a diuretic, iron, a bronchodilator, an anticholinergic, a corticosteroid, an antibiotic, a nonsteroidal anti-inflammatory drug (NSAID), an immunosuppressant, an HMG-CoA reductase inhibitor, an antidepressant, an anticancer therapy, or a topical agent.

[0107] In one embodiment of the invention, the additional therapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), 6-mercaptopurine, a combination of atorvastatin and amlodipine, a combination of lovastatin and niacin, a combination of simvastatin and ezetimibe, atropine, adalimumab, albuterol, alefacept, alemtuzumab, amitriptyline, anamorelin, arformoterol, aspirin, atorvastatin, atropine, azathioprine, azithromycin, benazepril, betamethasone, budesonide, bumetanide, bufenox ... nin, bupropion, captopril, carboplatin, celecoxib, certolizumab pegol, chlorothiazide, chlorthalidone, cyclosporine, citalopram, clenbuterol, coal tar, coconut oil, corticosteroids, cyproheptadine, cyclophosphamide, dabrafenib, daclizumab, desoximetasone, desvenlafaxine, dexamethasone, diclofenac, dicyclomine, diflunisal, dimebolin, dimethyl fumarate, dithranol, docetaxel, dopexamine, doxorubicin, dronabinol , duloxetine, efalizumab, enalapril, enobosarm, epinephrine, epirubicin, erythromycin, escitalopram, etanercept, etacrinate, etodolac, fentoterol, fingolimod, flunisolide, fluocinonide, fluocortolone, fluoxetine, fluvastatin, formoterol, fosinopril, furosemide, glatiramer acetate, golimumab, hydralazine, hydrochlorothiazide, hydrocortisone, hydrocortisone-17-butyrate, hydrocortisone-17-butyrate Relate, hydroxycarbamide, hyoscyamine, hyoscyamine, ibuprofen, indapamide, indomethacin, infliximab, interferon beta-1a, interferon beta-1b, interleukin-2, ipilimumab, isoetharine, isoprenaline, isoproterenol, isosorbide dinitrate, JX-594, ketoprofen, levosalbutamol, lisinopril, lovastatin, megestrol, megestrol acetate, mepenzolate, mesalazine, methotrexate, methotrexate, methyclothiazide, metolazone,Mevastatin, mitoxantrone, moexipril, nabilone, naproxen, natalizumab, nivolumab, nortriptyline, ocrelizumab, ofatumumab, oral nutritional supplements (e.g. Protibis cookies or dairy-based nutritional supplements), orciprenaline, oxaprozin, paclitaxel, para-aminobenzoic acid, parenteral iron, paroxetine, pembrolizumab, perindopril, phenobarbital, phenobarbital and scopolamine, pirbuterol, piroxicam, pitavastatin, pravastatin, prednisolone, prednisone, procaterol, Any one or more of proline-rich peptide (PRP)-1, psoralen, quinapril, ramapril, ritodrine, rituximab, rosuvastatin, salbutamol, salsalate, scopolamine, sertraline, simvastatin, sorafenib, sulindac, terbutaline, teriflunomide, theophylline, tiotropium, tolmetin, torsemide, trametinib, trandolapril, trazodone, triamcinolone acetonide, triamcinolone alcohol, vemurafenib, venlafacine, venlafaxine, vitamin D3, or vitamin D analogs.

[0108] The present invention also encompasses embodiments in which the LEPR / GDF8 / ActA combination is not associated with additional therapeutic agents and / or treatments. EXAMPLES

[0109] These examples are intended to illustrate, but not limit, the invention. The compositions, e.g., LEPR / GDF8 / ActA combinations, and methods described in the examples form part of the invention.

[0110] Example 1: In vivo efficacy testing of LEPR antagonist antibodies (H4H17322P2, H4H18457P2, and H4H18464P2) in humanized LEPR mice The effects of the three specific antagonist anti-LEPR antibodies of the invention, H4H17322P2, H4H18457P2, and H4H18464P2, on food intake, body weight, and adiposity were examined in monocultured genetically engineered LEPR mice expressing a leptin receptor composed of the human LEPR extracellular domain sequence instead of the mouse LEPR extracellular domain sequence. Hu / Hu Determined in mice.

[0111] Baseline daily food intake was measured 5–1 days before treatment (days −5 and −1). Body composition, including adiposity, was quantified by μCT 4 days before treatment and 6 days after treatment (days −4 and 6). On day 0, 32 12–13 week-old male LEPRs were Hu / Hu Mice were randomized into 4 groups of 8 mice each based on body weight 1 day pretreatment (day -1). On day 0, each group received a single dose of either 30 mg / kg isotype control antibody, 30 mg / kg H4H17322P2, 30 mg / kg H4H18457P2, or 30 mg / kg H4H18464P2 via subcutaneous injection. The isotype control antibody does not bind to any known mouse protein. Body weight was measured for each animal throughout the study period (Table 1A). The percent change in body weight from day 0 was calculated for each animal at each time point. Table 1B summarizes the mean fat and lean mass of animals in each antibody treatment group, quantified by μCT, 6 days before and 6 days after antibody treatment. All results are expressed as mean ± SEM. Additionally, plasma leptin was quantified both pre-dose and on day 6 (Table 1C).

[0112] Mice treated with anti-LEPR antagonist antibodies demonstrated an increase in the percent change in food intake (data not shown) and change in body weight (Table 1A). These increases were not observed with isotype control antibody treatment. Mice treated with 30 mg / kg H4H17322P2 showed a significant increase in food intake starting one day after treatment (day 1) and at subsequent time points compared to mice injected with isotype control antibody. Mice treated with 30 mg / kg H4H18457P2 showed a significant increase in food intake starting on day 2 and at subsequent time points (except day 4) compared to mice injected with isotype control antibody. Mice treated with 30 mg / kg H4H18464P2 showed a significant increase in food intake starting on day 2 and at subsequent time points (except day 4) compared to mice injected with isotype control antibody. Mice treated with 30mg / kg H4H17322P2 showed a significant increase in weight change rate starting 4 days after treatment (day 4) and at subsequent time points compared to mice injected with isotype control antibody. Mice treated with 30mg / kg H4H18457P2 showed a significant increase in weight change rate starting on day 3 and at subsequent time points compared to mice injected with isotype control antibody. Mice treated with 30mg / kg H4H18464P2 showed a significant increase in weight change rate starting on day 4 and at subsequent time points compared to mice injected with isotype control antibody. As shown in Table 1B, there was no difference in fat mass between groups before treatment (day -4). Mice treated with 30 mg / kg H4H17322P2, H4H18457P2, and H4H18464P2 antibodies showed a significant increase in fat mass and leptin levels after 6 days of treatment (day 6) compared to isotype control antibody (Table 1C). In conclusion, treatment with LEPR antagonist antibodies, but not isotype control antibody, increased food intake, body weight, fat storage, and leptin levels in mice.

[0113] (Table 1A) Weight (difference from baseline (g)) TIFF0007660507000013.tif58161REGN1945: Anti-Fel d1(IgG4) SEM: Standard error of the mean

[0114] (Table 1B) Body composition TIFF0007660507000014.tif26166

[0115] Table 1C: Plasma leptin (pg / mL) TIFF0007660507000015.tif24164

[0116] Example 2: Anti-GDF8 mAb (REGN1033), anti-activin A mAb (REGN2477), and LEPR antagonist mAb (H4H1845) in 20-24 week old male mice 7P 2) Combined treatment The effects of the specific antagonist anti-LEPR antibody, H4H18457P2, in combination with the anti-MSTN (also called anti-GDF8) and anti-INHBA (also called anti-activin A) blocking antibodies, H4H1657N2 (REGN1033) and H4H10446P2 (REGN2477), respectively, of the present invention on food intake, body weight, body composition, individual tissue weights, and ex vivo muscle force development were examined in singly housed, 20-24 week old, male genetically engineered LEPR mice expressing a leptin receptor composed of the human LEPR extracellular domain sequence instead of the mouse LEPR extracellular domain sequence. Hu / Hu Determined in mice.

[0117] Baseline daily food intake was measured on days -8 to 0. Baseline total body lean mass and fat mass were quantified by NMR (nuclear magnetic resonance) on days -5 or -1. On day 0, mice were stratified into 4 groups of 11-12 mice each based on body composition on day -5 and body weight on day 0. Beginning on day 0, each group received their respective antibody treatment dose via subcutaneous injection. REGN1033, REGN2477, and their respective IgG4 PmAb at 10 mg / kg twice weekly, as well as H4H18457P2 and its respective IgG4 P mAbs were administered at 30 mg / kg once a week. Thus, any dose of test antibody administered to test mice was administered in parallel with a corresponding dose of control antibody in control mice. Isotype control (IgG4 P )( p indicates S228P mutation, Eu numbering) The antibody did not bind to any known mouse protein. The isotype control antibody was REGN1945.

[0118] Treatment Group a) IgG4 P Control (10mg / kg+10mg / kg, 2x / week; 30mg / kg, 1x / week), N=11 b) REGN1033+REGN2477 (10mg / kg+10mg / kg, 2× / week), N=11 c) H4H18457P2 (30mg / kg, 1× / week), N=11 d) REGN1033+REGN2477+H4H18457P2 (10mg / kg+10mg / kg, 2× / week; 30mg / kg, 1× / week).

[0119] Food intake (Table 2A) and body weight (Table 2B) were measured for each animal throughout the study period. Body composition (Tables 2C and 4A-4D) was quantified on days 6 or 7 and 13 or 14. On days 22, 23, or 24, animals were euthanized and ex vivo force measurements were performed on isolated tibialis anterior muscles (Tables 3A and 3B). Individual organ and skeletal muscle weights were also quantified (Tables 5-7).

[0120] Mice treated with H4H18457P2 alone or in combination with REGN1033 and REGN2477 showed a significant increase in cumulative food intake starting 7 days after treatment (day 7) and at subsequent time points compared to mice injected with isotype control antibody (Table 2A). Mice treated with REGN1033 and REGN2477 showed similar cumulative food intake compared to mice injected with isotype control antibody (Table 2A). Mice treated with H4H18457P2 alone or in combination with REGN1033 and REGN2477 showed an increase in body weight starting 7 days after administration (day 7) and at subsequent time points compared to mice treated with isotype control antibody and compared to mice treated with REGN1033 and REGN2477 (Table 2B). Mice treated with REGN1033 and REGN2477 did not show a significant change in body weight compared to mice treated with isotype control antibody (Table 2B). A significant increase in lean mass from baseline (day -1) was exhibited by mice in each treatment group on days 6, 13, and 20 compared to mice injected with isotype control (Table 4C). Mice treated with H4H18457P2, REGN1033, and REGN2477 showed increased lean mass gain from baseline on days 6, 13, and 20 compared to mice treated with REGN1033 and REGN2477 (Table 4C). Mice treated with REGN1033 and REGN2477 showed a significant decrease in fat mass change from baseline compared to mice administered isotype control antibody. Mice treated with H4H18457P2, either alone or in combination with REGN1033 and REGN2477, showed increased fat mass change from baseline on days 6, 13, and 20 compared to mice receiving an isotype control antibody and compared to mice treated with REGN1033 and REGN2477 (Table 4A).Mice treated with H4H18457P2 in combination with REGN1033 and REGN2477 showed significantly reduced fat mass gain from baseline on days 6, 13, and 20 compared to mice treated with REGN1033 and REGN2477 (Table 4A). Compared to mice receiving an isotype control antibody, mice treated with REGN1033 and REGN2477, as well as mice treated with H4H18457P2, REGN1033, and REN2477, showed increased skeletal muscle twitch force and peak tetanic force of isolated tibialis anterior (Table 3A). Thus, mice treated with REGN1033 and REGN2477, as well as mice treated with H4H18457P2, REGN1033, and REGN3477, showed increased weights of skeletal muscles (quadriceps, tibialis anterior, and gastrocnemius) compared to mice administered isotype control antibody (Table 5). Mice treated with H4H18457P2 alone and in combination with REGN1033 and REGN2477 showed increased weights of inguinal tissue, gonadal tissue, and brown adipose tissue compared to mice administered isotype control antibody (Table 5). Brown adipose tissue weight (Table 5) was significantly increased in mice treated with H4H18457P2 in combination with REGN1033 and REGN2477 compared to mice treated with REGN1033 and REGN2477. No significant changes in heart and liver weights were detected between treatment groups compared to isotype control administration (Table 5). Collectively, these data demonstrate that LEPR antagonist H4H18457P2 increases food intake, body weight, and fat mass in mice. Furthermore, treatment with LEPR antagonist H4H18457P2 in combination with anti-MSTN (REGN1033) and anti-ActA (REGN2477) blocking antibodies results in increased food intake, body weight, lean mass, fat mass, adipose tissue weight, skeletal muscle weight, and skeletal muscle strength.Compared to REGN1033 and REGN2477 treatment, combined treatment with H4H18457P2, REGN1033, and REGN2477 results in a greater increase in lean mass from baseline and a smaller increase in fat mass from baseline.

[0121] (Table 2A) Food intake (g) TIFF0007660507000016.tif103142

[0122] (Table 2B) Weight (difference from baseline (g)) TIFF0007660507000017.tif103140

[0123] (Table 2C) Change in body composition from baseline TIFF0007660507000018.tif36134

[0124] Table 3A. Twitch force, peak tetanic force, and specific force (mN) TIFF0007660507000019.tif32164

[0125] (Table 3B) Isometric tetanic force (mN) TIFF0007660507000020.tif53148

[0126] (Table 4A) Fat amount (g) TIFF0007660507000021.tif39147

[0127] Table 4B. Fat mass (difference from baseline (g)) TIFF0007660507000022.tif39148

[0128] (Table 4C) Fat-free mass (g) TIFF0007660507000023.tif39146

[0129] Table 4D. Fat-free mass (difference from baseline (g)) TIFF0007660507000024.tif39144

[0130] (Table 5) Organ mass, muscle mass, and fat mass (mg) TIFF0007660507000025.tif62155TA: tibialis anterior muscle, GA: gastrocnemius muscle, WAT: white adipose tissue, BAT: brown adipose tissue.

[0131] Table 6. Organ mass, muscle mass, and fat mass (percentage of starting body weight) TIFF0007660507000026.tif62151

[0132] Table 7. Organ mass, muscle mass, and fat mass (% change from control) TIFF0007660507000027.tif64159

[0133] Example 3: Combined treatment of anti-GDF8 mAb (REGN1033), anti-activin A mAb (REGN2477), and LEPR antagonist mAb (H4H18457P2) in 12-14 week old male mice (+ additional treatment group) The effects of the specific antagonist anti-LEPR antibody, H4H18457P2, in combination with the anti-MSTN (also called anti-GDF8) and anti-INHBA (also called anti-activin A) blocking antibodies of the invention, H4H1657N2 (REGN1033) and H4H10446P2 (REGN2477), respectively, on food intake, body weight, body composition, individual tissue weights, and ex vivo muscle force development were examined in singly housed, 12-14 week old, male genetically engineered LEPR mice expressing a leptin receptor composed of the human LEPR extracellular domain sequence instead of the mouse LEPR extracellular domain sequence. Hu / Hu Determined in mice.

[0134] Baseline daily food intake was measured on days -8 to 0. Baseline total body lean mass and fat mass were quantified by NMR on day -1. On day 0, mice were stratified into 6 groups of 7-8 mice each based on body composition on day -1 and body weight on day 0. Beginning on day 0, each group received their respective antibody treatment dose via subcutaneous injection. REGN1033, REGN2477, and their respective IgG4 P The mAbs are administered at 10 mg / kg twice weekly. P The mAb is administered at 30 mg / kg once a week. Isotype control (IgG4 P ) antibody (REGN1945) does not bind to any known mouse proteins.

[0135] Treatment Group a) IgG4P control (10 mg / kg+10 mg / kg, 2x / week; 30 mg / kg, 1x / week), N=7 b) REGN1033 (10mg / kg, 2× / week), N=7 c) REGN1033+REGN2477 (10mg / kg+10mg / kg, 2× / week), N=7 d) H4H18457P2 (30mg / kg, 1× / week), N=7 e) REGN1033+H4H18457P2 (10mg / kg, 2× / week; 30mg / kg, 1× / week), N=8. f) REGN1033+REGN2477+H4H18457P2 (10mg / kg+10mg / kg, 2× / week; 30mg / kg, 1× / week), N=8.

[0136] Food intake (Table 8B) and body weight (Table 8A) were measured for each animal throughout the study. Body composition was quantified on days 6, 13, and 20 (Tables 8C and 8D). Animals were euthanized on days 21 or 22 for additional analyses including skeletal muscle fiber count and fiber cross-sectional area (Tables 9-11).

[0137] Compared to isotype control antibody, LEPR antagonist H4H18457P2 significantly increased body weight and cumulative food intake when administered alone or in combination with REGN1033 or REGN1033 and REGN2477. Mice treated with H4H18457P2 showed a significant increase in body weight and cumulative food intake starting from day 13 and day 8, respectively, and at subsequent time points, compared to mice administered with isotype control antibody (Tables 8A and 8B). Body weight and cumulative food intake significantly increased starting from day 7 and at subsequent time points with H4H18457P2 and REGN1033 treatment, compared to isotype control antibody administration. Mice treated with H4H18457P2, REGN1033, and REGN2477 also showed an increase in body weight and food intake starting from day 8 and day 7, respectively, until the end of the study, compared to mice administered with isotype control antibody. Mice treated with REGN1033 or REGN1033 and REGN2477 showed no significant changes in body weight or cumulative food intake compared to mice receiving a control antibody.

[0138] Mice treated with H4H18457P2 alone or in combination with REGN1033 or REGN1033 and REGN2477 showed an increase in fat mass from baseline at all measurement time points (days 6, 13, and 20) compared to mice administered an isotype control antibody (Table 8D). Mice treated with H4H18457P2, REGN1033, and REGN2477 showed less increase in fat mass from baseline on days 13 and 20 than mice treated with H4H18457P2 alone or in combination with REGN1033 (Table 8D). Mice treated with H4H18457P2, REGN1033, and REGN2477 also showed a significant increase in lean mass increase from baseline on days 13 and 20 compared to mice administered an isotype control antibody (Table 8C).

[0139] Histological analysis revealed no significant effect of either treatment group on muscle fiber number in the tibialis anterior or gastrocnemius muscles compared to isotype control antibody administration (Tables 9 and 10). Compared to isotype control antibody delivery, mice treated with REGN1033 in combination with REGN2477 and H4H18457P2 showed increased muscle fiber number in the soleus muscle (Table 11). In all three muscles examined (tibialis anterior, gastrocnemius, and soleus), H4H18457P2 treatment did not affect muscle fiber area compared to isotype control antibody administration (Tables 9, 10, and 11). Mice treated with REGN1033, REGN1033 and REGN2477, REGN1033 and H4H18457P2, or REGN1033, REGN2477 and H4H18457P2 showed increased muscle fiber area in the tibialis anterior and gastrocnemius muscles compared to mice receiving an isotype control antibody (Tables 9 and 10). In the soleus muscle, only mice treated with the triple combination of H4H18457P2, REGN1033, and REN2477 showed increased muscle fiber area compared to mice receiving an isotype control antibody (Table 11).

[0140] In summary, LEPR antagonist antibody treatment alone increases body weight, food intake, and fat accumulation, while combination with anti-ActA and / or anti-MSTN blocking antibodies leads to further increases in muscle fiber area or number, respectively.

[0141] (Table 8A) Weight (g) TIFF0007660507000028.tif99170

[0142] Table 8B: Cumulative food intake TIFF0007660507000029.tif96170

[0143] Table 8C. Lean Mass (difference from baseline (g)) TIFF0007660507000030.tif38170

[0144] Table 8D. Fat mass (difference from baseline (g)) TIFF0007660507000031.tif37170

[0145] Table 9. Area and number of TA muscle fibers TIFF0007660507000032.tif32170

[0146] Table 10. Area and number of GA muscle fibers TIFF0007660507000033.tif32170

[0147] Table 11. Area and number of soleus muscle fibers TIFF0007660507000034.tif31170

[0148] Example 4: Anti-GDF8 mAb (REGN1033), anti-activin A mAb (REGN2477), and LEPR antagonist mAb (H4H1845) in male mice 7P 2) Combined treatment The effects of the specific antagonist anti-LEPR antibody, H4H18457P2, in combination with anti-MSTN (also known as GDF8) and anti-INHBA (also known as activin A) blocking antibodies, H4H1657N2 (REGN1033) and H4H10446P2 (REGN2477), respectively, on food intake, body weight, body composition, individual tissue weights, and ex vivo muscle force development were examined in singly housed, 12- to 14-week-old male genetically engineered LEPR mice expressing a leptin receptor composed of the human LEPR extracellular domain sequence instead of the mouse LEPR extracellular domain sequence. Hu / Hu Determined in mice.

[0149] Baseline daily food intake was measured on days -8 to 0. Baseline total body lean mass and fat mass were quantified by NMR on day -1. On day 0, mice were stratified into 6 groups of 7-8 mice each based on body composition on day -1 and body weight on day 0. Beginning on day 0, each group received their respective antibody treatment dose via subcutaneous injection. REGN1033, REGN2477, and their respective IgG4 P The mAbs were administered at 10 mg / kg twice weekly. P The mAb was administered at 30 mg / kg once a week. Isotype control (IgG4 P ) The antibody does not bind to any known mouse proteins.

[0150] Treatment Group a) IgG4P control (10 mg / kg+10 mg / kg, 2x / week; 30 mg / kg, 1x / week), N=7 b) REGN1033 (10mg / kg, 2× / week), N=7 c) REGN1033+REGN2477 (10mg / kg+10mg / kg, 2× / week), N=7 d) H4H18457P2 (30mg / kg, 1× / week), N=7 e) REGN1033+H4H18457P2 (10mg / kg, 2× / week; 30mg / kg, 1× / week), N=8. f) REGN1033+REGN2477+H4H18457P2 (10mg / kg+10mg / kg, 2× / week; 30mg / kg, 1× / week), N=8.

[0151] Food intake and body weight were measured for each animal throughout the study. Body composition was quantified on days 6, 13, and 20. On days 21 or 22, animals were euthanized and individual organs and skeletal muscle tissue were weighed and harvested for additional analyses, including liver triglyceride quantification and skeletal muscle fiber number and cross-sectional area.

[0152] Quantification of liver triglyceride content revealed that mice treated with H4H18457P2 alone or in combination with REGN1033 showed increased liver triglyceride content compared to mice administered isotype control antibody (Table 12). In contrast, liver triglyceride content was not increased in mice treated with H4H18457P2 in combination with REGN1033 and REGN2477 compared to mice administered isotype control antibody (Table 12). Mice treated with H4H18457P2 in combination with REGN1033 and REGN2477 showed decreased liver triglyceride content compared to mice treated with H4H18457P2 in combination with REGN1033 (Table 12). Taken together, LEPR antagonist antibody treatment increased hepatic triglyceride content that was attenuated by combined treatment with LEPR antagonist, anti-ActA, and anti-MSTN blocking antibodies.

[0153] (Table 12) Liver triglycerides TIFF0007660507000035.tif37170**Data from 2 of 7 mice not included in values ​​shown. *Data from 1 of 7 mice not included in values ​​shown.

Claims

1. A composition comprising an antibody or antigen-binding fragment thereof that specifically binds to a leptin receptor (LEPR), the antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2; A composition comprising an antibody or antigen-binding fragment thereof that specifically binds to growth and differentiation factor-8 (GDF8), comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10; and A composition comprising an antibody or antigen-binding fragment thereof that specifically binds to activin A, the antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

18. A pharmaceutical composition for increasing lean muscle mass, comprising the steps of:

2. The method of claim 1 , wherein at least two of the compositions are co-formulated.

3. The pharmaceutical of claim 1 or 2, wherein the compositions are in separate compositions.

4. The pharmaceutical agent according to any one of claims 1 to 3, which is used in combination with a further therapeutic agent.

5. 5. The pharmaceutical composition of claim 4, wherein the additional therapeutic agent is one or more selected from appetite stimulants, cannabinoids, angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers, smooth muscle relaxants, nitrates, diuretics, iron, bronchodilators, anticholinergics, corticosteroids, antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), immunosuppressants, HMG-CoA reductase inhibitors, antidepressants, anticancer therapies, or topical agents.

6. An injectable device or container used to increase lean muscle mass, comprising a composition according to any one of claims 1 to 5.

7. 7. The device or container of claim 6, wherein the container is a vial.

8. 7. The device or container of claim 6, which is an injection device that is a hypodermic needle and syringe, an autoinjector, or a pre-filled syringe.

9. 1. A pharmaceutical composition for increasing lean muscle mass by administering to a subject, comprising a leptin receptor (LEPR) antagonist in combination with a growth and differentiation factor-8 (GDF8) antagonist and an activin A antagonist, the composition comprising: The LEPR antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:2; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

18. The said medicine.

10. The pharmaceutical composition according to claim 9, wherein the administration is parenteral administration.

11. 1. A pharmaceutical composition for increasing lean muscle mass in a subject by inhibiting leptin receptor (LEPR), growth and differentiation factor-8 (GDF8), and activin A in combination with a GDF8 antagonist and an activin A antagonist, including a leptin receptor antagonist, comprising: The LEPR antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:2; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

18. The said medicine.

12. 1. A medicament for use in combination with a growth and differentiation factor-8 (GDF8) antagonist, including a leptin receptor (LEPR) antagonist, and an activin A antagonist, for increasing food intake, fat deposition, body weight, muscle strength, muscle fiber size, and / or lean mass in a subject in need thereof, comprising: The LEPR antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:2; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

18. The said medicine.

13. The method of claim 12, wherein the increase is for increasing lean mass at the expense of fat mass.

14. 1. A medicament for use in combination with a growth and differentiation factor-8 (GDF8) antagonist, including a leptin receptor (LEPR) antagonist, and an activin A antagonist for improving exercise performance in a subject in need thereof, comprising: The LEPR antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:2; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

18. The said medicine.

15. The method of claim 14, wherein the subject is undergoing physical therapy.

16. The object is The patient is suffering from one or more of the following: malnutrition, failure to thrive, inadequate food intake, eating disorders, cachexia, muscle atrophy or wasting, and muscle damage; and / or undergoing stroke rehabilitation, The pharmaceutical composition according to any one of claims 9 to 15.

17. 1. A medicament for treating or preventing malnutrition, cachexia, failure to thrive, eating disorders characterized by inadequate caloric intake, muscle atrophy, age-related sarcopenia, or muscle damage in a subject in need thereof, characterized in that the medicament is used in combination with a growth and differentiation factor-8 (GDF8) antagonist, including a leptin receptor (LEPR) antagonist, and an activin A antagonist, The LEPR antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:2; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

18. The said medicine.

18. 1. A pharmaceutical composition for reducing an increase in hepatic triglyceride content in a subject administered a leptin receptor (LEPR) antagonist, comprising said leptin receptor antagonist in combination with a growth and differentiation factor-8 (GDF8) antagonist and an activin A antagonist, comprising: The LEPR antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:2; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

18. The said medicine.

19. The pharmaceutical composition according to any one of claims 1 to 5, which is formulated for injection.

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