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 of antagonists of leptin receptor, GDF8 and activin A and methods of using the same. The composition is effective, for example, to increase lean body mass at least in part at the expense of fat mass. Methods of treating malnutrition, cachexia and other conditions characterized by nutritional deficiencies and weight loss are also provided.

Description

[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. Technical Field

[0002] 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

[0004] An official copy of the sequence listing is submitted electronically via EFS-Web in an ASCII formatted sequence listing concurrently with the specification, with the file name "10547WOseqlist_ST25.txt", creation date December 17, 2019, and size of approximately 26KB. The sequence listing contained in this ASCII formatted file is part of the specification and is incorporated herein by reference in its entirety. Background Art

[0005] Growth and differentiation factor-8 (GDF8, also known as myostatin) and activin A are two important regulators of the development and maintenance of skeletal muscle and muscle mass.

[0006] GDF8 is a secretory ligand belonging to the transforming growth factor-β (TGF-β) superfamily of growth factors, which acts as a negative regulator of muscle mass. GDF8 antagonists have been used in adult mice and have a considerable positive effect on skeletal muscle mass. The receptor that binds to GDF8 and negatively regulates muscle mass is the type IIB activin receptor (ACVR2B). GDF8 is not the only negative regulator of muscle mass that works 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" by GDF8, "Nature Communications (Nature Comm.)" 8: 15153 (2017)). 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 synergistically with GDF8 to regulate skeletal muscle mass, and that combined GDF8 and activin A inhibition can effectively treat muscle atrophy in patients with muscle wasting disorders (Latres et al. (2017)).

[0007] Leptin is a polypeptide hormone 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 by 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. Alterations in signaling by leptin or LEPR or both may result in a variety of conditions, including, but not limited to, anorexia or other psychiatric eating disorders, cachexia, autoimmune conditions, cardiovascular disease, and neurodegenerative conditions. Summary of the invention

[0008] The present invention relates to compositions suitable not only for increasing total body mass but also for increasing body mass in a manner that produces a more desirable total body composition. As mentioned, administration of antagonistic anti-LEPR antibodies, anti-GDF8 antibodies, and anti-ActA antibodies (triplet combination) causes an increase in total body weight compared to the total body weight observed with anti-LEPR antibodies or anti-GDF8 antibodies and anti-ActA antibodies alone. Additional beneficial effects were observed with respect to the lean mass of individuals receiving the triple combination. The total lean mass in these individuals increased and the ratio of fat mass to lean mass decreased (relative to anti-LEPR antibodies or anti-GDF8 antibodies and anti-ActA antibodies alone). For example, this increase in lean mass was confirmed when individuals receiving the triple combination exhibited higher muscle mass and larger muscle fiber size (area) in some specific muscle structures analyzed.

[0009] The present invention provides a combination comprising: a leptin receptor (e.g., human leptin receptor) antagonist and a GDF8 (e.g., human GDF8) antagonist and an activin A (e.g., human activin A) antagonist (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2); and optionally a pharmaceutically acceptable carrier. For example, the combination may include a co-formulation comprising at least two antagonists selected from the following: 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 the leptin receptor, GDF8, and / or activin A, respectively. In one embodiment of the present 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 present invention, the LEPR antagonist is An antibody or antigen-binding fragment thereof that specifically binds to LEPR, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising CDR-H1, CDR-H2, CDR-H3 of a heavy chain variable region selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2, and H4H18508P2 and CDR-L1, CDR-L2, CDR-L3 of a light chain variable region selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2 and H4H18508P2, or binds to the same epitope on LEPR as the antibody or fragment and / or competes with the antibody or fragment for binding to LEPR.In one embodiment of the present invention, the GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, wherein the antibody or the antigen-binding fragment thereof comprises: a CDR-H1, a CDR-H2, a CD3-64 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 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, or binds to the same epitope on GDF8 as the antibody or fragment and / or competes with the antibody or fragment for binding to GDF8.In one embodiment of the present invention, the activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, wherein the antibody or the antigen-binding fragment thereof comprises: a peptide selected from the group consisting of H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, The CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and H2aM10965N The heavy chain variable region of R-H3, and a heavy chain variable region selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10443 CDR-L1, CDR-L2, CDR-L3 of the light chain variable region of 10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2 and H2aM10965N, or bind to the same epitope on activin A as the antibody or fragment and / or compete with the antibody or fragment for binding to activin A. In one embodiment of the present invention, the LEPR antagonist is an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof 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 The light chain variable region of H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2 and H4H18508P2, or binds to the same epitope on LEPR as the antibody or fragment and / or competes with the antibody or fragment for binding to LEPR.In one embodiment of the present invention, the GDF8 antagonist is an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable 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. region; and 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, or binds to the same epitope on GDF8 as the antibody or fragment and / or competes with the antibody or fragment for binding to GDF8. In one embodiment of the present invention, the activin A antagonist is an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region; and a light chain variable region selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2 and H2aM10965N, or binds to the same epitope on activin A as the antibody or fragment and / or competes with the antibody or fragment for binding to activin A.The combination optionally includes 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 infusion device (eg, a hypodermic needle and syringe, an automatic injector, or a prefilled syringe) or a container (eg, a vial) comprising a combination of the invention (eg, H4H18457P2 / H4H1657N2 / H4H10446P2).

[0011] The present invention also provides a method of administering the combination of the present invention to an individual (e.g., a human), the method comprising introducing the components of the combination, e.g., parenterally, e.g., by injection using an injection device of the present invention, into the body of the individual. In one embodiment of the present invention, the individual suffers from malnutrition, developmental delay, 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 of inhibiting LEPR, GDF8 and activin A in the body of an individual (e.g., a human), the method comprising administering to the individual, e.g., parenterally, e.g., by infusion using an infusion device of the present invention, a therapeutically effective amount of a combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). In one embodiment of the present invention, the individual suffers from malnutrition, developmental delay, insufficient food intake, eating disorders, cachexia, muscle atrophy or wasting and muscle damage, and / or is undergoing physical therapy.

[0013] The present invention also provides a method of increasing food intake, fat mass, body weight, muscle strength, muscle fiber size or lean mass (e.g., at the expense of fat mass) in an individual in need thereof, the method comprising administering to the individual (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 individual suffers from malnutrition, developmental delay, inadequate food intake, eating disorders, cachexia, muscle atrophy or wasting and muscle damage, and / or is undergoing physical therapy.

[0014] The present invention also provides a method for enhancing the athletic performance of an individual (e.g., a human) in need thereof, the method comprising administering to the individual a therapeutically effective amount of a combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). In one embodiment of the present invention, the individual suffers from malnutrition, developmental delay, inadequate food intake, eating disorders, cachexia, muscle atrophy or wasting, and muscle damage, and / or is undergoing physical therapy (e.g., stroke rehabilitation).

[0015] The present invention provides a method of reducing increased hepatic triglyceride levels in a subject administered a leptin receptor antagonist, the method comprising administering to the subject a GDF8 antagonist and an activin A antagonist (eg, H4H18457P2 / H4H1657N2 / H4H10446P2) in combination with the leptin receptor antagonist.

[0016] The present invention also provides a method for treating or preventing malnutrition, cachexia, developmental delay, eating disorders characterized by inadequate caloric intake, muscle atrophy, age-related sarcopenia, or muscle damage in an individual (e.g., a human) in need thereof, the method comprising administering to the individual a therapeutically effective amount of a combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). For example, in one embodiment of the invention, (i) an individual with malnutrition has hospital malnutrition, childhood developmental delay, an eating disorder characterized by inadequate caloric intake, anorexia and / or bulimia; (ii) an individual with cachexia has or is experiencing anorexia, bulimia, an eating disorder, lung disease, chronic obstructive pulmonary disease (COPD), chronic kidney disease, infectious disease, HIV infection, acquired immunodeficiency syndrome (AIDS), congestive heart failure, radiation therapy, cancer, hepatocellular carcinoma, melanoma, breast cancer, autoimmune disorders, inflammatory bowel disease, lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Crohn's disease, psoriasis, cystic fibrosis, cardiovascular disease, elevated blood pressure, depression, and / or a neurodegenerative disorder; and / or (iii) an individual with muscle wasting or wasting has or is experiencing sepsis, AIDS, kidney failure, heart failure, glucocorticoid overdose, Cushing's syndrome, syndrome), trauma, muscle disuse, immobility, bed rest, injury, hip fracture, hip replacement, knee replacement, and / or mechanical ventilation.

[0017] Methods of making the combinations of the invention (eg, H4H18457P2 / H4H1657N2 / H4H10446P2) comprising co-formulating a LEPR antagonist, a GDF8 antagonist, and an activin A antagonist and a pharmaceutically acceptable carrier are also part of the invention.

[0018] The invention also provides a method of making a device or container comprising a combination of the invention (eg H4H18457P2 / H4H1657N2 / H4H10446P2), the method comprising introducing the components of the combination into the container or device. DETAILED DESCRIPTION

[0019] The "LEPR / GDF8 / ActA" combination of the present invention includes a composition or a kit, such as a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more LEPR antagonists, one or more GDF8 antagonists and one or more activin A antagonists in combination with each other, and optionally one or more additional therapeutic agents. In one embodiment of the present invention, the LEPR / GDF8 / ActA combination comprises anti-LEPR antibody H4H18457P2 or an antigen-binding fragment thereof (or a variant thereof), anti-GDF8 antibody H4H1657N2 or an antigen-binding fragment thereof (or a variant thereof), and anti-ActA antibody H4H10446P2 or an antigen-binding fragment thereof (or a variant thereof) (H4H18457P2 / H4H1657N2 / H4H10446P2).

[0020] The term "and (in association with)" indicates that the components of the LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) of the present invention are matched. For example, the LEPR antagonist, the GDF8 antagonist, and the activin A antagonist can be formulated into a single composition, for example, for simultaneous delivery, or separately formulated into two or more compositions (e.g., and included in a kit). For example, the combination can be a first composition having a LEPR antagonist co-formulated with a GDF8 antagonist, which is matched with a second composition having a separately formulated activin A antagonist. Alternatively, the combination can be three separately formulated compositions—a first LEPR antagonist composition, a second GDF8 antagonist composition, and a third activin A antagonist composition. When separately formulated, each component of the combination can be administered to an individual at a different time than when the other components are administered; for example, each administration can be given at different times (e.g., separately or sequentially) at certain intervals within a given time period in a treatment regimen. Furthermore, the separate components may be administered to an individual by the same or different routes.

[0021] The present invention refers to anti-LEPR antibodies, anti-GDF8 antibodies and / or anti-ActA antibodies or antigen-binding fragments thereof whose sequences appear in previous publications. In one embodiment of the present invention, the antibody or fragment comprises at least one heavy chain variable domain (V H ) and / or at least one light chain variable region (V L), the sequences of the domains and / or regions 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. The anti-LEPR antibody, anti-GDF8 antibody and / or anti-ActA antibody or fragment may include V H The heavy chain CDRs (HCDRs) of the present invention 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; and / or the anti-LEPR antibody, anti-GDF8 antibody and / or anti-ActA antibody or fragment may include V L The light chain CDR (HCDR) of the present invention is 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. Antibodies or fragments comprising such mutations may be referred to herein as "variants". "Variants" of polypeptides (e.g., V L 、V H , HCDR or LCDR) can comprise 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 the referenced sequence; for example, when the comparison is performed by the BLAST algorithm, wherein the parameters of the algorithm are selected to give maximum match between corresponding sequences over the full length of the corresponding reference sequence (e.g., expectation threshold: 10; word length: 3; maximum match within query range: 0; BLOSUM 62 matrix; gap cost: existence 11, extension 1; conditional combination score matrix adjustment).

[0022] Sequence identity refers to the degree to which the amino acids in two polypeptides at equivalent positions are identical when the two sequences are optimally aligned. Sequence similarity includes identical residues and biochemically related amino acids that are not identical. Biochemically related amino acids that share similar properties and are interchangeable are discussed above.

[0023] In one embodiment of the present invention, the leptin receptor (LEPR, OB receptor, OB-R, CD295 or WSX) is a human leptin receptor comprising the amino acid sequence as set forth in UniProtKB / Swiss-Prot Accession No. P48357.

[0024] GDF8 (growth and differentiation factor-8, MSTN or myostatin) includes the protein having the amino acid sequence set forth under UniProtKB / Swiss-Prot accession number 014793.

[0025] Activins are homodimeric and heterodimeric molecules comprising β subunits, i.e., inhibin βA, inhibin βB, inhibin βθ and / or inhibin βE. Activin A is a homodimer with two βA subunits; activin B is a homodimer with two βB subunits; activin AB is a heterodimer with one βA subunit and one βB subunit; and activin AC is a heterodimer with one βA subunit and one βθ subunit. In one embodiment of the present invention, the inhibin βA chain amino acid sequence is set forth under UniProtKB / Swiss-Prot accession number P08476.

[0026] In one embodiment of the invention, lean mass is measured using micro-computed tomography (μCT) or dual-energy X-ray absorptiometry (DXA).

[0027] General Methods

[0028] Standard molecular biology methods are described by Sambrook, Fritsch and Maniatis (2nd edition, 1982 and 1989, 3rd edition, 2001) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd Supplement, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.). Standard methods also appear in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0029] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization are described (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley & Sons, New York). Chemical analysis, chemical modifications, post-translational modifications, fusion protein production, protein glycosylation are described (see, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley & Sons, New York; Ausubel, et al. (2001) Modern Experimental Techniques in Molecular Biology, Vol. 3, John Wiley & Sons, New York, 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 are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley & Sons, New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley & Sons, New York).

[0030] Monoclonal antibodies, polyclonal antibodies, 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). 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. (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).

[0031] An alternative to humanization is to use human antibody repertoires presented 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). Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnology 17:397-399). Single-chain antibodies and bifunctional antibodies are 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. Pat. No. 4,946,778).Bifunctional antibodies are provided (see, 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) Prot. Eng. 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. 272:276-23; Morrison (1985) Science 229:1202-1207; Traunecker et al. (1991) EMBO J. J.) 10:3655-3659; and U.S. Pat. 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., Veloci Mouse: 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. Pat. Nos. 7,576,259; 7,659,442; or 7,294,754 and US 2008 / 0078000A1.

[0032] Antigen purification is usually not required for antibody production. Animals can be immunized with cells carrying the antigen of interest. Subsequently, spleen cells can be isolated from immunized animals, and spleen cells can be fused with myeloma cell lines to produce hybrid tumors (see, for example, Meyaard et al. (1997) "Immunity" 7:283-290; Wright et al. (2000) "Immunity" 13:233-242; Preston et al., same document; Kaithamana et al. (1999) "Journal of Immunology" 163:5157-5164).

[0033] The antibody can be conjugated to, for example, a small drug molecule, an enzyme, a liposome, polyethylene glycol (PEG). The antibody is suitable for use in treatment, diagnosis, kits, or other purposes, and includes antibodies coupled to, for example, dyes, radioisotopes, enzymes, or metals (e.g., colloidal gold) (see, e.g., 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).

[0034] Flow cytometric methods including fluorescence activated cell sorting (FACS) are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley & Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd Supplement; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley & Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, polypeptides and antibodies including nucleic acid primers and probes for use as, for example, diagnostic reagents are available (Molecular Probes (2003) Catalog, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalog, St. Louis, Mo.).

[0035] Standard immunohistochemical methods are 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, Philadelphia, Pa.; Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0036] Software packages and libraries for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments are available (see, e.g., GenBank, Vector NTI.RTM. Suite (Informax, Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, San Diego, Calif.); DeCypher.RTM. (TimeLogic, 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 in Biomedicine. (1983) European Journal of Biochemistry (Eur. J. Biochem.) 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0037] Leptin receptor antagonists

[0038] The present invention includes LEPR / GDF8 / ActA combinations including one or more LEPR antagonists (eg, H4H18457P2 / H4H1657N2 / H4H10446P2). In one embodiment of the present invention, the LEPR antagonist is an anti-LEPR antibody or antigen-binding fragment thereof that does not compete with leptin for binding to LEPR.

[0039] LEPR antagonists include antibodies and antigen-binding fragments thereof that specifically bind to LEPR and antagonize one or more biological activities of LEPR, as well as other substances (e.g., peptides and small molecules). Molecules that specifically bind to LEPR can be referred to as "anti-LEPR". In one embodiment of the present invention, LEPR antagonists inhibit LEPR signaling, for example, by binding to human LEPR and antagonizing the activation of LEPR-dependent intracellular signaling cascades. In one embodiment of the present invention, LEPR antagonism can be achieved by blocking LEPR / leptin binding, for example, by forming a complex between an antagonist and leptin or LEPR or both. LEPR signaling antagonism includes, for example, a reduction in LEPR-dependent transcriptional activation of STAT3. See, for example, Villanueva and Myers, International Journal of Obesity (Int. J. Obes.) 32 (Supplement 7): S8-12 (2008) and Park and Ahima, F1000 Report (F1000 Prime Reports) 6: 73 (2014).

[0040] 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, wherein the LDFI hydrophobic binding site is modified so that two to four amino acid residues of the hydrophobic binding site are replaced by different amino acid residues so that the site becomes less hydrophobic, and the modified mammalian leptin polypeptide is a leptin antagonist; or a fragment of the modified mammalian leptin polypeptide comprises the altered hydrophobic binding site, wherein the fragment itself is a leptin antagonist. For example, wherein two or more amino acids of the LDFI motif are replaced by alanine, arginine, aspartic acid, glutamic acid, glycine, lysine, or serine, such as with mutations L39A / D40A / F41A / I42A. The leptin mutation can be pegylated, for example, with one or more 4000-6000 Dalton PEG molecules. See U.S. Patent No. 7307142.

[0041] In one embodiment of the present invention, the LEPR antagonist is an antibody or antigen-binding fragment selected from H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2 and H4H18508P2; or any other antibody or antigen-binding fragment described in WO2018 / 089532.

[0042] In one embodiment of the present invention, the anti-LEPR antibody is H4H18457P2.

[0043] In one embodiment of the present invention, the anti-LEPR antibody or fragment of the present invention comprises:

[0044] (i) a HCDR (HCDR1, HCDR2 and HCDR3) (or a variant having one or more of the HCDRs) in the heavy chain variable region and / or a LCDR (LCDR1, LCDR2 and LCDR3) (or a variant having one or more of the LCDRs) in the light chain variable region of an antibody selected from the group consisting of H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2 and H4H18508P2; and / or

[0045] (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;

[0046] and / or characterized by:

[0047] (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);

[0048] and / or

[0049] (iv) binds to LEPR at the same epitope as H4H17322P2, H4H18437P2, H4H18439P2, H4H18440P2, H4H18457P2, H4H18462P2, H4H18464P2, H4H18466P2 and / or H4H18508P2, e.g., wherein the epitope is a LEPR extracellular domain, a LEPR CRH (cytokine receptor homology) domain 2, a CRH2 domain, a FNIII (fibronectin type III) domain, or an Ig(D3) domain.

[0050] See International Patent Application Publication No. WO2018 / 89532.

[0051] In one embodiment of the present invention, the 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 κ and λ.

[0052] In one embodiment of the present invention, the LEPR antagonist is antibody H4H18457P2 or an antigen-binding fragment thereof.

[0053] In one embodiment of the present invention, the LEPR antagonist is an antibody or an antigen-binding fragment thereof comprising:

[0054] (1) V comprising the following amino acid sequence H :

[0055]

[0056] (SEQ ID NO: 1; or a variant thereof)

[0057] and / or

[0058] V comprising the following amino acid sequence L :

[0059] DIQMTQSPSSLSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLI

[0060] Y AAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPPIT FGQGTRLEIK (SEQ ID NO: 2; or a variant thereof);

[0061] and / or (2)

[0063] Containing its CDR-L, such as the following V L :

[0064] A CDR-L1 comprising the following amino acid sequence: Gln Ser Ile Ser Ser Tyr (SEQ ID NO: 3; or a variant thereof);

[0065] a CDR-L2 comprising the following amino acid sequence: Ala Ala Ser (SEQ ID NO: 4; or a variant thereof); and

[0066] a CDR-L3 comprising the following amino acid sequence: Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr (SEQ ID NO: 5; or a variant thereof); and / or

[0067] Containing its CDR-H, such as the following V H :

[0068] a CDR-H1 comprising the following amino acid sequence: Gly Phe Thr Phe Asp Asp Tyr Gly (SEQ ID NO: 6; or a variant thereof);

[0069] a CDR-H2 comprising the following amino acid sequence: Ile Ser Trp Asn Gly Gly Ile Thr (SEQ ID NO: 7; or a variant thereof); and

[0070] a CDR-H3 comprising the following amino acid sequence: Ala Arg Ala Arg Tyr Gly Gly Ala Asp Tyr (SEQ ID NO: 8; or a variant thereof);

[0071] and / or (3)

[0073] A heavy chain immunoglobulin comprising the following amino acid sequence:

[0074] EVQLVESGGSVVRPGESLRLSCAASGFTFDDYGMSWVRQAPGKGLEWV

[0075] SGISWNGGITVYADSVKGRFTVSRDNAKNSLYLQMNSLRAEDTALYHC

[0076] ARARYGGADYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC

[0077] LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

[0078] LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF

[0079] PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP

[0080] REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKA

[0081] KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP

[0082] ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 25) (or a variant thereof)

[0083] and

[0084] A light chain immunoglobulin comprising the following amino acid sequence:

[0085] DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI

[0086] YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPP

[0087] ITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE

[0088] AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26) (or a variant thereof).

[0089] In one embodiment of the present invention, the V L linked to a human kappa or lambda light chain immunoglobulin constant domain and / or the V H linked to a human IgG (eg, IgG1, IgG2, IgG3, or IgG4 (eg, S228P mutant IgG4)) heavy chain immunoglobulin constant domain.

[0090] GDF8 antagonists

[0091] The invention encompasses LEPR / GDF8 / ActA combinations that include one or more GDF8 antagonists (eg, H4H18457P2 / H4H1657N2 / H4H10446P2).

[0092] GDF8 antagonists include antibodies and antigen-binding fragments thereof and 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 SMAD-dependent activation of A204 cells that stably express Smad-dependent (CAGA12) luciferase.

[0093] 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).

[0094] In one embodiment of the invention, the GDF8 antagonist specifically binds to GDF8, but does not bind to other ActRIIB ligands, such as GDF3, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, Activin A, Activin B, Activin AB and / or Node, for example.

[0095] In one embodiment of the present invention, the GDF8 antagonist is an anti-GDF8 antibody or an antigen-binding fragment thereof. Anti-GDF8 antibodies are mentioned in, for example, U.S. Patent Nos. 6,096,506; 7,320,789; 7,261,893; 7,807,159; 7,888,486; 7,635,760; 7,632,499, U.S. Patent Application Publication Nos. 2007 / 0178095, 2010 / 0166764 and 2009 / 0148436, and International Patent Application Publication No. WO2010 / 070094.

[0096] Anti-GDF8 antibodies are also described in U.S. Patent Application No. 13 / 115,170, filed May 25, 2011 and published as US2011 / 0293630, now U.S. Patent No. 8,840,894, or International Patent Application No. PCT / US2012 / 064911 (WO2013 / 074557), filed November 14, 2012, including those designated 21-E5, 2 Antibodies or antigen-binding fragments thereof of 1-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.

[0097] In one embodiment of the present 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.

[0098] In one embodiment of the present invention, the anti-GDF8 antibody is H4H1657N2.

[0099] In one embodiment of the present invention, the anti-GDF8 antibody or fragment of the present invention comprises:

[0100] (i) a HCDR (HCDR1, HCDR2 and HCDR3) (or a variant having one or more of the HCDRs) in the heavy chain variable region and / or a LCDR (LCDR1, LCDR2 and LCDR3) (or a variant having one or more of the LCDRs) in 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

[0101] (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;

[0102] and / or characterized by:

[0103] (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;

[0104] and / or

[0105] (iv) binds to GDF8 at the same epitope as 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, e.g., wherein 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 a peptide consisting of said amino acids (e.g., comprising a C-terminal tag, e.g., biotin).

[0106] See published US patent application No. US2011 / 0293630. In one embodiment of the present invention, the 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 κ and λ.

[0107] Antibody H4H1657N2 may be referred to as REGN1033 or trevogrumab.

[0108] In one embodiment of the present invention, the GDF8 antagonist is an antibody or an antigen-binding fragment thereof comprising: (1)

[0110] V comprising the following amino acid sequence H :

[0111]

[0112] (SEQ ID NO:9; or a variant thereof);

[0113] and

[0114] V comprising the following amino acid sequence L :

[0115]

[0116] (SEQ ID NO: 10; or a variant thereof)

[0117] and / or (2)

[0119] Containing its CDR-L, such as the following V L :

[0120] CDR-L1 comprising the following amino acid sequence: Gln Asp Ile Ser Asp Tyr (SEQ ID NO: 11; or a variant thereof)

[0121] CDR-L2 comprising the following amino acid sequence: Thr Thr Ser (SEQ ID NO: 12; or a variant thereof)

[0122] CDR-L3 comprising the following amino acid sequence: Gln Lys Tyr Asp Ser Ala Pro Leu Thr (SEQ ID NO: 13; or a variant thereof)

[0123] and / or

[0124] Containing its CDR-H, such as the following V H :

[0125] CDR-H1 comprising the following amino acid sequence: Gly Phe Thr Phe Ser Ala Tyr Ala (SEQ ID NO: 14; or a variant thereof)

[0126] CDR-H2 comprising the following amino acid sequence: Ile Ser Gly Ser Gly Gly Ser Ala (SEQ ID NO: 15; or a variant thereof)

[0127] a CDR-H3 comprising the following amino acid sequence: Ala Lys Asp Gly Ala Trp Lys Met Ser GlyLeu Asp Val (SEQ ID NO: 16; or a variant thereof); and / or (3)

[0129] A heavy chain immunoglobulin comprising the following amino acid sequence:

[0130] EVQVLESGGDLVQPGGSLRLSCAASGFTFSAYAMTWVRQAPGKGLEWVSAISGSGGSAYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKDGAWKMSGLDVWGQ GTTVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:27) (or a variant thereof),

[0131] and

[0132] A light chain immunoglobulin comprising the following amino acid sequence:

[0133] DIQMTQSPASSLSASVGDRVTITCRASQDISDYLAWYQQKPGKIPRLLIYTTSTLQSGVPSRFRGSGSGTDFTLTISSLQPEDVATYYCQKYDSAPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:28) (or its variants)

[0134] In one embodiment of the present invention, the V L linked to a human kappa or lambda light chain immunoglobulin constant domain and / or the V H linked to a human IgG (eg, IgG1, IgG2, IgG3, or IgG4 (eg, S228P mutant IgG4)) heavy chain immunoglobulin constant domain.

[0135] Activin A antagonists

[0136] The invention encompasses LEPR / GDF8 / ActA combinations that include one or more activin A antagonists (eg, H4H18457P2 / H4H1657N2 / H4H10446P2).

[0137] Activin A antagonists include antibodies and antigen-binding fragments thereof and 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 can be referred to as "anti-activin A" or "anti-ActA". In one embodiment of the present invention, the ActA antagonist:

[0138] (i) interfering with the interaction between activin A and activin A receptors (e.g., type IIA activin receptor, type IIB activin receptor, type I activin receptor, etc.);

[0139] (ii) interfering with the formation of the activin-activin receptor complex; and / or

[0140] (iii) causing inhibition of at least one biological function of activin A, such as phosphorylation and activation of type I activin receptor and phosphorylation of SMAD2 and SMAD3 proteins.

[0141] Activin A antagonists, such as antibodies and antigen-binding fragments thereof, and 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 activin A (βA / βA homodimer) and activin AB (βA / βB heterodimer). In one embodiment of the present invention, the activin A specific binding protein binds to activin A and activin AB (but not to activin B). Anti-activin A antibodies and antigen-binding fragments are mentioned in, for example, US2009 / 0234106. The specific anti-activin A antibody is named "MAB3381" and is commercially available from R&D Systems, Inc, Minneapolis, MN. MAB3381 specifically binds to activin A (homodimer) and activin AB (heterodimer).

[0142] In one embodiment of the present invention, the activin A antagonist is selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440 or any other antibody or antigen-binding fragment described in WO2015 / 017576.

[0143] In one embodiment of the present invention, the anti-activin A antibody is garetosmab.

[0144] In one embodiment of the present invention, the anti-activin A antibody or fragment of the present invention comprises:

[0145] (i) selected from H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2 HCDRs (HCDR1, HCDR2 and HCDR3) (or variants having one or more of the HCDRs) in the heavy chain variable region and / or LCDRs (LCDR1, LCDR2 and LCDR3) (or variants having one or more of the LCDRs) in the light chain variable region of antibodies of the type 10447P2, H4H10448P2, H4H10452P2, H4H10468P2 and H2aM10965N; and / or

[0146] (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 H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, or H2aM10965N;

[0147] and / or characterized by:

[0148] (iii) competes with H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, and / or H2aM10965N for binding to activin A;

[0149] and / or

[0150] (iv) binds to activin A at the same epitope as H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P22, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2 and / or H2aM10965N.

[0151] See Published International Patent Application Publication No. WO2015 / 017576. In one embodiment of the present invention, the 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 κ and λ.

[0152] Antibody H4H10446P2 may be referred to as REGN2477 or galactuzumab.

[0153] In one embodiment of the present invention, the activin A antagonist is an antibody or an antigen-binding fragment thereof comprising: (1)

[0155] V comprising the following amino acid sequence H :

[0156] QVQLQESGPGLVKPSETLSLTCTVS GGSFSSHF WSWIRQPPGKGLEWI

[0157] GY ILYTGGTSFNPSLKSRVSMSVGTSKNQFSLKLSSVTAADTAVYYC ARARSGITFTGI IVPGSFDI WGQGTMVTVSS (SEQ ID NO: 17; or a variant thereof);

[0158] and

[0159] V comprising the following amino acid sequence L :

[0160] EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLL

[0161] IY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QYGSSPWT FGQGTKVEIK (SEQ ID NO: 18; or a variant thereof); and / or (2)

[0163] Containing its CDR-L, such as the following V L :

[0164] A CDR-L1 comprising the following amino acid sequence: Gln Ser Val Ser Ser Ser Tyr (SEQ ID NO: 19; or a variant thereof);

[0165] A CDR-L2 comprising the following amino acid sequence: Gly Ala Ser (SEQ ID NO: 20; or a variant thereof); and

[0166] A CDR-L3 comprising the following amino acid sequence: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO: 21; or a variant thereof);

[0167] and

[0168] Containing its CDR-H, such as the following V H :

[0169] a CDR-H1 comprising the following amino acid sequence: Gly Gly Ser Phe Ser Ser His Phe (SEQ ID NO: 22; or a variant thereof);

[0170] a CDR-H2 comprising the following amino acid sequence: Ile Leu Tyr Thr Gly Gly Thr (SEQ ID NO: 23; or a variant thereof); and

[0171] a CDR-H3 comprising the following amino acid sequence: Ala Arg Ala Arg Ser Gly Ile Thr Phe ThrGly Ile Ile Val Pro Gly Ser Phe Asp Ile (SEQ ID NO: 24; or a variant thereof);

[0172] and / or (3)

[0174] A heavy chain immunoglobulin comprising the following amino acid sequence:

[0175] QVQLQESGPGLVKPSETLSLTCTVSGGSFSSHFWSWIRQPPGKGLEWIGYILYTGGTSFNPSLKSRVSMSVGTSKNQFSLKLSSVTAADTAVYYCARARSGITFTGIIVPGSFD IWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:29) (or variations thereof)

[0176] and

[0177] A light chain immunoglobulin comprising the following amino acid sequence:

[0178] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:30) (or a variant thereof).

[0179] In one embodiment of the present invention, the V L linked to a human kappa or lambda light chain immunoglobulin constant domain and / or the V H linked to a human IgG (eg, IgG1, IgG2, IgG3, or IgG4 (eg, S228P mutant IgG4)) heavy chain immunoglobulin constant domain.

[0180] Pharmaceutical composition

[0181] The present invention includes pharmaceutical formulations having a LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2), including, for example, one or more (e.g., 3) of its components, mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984). Methods for making the pharmaceutical formulations comprising mixing a pharmaceutically acceptable carrier or excipient with one or more components form part of the present invention, as do pharmaceutical compositions produced by the methods.

[0182] The scope of the present invention includes dried, such as freeze-dried, LEPR / GDF8 / ActA combinations of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) or one or more components thereof or pharmaceutical compositions thereof comprising a pharmaceutically acceptable carrier but substantially lacking water. In one embodiment of the present invention, the pharmaceutical formulation is aqueous (including water). In one embodiment of the present invention, the pharmaceutical formulation is sterile.

[0183] Pharmaceutical formulations of the therapeutic agent can be prepared by mixing with an acceptable carrier, excipient or stabilizer in the form of, for example, a lyophilized powder, slurry, aqueous solution or suspension (see, for example, Hardman et al. (2001) Goodman and Gilman, The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, New York; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets). Dosage Forms: Tablets, Marcel Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, New York; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, New York).

[0184] The mode of administration of the LEPR / GDF8 / ActA combination can vary. Routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal or intraarterial.

[0185] The present invention provides a method of administering a pharmaceutical formulation comprising a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) to an individual (e.g., a human), the method comprising introducing the formulation into the individual's body, e.g., into a vein, subcutaneous tissue, or muscle tissue of the individual. For example, the method comprises puncturing the individual's body with a needle of a syringe and injecting the formulation into the individual's body. The method includes introducing a formulation comprising all three components of the co-formulated combination into the individual's body; or, for example, introducing three separately formulated components of the combination into the individual's body.

[0186] The present invention provides one or more containers (e.g., plastic or glass vials, hollow bore needles, or cylindrical syringes, for example, with caps or chromatographic columns) containing a LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) or a pharmaceutical composition thereof containing a pharmaceutically acceptable carrier. The present invention includes methods for preparing one or more containers containing the combination, the methods comprising introducing the components of the combination into one or more containers, for example, a single container containing a co-formulated combination of components. In one embodiment of the present invention, the one or more containers are subsequently introduced into a kit.

[0187] The present invention also provides devices, such as injection devices and methods of use thereof, comprising a LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) or a pharmaceutical composition thereof. An injection device is a device for introducing a substance into a patient's body by a parenteral route, such as intramuscularly, subcutaneously, or intravenously. For example, the injection device can be, for example, a syringe (e.g., pre-filled with a pharmaceutical composition, such as an automatic injector, or filled at the point of use, such as by a user or clinician) comprising a cylinder or barrel for holding a fluid to be injected (e.g., comprising an antibody or fragment or a pharmaceutical composition thereof), a needle for puncturing the skin and / or a blood vessel to inject the fluid, and a plunger for pushing the fluid out of the cylinder and through the needle hole.

[0188] The pharmaceutical compositions disclosed herein can also be administered using a needle-free subcutaneous injection device, such as the devices disclosed in U.S. Pat. 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. The needle-free devices containing the pharmaceutical compositions and methods of use thereof are also part of the present invention.

[0189] The invention includes methods of preparing one or more infusion devices (e.g., prefilled syringes or automatic injectors) comprising a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2), the method comprising introducing the components of the combination into one or more of the devices, e.g., a single device comprising the co-formulated combination of components. In one embodiment of the invention, one or more infusion devices are subsequently introduced into a kit.

[0190] The present invention also includes a kit comprising a LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). In one embodiment of the present invention, the kit comprises each antagonist in a separate container or injection device (e.g., a prefilled syringe or an automatic injector); or all three antagonists co-formulated in a single container or injection device. The kit may include a drug instruction sheet including information about the pharmaceutical composition and dosage form in the kit. In general, the information helps patients and doctors to use the packaged pharmaceutical composition effectively and safely. For example, any of the following information about the combination of the present invention may be supplied in the instructions: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and uses, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, mode of supply, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0191] Treatment and administration

[0192] The LEPR / GDF8 / ActA combination has demonstrated a remarkable ability to cause an increase in lean mass in individuals administered the combination. The increase in lean mass is likely at the expense of an increase in fat mass. The LEPR / GDF8 / ActA combination causes a greater increase in lean mass than blockade of GDF8 and ActA alone, and the increase in fat mass is less than that observed with a single LEPR antagonist. This may be due to the heat generated by the fat that is now used to build additional muscle.

[0193] The method of administering the LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) of the present invention to an individual includes introducing the components of the combination in combination with each other into the body of the individual. The introduction can be carried out by any acceptable route, such as parenteral. The components can be co-formulated into a single composition or formulated into separate compositions. If the administration of one or more of the components in the separate composition is performed as part of a regimen, the administration can be separated in time. In one embodiment of the present invention, the method comprises injecting all three components into the individual at once; in another embodiment of the present invention, the GDF8 antagonist and the ActA antagonist are formulated together and injected once in a separate injection together with the LEPR antagonist; in another embodiment of the present invention, all three antagonists are injected separately in three separate injections (e.g., subcutaneously, intravenously or intramuscularly or a combination of two or three of the routes).

[0194] The scope of the invention provides a method of increasing food intake, fat mass, body weight (e.g., at the expense of fat mass), muscle strength, muscle fiber size, or lean mass (e.g., at the expense of fat mass) in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0195] In addition, the LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) can be used for:

[0196] - Treatment or prevention of a disease or condition that would be cured or ameliorated to any degree 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

[0197] - Treatment or prevention of a disease or condition that would be cured or improved to any degree by causing an increase or reversing a decrease in: food intake, fat mass, 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);

[0198] The above is carried out by administering a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) to an individual. The diseases or conditions that can be treated or prevented include, for example, malnutrition, developmental delay, insufficient food / caloric intake, eating disorders, cachexia, muscle atrophy / wasting, age-related sarcopenia, and muscle damage. The conditions and diseases are discussed in detail herein.

[0199] Malnutrition is an example of a condition in an individual that would benefit from LEPR / GDF8 / ActA combination therapy. Malnutrition is a term used to describe any nutritional imbalance; from overnutrition to undernutrition, such as seen in hospitals and residential care facilities. For purposes of the present invention, malnutrition refers to undernutrition (unless otherwise indicated). Therefore, the present invention provides a method of treating or preventing malnutrition in an individual, such as a person in a hospital or residential care facility, by administering a therapeutically effective amount of a LEPR / GDF8 / ActA combination to an individual in need thereof.

[0200] Malnutrition can develop as a result of:

[0201] - Inadequate dietary intake;

[0202] - increased requirements associated with a disease state (e.g., individuals infected with HIV and individuals with AIDS (acquired immunodeficiency syndrome) or cystic fibrosis may require increased dietary intake in order to maintain normal body weight) for which increased dietary intake is insufficient to compensate; and / or

[0203] - Complications of the underlying disease (e.g., patients with cirrhosis, chronic pancreatitis, lactase deficiency, pancreatic cancer, amyloidosis, celiac disease, Crohn's disease, radiation enteritis, and Addison's disease may suffer from malabsorption due to inadequate digestive agents; and patients with cancer or infectious diseases (or other diseases) may suffer from cachexia secondary to the underlying disease (see below)) for which increased dietary intake is insufficient to compensate.

[0204] Thus, the invention includes a method of reversing or halting malnutrition in an individual due to lack of dietary intake, increased requirements associated with a disease state (e.g., HIV or AIDS), and / or complications of an underlying disease, the method comprising administering to the individual a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0205] Malnutrition is associated with negative outcomes for patients, including higher rates of infection and complications, increased muscle loss, impaired wound healing, longer hospital stays, and increased morbidity and mortality. Thus, the invention includes methods of reducing negative outcomes associated with malnutrition in a subject (e.g., reducing the likelihood of infection or preventing impaired wound healing), the methods comprising administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0206] There are many nutrition screening and assessment tools for identifying risk of malnutrition and diagnosing malnutrition. For example, the Malnutrition Screening Tool (MST) is a simple, three-question tool that assesses recent weight and loss of appetite 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 Nutrition Assessment (MNA) was specifically developed for use among elderly patients (≥65 years) in hospitals, nursing homes, and the community, and is therefore limited to this demographic (Anthony, Nutrition screening tools for hospitalized patients, Clinical Nutrition Practice 2008; 23: 373-382; Gibson, Principles of Nutritional Assessment. 2nd ed. Oxford University Press, New York; New York, USA: 2005). The Nutrition Risk Screening (NRS-2002) uses recent weight loss, BMI reduction, and dietary intake reduction in combination with 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, Clinical Nutrition Practice 2008; 23: 373-382).The four-item Short Nutrition Assessment Questionnaire (SNAQ) was developed to diagnose malnutrition in hospitalized patients and to provide an indication for dietary referral and outline a nutritional treatment plan (Anthony, Nutritional Screening Tool for Hospitalized Patients. Clin Nutr Pract. 2008;23:373-382; Kruizenga et al., Development and validation of a hospital screening tool for malnutriton: the short nutritional assessment questionnaire (SNAQ). Clin Nutr. 2005;24:75-82). This has been validated for use in inpatients and outpatients, as well as residential 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 nutrition assessment tools and assesses nutritional status by completing a questionnaire that includes data on changes in weight, changes in dietary intake, gastrointestinal symptoms, changes in functional capacity associated with malnutrition, and 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).

[0207] Nosocomial malnutrition is a condition experienced by an individual upon admission to a hospital. The malnutrition state may pre-exist or develop during an individual's hospital stay. The present invention provides a method of treating or preventing nosocomial malnutrition in an individual, the method comprising administering to the individual a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0208] Malnutrition is also a common condition suffered by individuals with cystic fibrosis. Various related and unrelated complicating factors may contribute to energy imbalance in patients with cystic fibrosis. The net effect on growth potential varies significantly among patients, depending on differences in disease expression and as the disease progresses. The present invention provides methods for treating or preventing malnutrition in individuals with cystic fibrosis, the methods comprising administering to the individual a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0209] Stunting in childhood is a state of undernutrition resulting from insufficient caloric intake, insufficient caloric absorption, or excessive caloric expenditure. In the neonatal period, stunting may be associated with common underlying diseases, such as short bowel, volvulus, and intestinal resection following necrotizing enterocolitis, congenital absorption defects and structural defects of the small intestine, and inadequate food intake. In infancy (2-8 months of age), stunting may be associated with common underlying diseases, such as inadequate food intake, neglect, enteroallergy to cow's milk protein, esophagitis with gastroesophageal reflux, cystic fibrosis, eating disorders, and / or increased energy requirements in the setting of underlying cardiac, neurological, neoplastic, or renal disease, erosions, chronic diarrhea in the setting of immune system defects, autoimmune enteropathy, post-enteritis syndrome and malabsorption syndrome, and Munchausen syndrome by proxy. In early childhood (9-36 months), developmental delay may be associated with common underlying medical conditions such as inadequate food intake, neglect, chylomicron, cystic fibrosis, eating disorders and / or increased energy requirements in the setting of underlying cardiac, neurological, tumor or renal disease, chronic diarrhea in the setting of immune system deficiency and Munchausen syndrome by proxy. In childhood (3-16 years), developmental delay may be associated with common underlying medical conditions such as inadequate food intake, neglect, psychiatric disorders (e.g. anorexia nervosa), chronic inflammatory bowel disease, chylomicron, cystic fibrosis, eating disorders and / or increased energy requirements in the setting of underlying cardiac, neurological, tumor or renal disease, chronic diarrhea in the setting of immune system deficiency and giardiasis (lambliasis) and other chronic intestinal infections. The invention provides methods for treating or preventing childhood developmental delay (e.g., in a newborn, infant, toddler, or child) in a subject, for example, 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).

[0210] Inadequate food intake may be associated with symptoms such as loss of appetite, chronic vomiting, swallowing and chewing disorders, esophageal motility abnormalities, and shortness of breath associated with, for example, heart and lung disorders. The present invention provides methods for treating or preventing inadequate food intake in an individual, for example, associated with the symptoms discussed herein, comprising administering to the individual a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0211] Eating disorders characterized by insufficient caloric intake include anorexia and / or bulimia. The present invention provides a method for treating or preventing anorexia and / or bulimia in an individual, the method comprising administering a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) to the individual. In one embodiment of the present invention, anorexia is anorexia nervosa, anorexia senileis, or anorexia in patients receiving hemodialysis.

[0212] Cachexia is a complex metabolic syndrome often associated with underlying diseases and characterized by muscle loss with or without loss of fat mass. The prominent clinical features of cachexia are weight loss in adults (corrected for fluid retention) or growth failure in children (excluding endocrine disorders). Anorexia, inflammation, insulin resistance, and increased muscle protein breakdown are symptoms commonly associated with cachexia. The present invention provides, for example, at any stage described herein (e.g., refractory cachexia) and / or as defined by any benchmark described herein or in the art, a method for treating or preventing cachexia (or any symptom of cachexia) in an individual, the method comprising administering a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) to the individual.

[0213] Cachexia secondary to other diseases, conditions, or treatments include cachexia secondary to anorexia or other psychiatric eating disorders, pulmonary disease (e.g., chronic obstructive pulmonary disease (COPD)), chronic kidney disease, infectious diseases (e.g., HIV infection or acquired immunodeficiency 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, elevated blood pressure, depression, and / or neurodegenerative disorders. The present invention provides methods for treating or preventing cachexia secondary to any disease or condition in a subject, e.g., any disease or condition described herein (e.g., cancer), comprising administering to the subject a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0214] The International Consensus Statement on the Definition and Classification of Cancer Cachexia published in The Lancet Oncology in May 2011 established these benchmarks for diagnosing cachexia in patients with cancer.

[0215] (i) Weight loss greater than 5% in the past 6 months; or

[0216] (ii) BMI less than 20 and any weight loss greater than 2%; or

[0217] (iii) Limb skeletal muscle index consistent with sarcopenia (another wasting syndrome) and greater than 2% body weight loss. Fearon et al., Lancet Oncol. 2011 May;12(5):489-95.

[0218] The stages of cancer cachexia agreed upon by the group are:

[0219] Pre-cachexia: less than 5% weight loss and other symptoms such as impaired glucose tolerance or anorexia;

[0220] Cachexia: greater than 5% body weight loss or other symptoms and conditions consistent with cachexia diagnostic criteria; and

[0221] Refractory cachexia: Patients who are experiencing cachexia and no longer respond to cancer treatment have low performance scores and have a life expectancy of less than 3 months.

[0222] Muscle atrophy or loss 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 sarcopenia, decreased muscle mass and / or weakness, decreased muscle strength. These diseases and conditions include sepsis, AIDS, kidney and heart failure, excessive glucocorticoids (e.g., Cushing syndrome) and trauma, and muscle atrophy is also present in 80% of cancer patients. In addition, muscle atrophy or loss may be caused by or related to: disuse, immobility, bed rest, injury (e.g., hip fracture), neurodegenerative diseases related to motor neuron loss, medical treatment or surgical intervention (e.g., hip replacement, knee replacement, etc.) or mechanical ventilation inevitably. The present invention provides methods for treating or preventing muscle atrophy or wasting in an individual (e.g., muscle atrophy or wasting secondary to a disease or condition such as AIDS or cancer), the method comprising administering to the individual a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2). In one embodiment of the invention, the muscle atrophy is sarcopenia, such as age-related sarcopenia or sarcopenia in an individual receiving hemodialysis and / or suffering from chronic kidney disease cachexia. Age-related sarcopenia is a degenerative loss of skeletal muscle mass (e.g., a loss of about 0.5%-1% per year after age 50), a degenerative loss of skeletal muscle quality, and a degenerative loss of skeletal muscle strength associated with aging. Therefore, the present invention includes methods for treating or preventing muscle atrophy due to age-related sarcopenia.

[0223] Muscle damage may be caused by strains such as strains or tears from excessive exertion or sudden twisting. Muscle tears may cause swelling, pain, and severe bleeding, which may lead to blood clots. Severe tears may require surgery. The muscle stimulating properties of the LEPR / GDF8 / ActA combination make it suitable for treating or preventing muscle damage. The present invention provides a method for treating or preventing muscle damage in an individual, the method comprising administering a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) to the individual.

[0224] Other conditions that can be treated or prevented using the LEPR / GDF8 / ActA combinations of the invention include amyotrophic lateral sclerosis, arthritis, autoimmune disorders, benign and malignant pheochromocytomas, breast cancer, cancer, cardiovascular disease, chronic heart failure, chronic obstructive pulmonary disease, depression, diabetes, elevated blood pressure, glucocorticoid-induced myopathy, hepatocellular carcinoma, inflammatory bowel disease, keloids and hypertrophic scars, lupus erythematosus, melanoma, metabolic syndrome, multiple sclerosis, muscular dystrophy (e.g., myotonic, Duchenne, Becker, acrofacial, facioscapulohumeral (FSHD, also known as Landouzy-Dejerine), congenital, oculopharyngeal, distal, Emery-Dreifuss, etc.), neurodegenerative disorders, organ atrophy, osteoarthritis, osteopenia, osteoporosis, Parkinson's disease (Parkinson's disease), preeclampsia, psoriasis, pulmonary hypertension, sarcopenia, sepsis, and uterine fibroids / leiomyomata.

[0225] The LEPR / GDF8 / ActA combination of the present invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) effectively increases lean muscle mass and muscle strength and thus effectively enhances athletic performance. Therefore, the present invention provides a method for enhancing the athletic performance of an individual in need, the method comprising administering a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) to the individual. Athletic performance includes walking speed, running speed, the ability to sit and stand per unit time (e.g., more than 30 seconds), walking distance per unit time (e.g., more than 6 minutes), biceps curl weight, chest press weight, stair climbing speed (e.g., time to climb 4 steps), and / or grip strength (e.g., as measured using manual dynamometer). In one embodiment of the present invention, the individual is undergoing stroke rehabilitation (e.g., stroke hemiplegia rehabilitation) or physical therapy. Thus, the LEPR / GDF8 / ActA combination of the invention can be used as an adjuvant in any therapeutic procedure in which enhanced athletic performance is desired, such as physical therapy, such as stroke rehabilitation or surgical recovery (e.g., knee surgery or knee replacement to repair tendon or ligament damage) or recovery from physical injury.

[0226] An individual (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 reducing 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 a LEPR antagonist. For example, the method may comprise administering a therapeutically effective amount of a LEPR / GDF8 / ActA combination (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2).

[0227] A "subject" is a mammal, eg, a human, dog, cat, horse, cow, mouse, rat, monkey (eg, a cynomolgus monkey, eg, a rhesus monkey), or rabbit.

[0228] An effective 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 (all three antibodies or antigen-binding fragments), for example to treat or prevent any of the diseases or conditions discussed herein (e.g., cachexia).

[0229] In particular embodiments, the LEPR / GDF8 / ActA combinations of the invention (e.g., H4H18457P2 / H4H1657N2 / H4H10446P2) can be used alone or in combination with any other additional therapeutic agents and / or therapeutic procedures, which are, for example, useful for increasing an individual's food intake, fat mass, body weight, lean mass (e.g., muscle mass) and / or strength and / or useful for treating or preventing any of the diseases or conditions discussed herein, e.g., cachexia.

[0230] In one embodiment of the invention, the therapeutic procedure is nasogastric tube feeding.

[0231] In one embodiment of the invention, the additional therapeutic agent is any one or more of the following: 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.

[0232] In one embodiment of the present invention, the other therapeutic agent is any one or more of the following: 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 anamorelin, arformoterol, aspirin, aspirin, atorvastatin, atropine, azathioprine, azithromycin, benazepril, betamethasone, budesonide, bumetanide, buphenine, bupropion, captopril, carboplatin, celecoxib, certolizumab pegol pegol), chlorothiazide, chlorthalidone, cyclosporine, citalopram, clenbuterol, coal tar, coconut oil, corticosteroids, diphenhydramine, cyclophosphamide, dabrafenib, daclizumab, desoximetasone, desvenlafaxine, dexamethasone, diclofenac, dicyclomine, diflunisal, dimebolin, dimethyl fumarate, dimethyl fumarate, anthracene, docetaxel, dopexamine xamine), doxorubicin, dronabinol, duloxetine, efalizumab, enalapril, enobosarm, epinephrine, epirubicin, erythromycin, escitalopram, etanercept, ethacrynate, etodolac, fentoterol,fingolimod, flunisolide, fluocinonide, fluocortolone, fluoxetine, fluvastatin, formoterol, fosinopril, furosemide, glatiramer acetate acetate), golimumab, hydralazine, hydrochlorothiazide, hydrocortisone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, hydroxyurea, hyoscyamine, hyoscyamine, ibuprofen, indapamide, indomethacin, infliximab, interferon beta-1a, interferon beta-1b, interleukin-2, ipilimumab, isoetarine, isoproterenol, isosorbide dinitrate, JX -594, ketoprofen, levalbuterol, lisinopril, lovastatin, megestrol acetate, mepenzolate, mesalazine, methotrexate, methotrexate, methylchlorothiazide, metolazone, mevastatin, mitoxantrone, moexipril, nabilone, naproxen, natalizumab, nivolumab olumab, nortriptyline, ocrelizumab, ofatumumab, oral nutritional supplements (such as Protibis cookies or dairy-based supplements), orciprenaline, oxaprozin, paclitaxel, para-aminobenzoic acid, parenteral iron, paroxetine, pembrolizumab, perindopril ), phenobarbital, phenobarbital, phenobarbital and scopolamine, pirbuterol, piroxicam, pitavastatin, pravastatin, prednisolone, prednisone, prednisone, procaterol, proline-rich peptide (PRP)-1, psoralen, quinapril, ramapril,Ritodrine, rituximab, rosuvastatin, salbutamol, salicylate, scopolamine, sertraline, simvastatin, sorafenib, sulindac, terbutaline, teriflunomide, theophylline, tiotropium, tolmetin, torsemide, trametinib, trandolapril, trazodone, triamcinolone acetonide, triamcinolone alcohol, vemurafenib, venlafacine, venlafaxine, vitamin D3, or a vitamin D analog.

[0233] The invention also encompasses embodiments in which the LEPR / GDF8 / ActA combination is not combined with another therapeutic agent and / or procedure.

[0234] Examples

[0235] These examples are intended to illustrate the invention and not to limit it. The compositions, such as LEPR / GDF8 / ActA combinations and methods described in the examples form part of the present invention.

[0236] Example 1 : In vivo efficacy testing of LEPR antagonist antibodies (H4H17322P2, H4H18457P2, and H4H18464P2) in humanized LEPR mice

[0237] Effects of three specific antagonist anti-LEPR antibodies H4H17322P2, H4H18457P2 and H4H18464P2 of the present invention on food intake, body weight and fat mass in genetically engineered LEPR mice expressing leptin receptor raised alone Hu / Hu The leptin receptor was constructed by replacing the murine LEPR ectodomain sequence with the human LEPR ectodomain sequence.

[0238] Baseline daily food intake was measured between 5 days and 1 day before treatment (Days -5 and -1). Body composition, including fat mass, was quantified by μCT four days before treatment and 6 days after treatment (Days -4 and 6). On Day 0, thirty-two 12- to 13-week-old male LEPR mice were placed on a 4-well plate based on body weight from 1 day before treatment (Day -1).Hu / Hu Mice were randomly divided into four groups, 8 in each group. On the 0th day, each group received a single dose of 30mg / kg isotype control antibody, 30mg / kg H4H17322P2, 30mg / kg H4H18457P2 or 30mg / kg H4H18464P2 by subcutaneous injection. The isotype control antibody does not bind to any known mouse protein. The body weight of each animal during the study was measured (Table 1A). The percentage change in body weight from the 0th day was calculated for each animal at each time point. Table 1B summarizes the average fat mass and lean mass of animals in each antibody treatment group quantified by μCT 6 days before antibody treatment and 6 days after antibody treatment. All results are represented by mean ± SEM. In addition, the plasma leptin (Table 1C) before and on the 6th day of quantitative administration was measured.

[0239] Mice treated with anti-LEPR antagonist antibodies showed an increase in food intake percentage change (data not shown) and an increase in weight change percentage (Table 1A). These increases were not observed in the case of isotype control antibody treatment. Compared with mice injected with isotype control antibodies, mice treated with 30mg / kg H4H17322P2 began to show a considerable increase in food intake one day after treatment (day 1) and at subsequent time points. Compared with mice injected with isotype control antibodies, mice treated with 30mg / kg H4H18457P2 began to show a considerable increase in food intake at subsequent time points at the 2nd day. Compared with mice injected with isotype control antibodies, mice treated with 30mg / kg H4H18464P2 began to show a considerable increase in food intake at the 2nd day and at subsequent time points, but not at the 4th day. Compared with mice injected with isotype control antibodies, mice treated with 30mg / kg H4H17322P2 began to show a considerable increase in weight change percentage at subsequent time points four days after treatment (day 4). Compared with mice injected with isotype control antibodies, mice treated with 30 mg / kg H4H18457P2 showed a considerable increase in percentage of weight change starting on day 3 and at subsequent time points. Compared with mice injected with isotype control antibodies, mice treated with 30 mg / kg H4H18464P2 showed a considerable increase in percentage of weight change starting on day 4 and at subsequent time points. As depicted in Table 1B, there was no difference in fat mass between the groups before treatment (day -4). Compared with isotype control antibodies, mice treated with 30 mg / kg H4H17322P2, H4H18457P2 and H4H18464P2 antibodies showed a considerable increase in fat mass and leptin content 6 days after treatment (day 6) (Table 1C). In summary, treatment with LEPR antagonist antibodies rather than isotype control antibodies increases food intake, body weight, fat mass and leptin content in mice.

[0240] Table 1A. Body weight (difference from baseline, g)

[0241]

[0242] REGN1945: Anti-Fel d1 (IgG4)

[0243] SEM: standard error of the mean

[0244] Table 1B. Body composition

[0245]

[0246] Table 1C. Plasma Leptin (pg / mL)

[0247]

[0248] Example 2 : Combination treatment of anti-GDF8 mAb (REGN1033), anti-activin A mAb (REGN2477), and LEPR antagonist mAb (H4H184572P2) in 20-24 week old male mice

[0249] Effects of the specific antagonist anti-LEPR antibody of the invention (H4H18457P2) and anti-MSTN (also known as anti-GDF8) and anti-INHBA (also known as anti-activin A) blocking antibodies (H4H1657N2 (REGN1033) and H4H10446P2 (REGN2477), respectively) on food intake, body weight, body composition, individual tissue weights, and isolated muscle force generation in individually housed 20- to 24-week-old genetically engineered male LEPR expressing the leptin receptor Hu / H u mice, wherein the leptin receptor is composed of the human LEPR ectodomain sequence instead of the murine LEPR ectodomain sequence.

[0250] Baseline daily food intake was measured between day 18 and day 0. Baseline whole body lean mass and fat mass were quantified by NMR (nuclear magnetic resonance) on day 15 or day 11. On day 0, mice were stratified into four groups of 11 to 12 mice each based on body composition from day 15 and body weight from day 0. Starting from day 0, each group received the corresponding antibody treatment dose by subcutaneous injection. REGN1033, REGN2477 and the corresponding IgG4 were administered at 10 mg / kg twice a week. P mAb; and H4H18457P2 and its corresponding IgG4 were administered once a week at 30 mg / kg P mAb; therefore, any dose of test antibody given to test mice was given in parallel with the corresponding dose of control antibody in control mice. Isotype control (IgG4P ) (p = S228P mutation, Eu numbering) The antibody does not bind to any known mouse protein. The isotype control antibody is REGN1945.

[0251] Treatment Group

[0252] a) IgG4 P Control (10 mg / kg + 10 mg / kg, 2× / week; 30 mg / kg, 1× / week), number = 11

[0253] b) REGN1033+REGN2477 (10 mg / kg+10 mg / kg, 2× / week), number N=11

[0254] c) H4H18457P2 (30 mg / kg, 1× / week), number = 11

[0255] d) REGN1033+REGN2477+H4H18457P2 (10 mg / kg+10 mg / kg, 2× / week; 30 mg / kg, 1× / week).

[0256] Food intake (Table 2A) and body weight (Table 2B) were measured for each animal during the study. Body composition was quantified on days 6 or 7 and 13 or 14 (Tables 2C and 4A-4D). On days 22, 23 or 24, animals were euthanized and isolated tibialis anterior muscle was subjected to ex vivo force measurements (Tables 3A and 3B). Individual organ and skeletal muscle weights were also quantified (Tables 5-7).

[0257] Compared to mice injected with isotype control antibodies, mice treated with H4H18457P2 alone or in combination with REGN1033 and REGN2477 began seven days after treatment (day 7) and showed a considerable increase in cumulative food intake at subsequent time points (Table 2A). Compared to mice injected with isotype control antibodies, mice treated with REGN1033 and REGN2477 showed similar cumulative food intake (Table 2A). Compared to mice treated with isotype control antibodies and when compared to mice treated with REGN1033 and REGN2477, mice treated with H4H18457P2 alone or in combination with REGN1033 and REGN2477 began 7 days after administration (day 7) and showed weight gain at subsequent time points (Table 2B). Compared to mice treated with isotype control antibodies, mice treated with REGN1033 and REGN2477 did not show obvious weight changes (Table 2B). When compared to mice injected with isotype control, mice in each treatment group demonstrated substantial lean mass gains relative to baseline (day -1) at days 6, 13, and 20 (Table 4C). Compared to mice treated with REGN1033 and REGN2477, mice treated with H4H18457P2, REGN1033, and REGN2477 demonstrated increased lean mass gains relative to baseline at days 6, 13, and 20 (Table 4C). Mice treated with REGN1033 and REGN2477 showed substantial reductions in fat mass changes relative to baseline when compared to mice administered with isotype control antibodies. Mice treated with H4H18457P2 alone or in combination with REGN1033 and REGN2477 showed increased fat mass change relative to baseline on days 6, 13, and 20 when compared to mice administered an isotype control antibody and when compared to mice treated with REGN1033 and REGN2477 (Table 4A). Mice treated with H4H18457P2 and REGN1033 and REGN2477 showed considerable reductions in fat mass gain relative to baseline on days 6, 13, and 20 when compared to mice treated with REGN1033 and REGN2477 (Table 4A). Compared to mice administered with isotype control antibodies, mice treated with REGN1033 and REGN2477 and mice treated with H4H18457P2, REGN1033 and REGN2477 exhibited increased twitch force and peak tonic tension of isolated tibialis anterior skeletal muscle (Table 3A). Thus, when compared to mice administered with isotype control antibodies, mice treated with REGN1033 and REGN2477 and mice treated with H4H18457P2, REGN1033 and REGN3477 exhibited increased skeletal muscle (quadriceps, tibialis anterior and gastrocnemius) weights (Table 5).Compared with mice administered with isotype control antibodies, mice treated with H4H18457P2 alone and in combination with REGN1033 and REGN2477 showed increased inguinal, gonadal and brown adipose tissue weights (Table 5). Compared with mice treated with REGN1033 and REGN2477, brown adipose tissue weights (Table 5) in mice treated with H4H18457P2 and REGN1033 and REGN2477 were significantly increased. When compared with isotype control administration, no significant changes in heart and liver weights were detected among the treatment groups (Table 5). In summary, these data show that the LEPR antagonist H4H18457P2 increases food intake, body weight and fat mass in mice. In addition, treatment with the LEPR antagonist H4H18457P2 in combination with anti-MSTN (REGN1033) and anti-ActA (REGN2477) blocking antibodies resulted in increases in food intake, body weight, lean mass, fat mass, adipose tissue weight, skeletal muscle weight, and skeletal muscle strength. When compared to treatment with REGN1033 and REGN2477, combined treatment with H4H18457P2, REGN1033, and REGN2477 resulted in increases in lean mass again relative to baseline and in lesser increases in fat mass relative to baseline.

[0258] Table 2A. Food intake (g)

[0259]

[0260]

[0261] Table 2B. Body weight (difference from baseline, g)

[0262]

[0263] Table 2C. Changes in Body Composition from Baseline

[0264]

[0265]

[0266] Table 3A. Twitch peak linear tension and specific force (mN

[0267]

[0268] Table 3B. Isometric Tetanic Tension (mN)

[0269]

[0270] Table 4A. Fat mass (g)

[0271]

[0272] Table 4B. Fat mass (difference from baseline g)

[0273]

[0274] Table 4C. Lean mass (g)

[0275]

[0276] Table 4D. Lean mass (difference from baseline g)

[0277]

[0278] Table 5. Organ mass, muscle mass, and fat mass (mg)

[0279]

[0280]

[0281] TA: tibialis anterior muscle; GA: gastrocnemius muscle; WAT: white adipose tissue; BAT: brown adipose tissue.

[0282] Table 6. Organ mass, muscle mass, and fat mass (percent of starting body weight)

[0283]

[0284] Table 7. Organ mass, muscle mass, fat mass (% change relative to control)

[0285]

[0286]

[0287] Example 3 : Combination treatment of anti-GDF8 mAb (REGN1033), anti-activin A mAb (REGN2477), and LEPR antagonist mAb (H4H18457P2) in 12- to 14-week-old male mice (+ additional treatment group).

[0288] Effects of the specific antagonist anti-LEPR antibody of the invention (H4H18457P2) and anti-MSTN (also known as anti-GDF8) and anti-INHBA (also known as anti-activin A) blocking antibodies (H4H1657N2 (REGN1033) and H4H10446P2 (REGN2477), respectively) on food intake, body weight, body composition, individual tissue weights, and isolated muscle force generation in individually housed 12- to 14-week-old genetically engineered male LEPR expressing the leptin receptorHu / H u mice, wherein the leptin receptor is composed of the human LEPR ectodomain sequence instead of the murine LEPR ectodomain sequence.

[0289] Baseline daily food intake was measured between day 18 and day 0. On day 11, baseline whole body lean mass and fat mass were quantified by NMR. On day 0, mice were stratified into six groups of 7 to 8 mice each based on body composition from day 11 and body weight from day 0. Starting on day 0, each group received the corresponding antibody treatment dose by subcutaneous injection. REGN1033, REGN2477 and the corresponding IgG4 were administered at 10 mg / kg twice a week. P mAb. H4H18457P2 and its corresponding IgG4 were administered at 30 mg / kg once a week P mAb. Isotype control (IgG4 P ) antibody (REGN1945) does not bind to any known mouse proteins.

[0290] Treatment Group

[0291] a) IgG4P control (10 mg / kg + 10 mg / kg, 2× / week; 30 mg / kg, 1× / week), number = 7

[0292] b) REGN1033 (10 mg / kg, 2× / week), number = 7

[0293] c) REGN1033+REGN2477 (10 mg / kg+10 mg / kg, 2× / week), number = 7

[0294] d) H4H18457P2 (30 mg / kg, 1× / week), number = 7

[0295] e) REGN1033+H4H18457P2 (10 mg / kg, 2× / week; 30 mg / kg, 1× / week), number = 8

[0296] f) REGN1033+REGN2477+H4H18457P2 (10 mg / kg+10 mg / kg, 2× / week; 30 mg / kg, 1× / week), number = 8

[0297] Food intake (Table 8B) and body weight (Table 8A) were measured for each animal during the study. Body composition was quantified on days 6, 13, and 20 (Tables 8C and 8D). On day 21 or 22, animals were euthanized for additional analysis including skeletal muscle fiber number and cross-sectional fiber area (Tables 9-11).

[0298] Compared to the isotype control antibody, the LEPR antagonist H4H18457P2 significantly increased body weight and cumulative food intake when administered alone or in combination with REGN1033 or REGN1033 and REGN2477. When compared to mice administered with the isotype control antibody, mice treated with H4H18457P2 showed considerable increases in body weight and cumulative food intake starting at day 13 and day 8 and at subsequent time points (Tables 8A and 8B). Compared to the administration of the isotype control antibody, body weight and cumulative food intake in the case of H4H18457P2 and REGN1033 treatment at subsequent time points increased significantly starting at day 7. When compared to mice administered with the isotype control antibody, mice treated with H4H18457P2, REGN1033, and REGN2477 also showed increased body weight and food intake at day 8 and day 7, respectively, to the end of the study. Mice treated with REGN1033 or REGN1033 and REGN2477 did not exhibit significant changes in body weight or cumulative food intake relative to mice administered a control antibody.

[0299] When compared to mice administered with an isotype control antibody, mice treated with H4H18457P2 alone or in combination with REGN1033 or REGN1033 and REGN2477 showed an increase in fat mass relative to baseline at all measured time points (days 6, 13, and 20) (Table 8D). Compared to mice treated with H4H18457P2 alone or in combination with REGN1033, mice treated with H4H18457P2, REGN1033, and REGN2477 showed less fat mass gain relative to baseline at days 13 and 20 (Table 8D). When compared to mice administered with an isotype control antibody, mice treated with H4H18457P2, REGN1033, and REGN2477 also showed a considerable increase in lean mass gain relative to baseline at days 13 and 20 (Table 8C).

[0300] Histological analysis revealed no significant effect on muscle fiber number in the tibialis anterior or gastrocnemius muscles in any treatment group compared to isotype control antibody administration (Tables 9 and 10). Mice treated with REGN1033 and REGN2477 or H4H18457P2 showed increased muscle fiber number in the soleus muscle compared to isotype control antibody delivery (Table 11). In all three muscles examined (tibialis anterior, gastrocnemius, and soleus), H4H18457P2 treatment did not affect muscle fiber area when compared to isotype control antibody administration (Tables 9, 10, and 11). When compared to mice administered with an isotype control antibody, mice treated with REGN1033, REGN1033 and REGN2477, REGN1033 and H4H18457P2, or REGN1033 and REGN2477 and H4H18457P2 showed increased muscle fiber area in the tibialis anterior and gastrocnemius muscles (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 relative to mice administered with an isotype control antibody (Table 11).

[0301] In conclusion, LEPR antagonist antibody treatment alone increased body weight, food intake and fat mass, and combination with anti-ActA and / or anti-MSTN blocking antibodies induced additional increases in muscle fiber area or number, respectively.

[0302] Table 8A. Body weight (g)

[0303]

[0304] Table 8B. Cumulative food intake (g)

[0305]

[0306] Table 8C. Lean mass (difference from baseline g)

[0307]

[0308] Table 8D. Fat mass (difference from baseline g)

[0309]

[0310] Table 9. TA muscle fiber area and number

[0311]

[0312] Table 10. GA muscle fiber area and number

[0313]

[0314] Table 11. Soleus muscle fiber area and number

[0315]

[0316] Example 4 : Combination therapy of anti-GDF8 mAb (REGN1033), anti-activin mAb (REGN2477), and LEPR antagonist mAb (H4H184572P2) in male mice

[0317] Effects of a specific antagonist anti-LEPR antibody (H4H18457P2) and 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 isolated muscle force generation in individually housed 12- to 14-week-old male LEPR mice genetically engineered to express the leptin receptor Hu / H u mice, wherein the leptin receptor is composed of the human LEPR ectodomain sequence instead of the murine LEPR ectodomain sequence.

[0318] Baseline daily food intake was measured between day 18 and day 0. On day 11, baseline whole body lean mass and fat mass were quantified by NMR. On day 0, mice were stratified into six groups of 7 to 8 mice each based on body composition from day 11 and body weight from day 0. Starting on day 0, each group received the corresponding antibody treatment dose by subcutaneous injection. REGN1033, REGN2477 and the corresponding IgG4 were administered at 10 mg / kg twice a week. P mAb. H4H18457P2 and its corresponding IgG4 were administered at 30 mg / kg once a week P mAb. Isotype control (IgG4 P ) antibodies do not bind to any known mouse proteins.

[0319] Treatment Group

[0320] a) IgG4P control (10 mg / kg + 10 mg / kg, 2× / week; 30 mg / kg, 1× / week), number = 7

[0321] b) REGN1033 (10 mg / kg, 2× / week), number = 7

[0322] c) REGN1033+REGN2477 (10 mg / kg+10 mg / kg, 2× / week), number = 7

[0323] d) H4H18457P2 (30 mg / kg, 1× / week), number = 7

[0324] e) REGN1033+H4H18457P2 (10 mg / kg, 2× / week; 30 mg / kg, 1× / week), number = 8

[0325] f) REGN1033+REGN2477+H4H18457P2 (10 mg / kg+10 mg / kg, 2× / week; 30 mg / kg, 1× / week), number = 8

[0326] Food intake and body weight were measured for each animal during the study. Body composition was quantified on days 6, 13, and 20. On day 21 or 22, animals were euthanized, and individual organs and skeletal muscle tissue were weighed and collected for additional analyses, including hepatic triglyceride quantification and skeletal muscle fiber number and cross-sectional fiber area.

[0327] Quantification of liver triglyceride content revealed that mice treated with H4H18457P2 alone or in combination with REGN1033 showed increased liver triglyceride content when compared to mice administered with an isotype control antibody (Table 12). In contrast, liver triglyceride content in mice treated with H4H18457P2 and REGN1033 and REGN2477 did not increase when compared to mice administered with an isotype control antibody (Table 12). Mice treated with H4H18457P2 and REGN1033 and REGN2477 showed reduced liver triglyceride content when compared to mice treated with H4H18457P2 and REGN1033 (Table 12). In summary, LEPR antagonist antibody treatment increased liver triglyceride content, and liver triglyceride content decreased in the case of combined treatment with LEPR antagonists, anti-ActA, and anti-MSTN blocking antibodies.

[0328] Table 12. Liver triglycerides

[0329]

[0330] **Data from 2 of 7 mice are not included in the values ​​shown

[0331] *Data from 1 of 7 mice are not included in the values ​​shown. Sequence Listing <110> Razerne Pharmaceuticals J.Altarejos J. Colomada <120> Compositions and methods for increasing body weight and lean muscle mass using antagonists to leptin receptor, GDF8 and activin A <130> 10547WO01 <140> TBD <141> 2019-12-17 <150> 62 / 781226 <151> 2018-12-18 <160> 30 <170> PatentIn 3.5 Edition <210> 1 <211> 117 <212> PRT <213> Homo sapiens <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Ser Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ile Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr His Cys 85 90 95 Ala Arg Ala Arg Tyr Gly Gly Ala Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 2 <211> 108 <212> PRT <213> Homo sapiens <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 3 <211> 6 <212> PRT <213> Homo sapiens <400> 3 Gln Ser Ile Ser Ser Tyr 1 5 <210> 4 <211> 3 <212> PRT <213> Homo sapiens <400> 4 Ala Ala Ser 1 <210> 5 <211> 10 <212> PRT <213> Homo sapiens <400> 5 Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 5 10 <210> 6 <211> 8 <212> PRT <213> Homo sapiens <400> 6 Gly Phe Thr Phe Asp Asp Tyr Gly 1 5 <210> 7 <211> 8 <212> PRT <213> Homo sapiens <400> 7 Ile Ser Trp Asn Gly Gly Ile Thr 1 5 <210> 8 <211> 10 <212> PRT <213> Homo sapiens <400> 8 Ala Arg Ala Arg Tyr Gly Gly Ala Asp Tyr 1 5 10 <210> 9 <211> 120 <212> PRT <213> Homo sapiens <400> 9 Glu Val Gln Val Leu Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ala Trp Lys Met Ser Gly Leu Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Ile Val Ser Ser 115 120 <210> 10 <211> 107 <212> PRT <213> Homo sapiens <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asp Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Arg Leu Leu Ile 35 40 45 Tyr Thr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Ala Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 6 <212> PRT <213> Homo sapiens <400> 11 Gln Asp Ile Ser Asp Tyr 1 5 <210> 12 <211> 3 <212> PRT <213> Homo sapiens <400> 12 Thr Thr Ser 1 <210> 13 <211> 9 <212> PRT <213> Homo sapiens <400> 13 Gln Lys Tyr Asp Ser Ala Pro Leu Thr 1 5 <210> 14 <211> 8 <212> PRT <213> Homo sapiens <400> 14 Gly Phe Thr Phe Ser Ala Tyr Ala 1 5 <210> 15 <211> 8 <212> PRT <213> Homo sapiens <400> 15 Ile Ser Gly Ser Gly Gly Ser Ala 1 5 <210> 16 <211> 13 <212> PRT <213> Homo sapiens <400> 16 Ala Lys Asp Gly Ala Trp Lys Met Ser Gly Leu Asp Val 1 5 10 <210> 17 <211> 126 <212> PRT <213> Homo sapiens <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Phe Ser Ser His 20 25 30 Phe Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Leu Tyr Thr Gly Gly Thr Ser Phe Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Ser Met Ser Val Gly Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Ser Gly Ile Thr Phe Thr Gly Ile Ile Val Pro Gly Ser 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 18 <211> 108 <212> PRT <213> Homo sapiens <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 19 <211> 7 <212> PRT <213> Homo sapiens <400> 19 Gln Ser Val Ser Ser Ser Tyr 1 5 <210> 20 <211> 3 <212> PRT <213> Homo sapiens <400> 20 Gly Ala Ser 1 <210> 21 <211> 9 <212> PRT <213> Homo sapiens <400> 21 Gln Gln Tyr Gly Ser Ser Pro Trp Thr 1 5 <210> 22 <211> 8 <212> PRT <213> Homo sapiens <400> 22 Gly Gly Ser Phe Ser Ser His Phe 1 5 <210> 23 <211> 7 <212> PRT <213> Homo sapiens <400> 23 Ile Leu Tyr Thr Gly Gly Thr 1 5 <210> 24 <211> 20 <212> PRT <213> Homo sapiens <400> 24 Ala Arg Ala Arg Ser Gly Ile Thr Phe Thr Gly Ile Ile Val Pro Gly 1 5 10 15 Ser Phe Asp Ile 20 <210> 25 <211> 444 <212> PRT <213> Homo sapiens <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Ser Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ile Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr His Cys 85 90 95 Ala Arg Ala Arg Tyr Gly Gly Ala Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 26 <211> 215 <212> PRT <213> Homo sapiens <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 27 <211> 447 <212> PRT <213> Homo sapiens <400> 27 Glu Val Gln Val Leu Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ala Trp Lys Met Ser Gly Leu Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 28 <211> 214 <212> PRT <213> Homo sapiens <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asp Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Arg Leu Leu Ile 35 40 45 Tyr Thr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Ala Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 29 <211> 453 <212> PRT <213> Homo sapiens <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Phe Ser Ser His 20 25 30 Phe Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Leu Tyr Thr Gly Gly Thr Ser Phe Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Ser Met Ser Val Gly Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Ser Gly Ile Thr Phe Thr Gly Ile Ile Val Pro Gly Ser 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 130 135 140 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185 190 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 195 200 205 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 210 215 220 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 325 330 335 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Leu Gly Lys 450 <210> 30 <211> 215 <212> PRT <213> Homo sapiens <400> 30 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. A combination of components comprising: A LEPR antagonist antibody or antigen-binding fragment thereof that specifically binds to a leptin receptor (LEPR), comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is as shown in SEQ ID NO:6, the amino acid sequence of the CDR-H2 is as shown in SEQ ID NO:7, and the amino acid sequence of the CDR-H3 is as shown in SEQ ID NO:8; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is as shown in SEQ ID NO:3, the amino acid sequence of the CDR-L2 is as shown in SEQ ID NO:4, and the amino acid sequence of the CDR-L3 is as shown in SEQ ID NO:5; and A GDF8 antagonist antibody or antigen-binding fragment thereof that specifically binds to growth and differentiation factor-8 (GDF8), comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is as shown in SEQ ID NO: 14, the amino acid sequence of the CDR-H2 is as shown in SEQ ID NO: 15, and the amino acid sequence of the CDR-H3 is as shown in SEQ ID NO: 16; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is as shown in SEQ ID NO: 11, the amino acid sequence of the CDR-L2 is as shown in SEQ ID NO: 12, and the amino acid sequence of the CDR-L3 is as shown in SEQ ID NO: 13; and An activin A antagonist antibody or its antigen-binding fragment that specifically binds to activin A, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of CDR-H1 is shown in SEQ ID NO:22, the amino acid sequence of CDR-H2 is shown in SEQ ID NO:23, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO:24; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of CDR-L1 is shown in SEQ ID NO:19, the amino acid sequence of CDR-L2 is shown in SEQ ID NO:20, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

21.

2. The combination according to claim 1, wherein said The LEPR antagonist antibody or antigen-binding fragment thereof that specifically binds to the leptin receptor (LEPR) is an antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; The GDF8 antagonist antibody or antigen-binding fragment thereof that specifically binds to growth and differentiation factor-8 (GDF8) is an antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10; and The activin A antagonist antibody or its antigen-binding fragment that specifically binds to activin A is an antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

18.

3. The combination according to claim 1, wherein said The LEPR antagonist antibody or antigen-binding fragment thereof that specifically binds to the leptin receptor (LEPR) is an antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26; The GDF8 antagonist antibody or antigen-binding fragment thereof that specifically binds to growth and differentiation factor-8 (GDF8) is an antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 28; and The activin A antagonist antibody or its antigen-binding fragment that specifically binds to activin A is an antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

30.

4. The combination according to claim 1, wherein the combination further comprises a pharmaceutically acceptable carrier or diluent.

5. The combination according to claim 1, wherein at least two of the antagonists selected from the group consisting of a leptin receptor antagonist, a GDF8 antagonist, and an activin A antagonist are co-formulated.

6. The combination of claim 1, wherein the LEPR antagonist, GDF8 antagonist and activin A antagonist are in separate compositions.

7. The combination according to claim 1, comprising another therapeutic agent.

8. The combination of claim 7, wherein the additional therapeutic agent is one or more selected from the group consisting of 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.

9. The combination according to any one of claims 1 to 8, wherein the combination is formulated for injection.

10. An injection device or container comprising the combination according to any one of claims 1 to 8.

11. The device or container according to claim 10, which is a container as a vial.

12. The device or container of claim 10 which is an injection device that is a hypodermic needle and syringe, an autoinjector, or a prefilled syringe.

13. Use of a combination of components comprising a leptin receptor antagonist and a growth and differentiation factor-8 (GDF8) antagonist and an activin A antagonist for the preparation of a medicament for increasing food intake, fat mass, body weight, muscle strength, muscle fiber size and / or lean mass in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the combination, wherein The leptin receptor (LEPR) antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is shown in SEQ ID NO:6, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:7, and the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:8; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:3, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:4, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:5; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is as shown in SEQ ID NO: 14, the amino acid sequence of the CDR-H2 is as shown in SEQ ID NO: 15, and the amino acid sequence of the CDR-H3 is as shown in SEQ ID NO: 16; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is as shown in SEQ ID NO: 11, the amino acid sequence of the CDR-L2 is as shown in SEQ ID NO: 12, and the amino acid sequence of the CDR-L3 is as shown in SEQ ID NO: 13; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is shown in SEQ ID NO:22, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:23, and the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:24; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:19, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:20, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:

21.

14. Use according to claim 13, for increasing lean mass, wherein the increase is at the expense of fat mass.

15. Use of a combination of components comprising a leptin receptor antagonist and a growth and differentiation factor-8 (GDF8) antagonist and an activin A antagonist for the preparation of a medicament for enhancing athletic performance in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the combination, wherein The leptin receptor (LEPR) antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is shown in SEQ ID NO:6, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:7, and the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:8; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:3, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:4, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:5; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is as shown in SEQ ID NO: 14, the amino acid sequence of the CDR-H2 is as shown in SEQ ID NO: 15, and the amino acid sequence of the CDR-H3 is as shown in SEQ ID NO: 16; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is as shown in SEQ ID NO: 11, the amino acid sequence of the CDR-L2 is as shown in SEQ ID NO: 12, and the amino acid sequence of the CDR-L3 is as shown in SEQ ID NO: 13; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is shown in SEQ ID NO:22, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:23, and the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:24; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:19, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:20, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:

21.

16. The use according to claim 15, wherein the individual is undergoing physical therapy.

17. The method of claim 13, wherein the subject suffers from one or more of the following: malnutrition, developmental delay, inadequate food intake, eating disorders, cachexia, muscle atrophy or wasting, and muscle damage, and / or Rehabilitation from a stroke is ongoing.

18. Use of a combination of components comprising a leptin receptor antagonist and a growth and differentiation factor-8 (GDF8) antagonist and an activin A antagonist in the preparation of a medicament for treating or preventing malnutrition, cachexia, developmental delay, eating disorders characterized by inadequate caloric intake, muscle atrophy, age-related sarcopenia or muscle damage in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the combination, wherein The leptin receptor (LEPR) antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is shown in SEQ ID NO:6, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:7, and the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:8; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:3, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:4, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:5; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is as shown in SEQ ID NO: 14, the amino acid sequence of the CDR-H2 is as shown in SEQ ID NO: 15, and the amino acid sequence of the CDR-H3 is as shown in SEQ ID NO: 16; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is as shown in SEQ ID NO: 11, the amino acid sequence of the CDR-L2 is as shown in SEQ ID NO: 12, and the amino acid sequence of the CDR-L3 is as shown in SEQ ID NO: 13; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is shown in SEQ ID NO:22, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:23, and the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:24; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:19, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:20, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:

21.

19. Use of a combination of components comprising a leptin receptor antagonist and a growth and differentiation factor-8 (GDF8) antagonist and an activin A antagonist in the preparation of a medicament for reducing increased hepatic triglyceride levels in an individual administered a leptin receptor antagonist, comprising administering to the individual a therapeutically effective amount of the combination, wherein The leptin receptor (LEPR) antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to LEPR, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is shown in SEQ ID NO:6, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:7, and the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:8; and the light chain variable region comprises: three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:3, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:4, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:5; The GDF8 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to GDF8, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is as shown in SEQ ID NO: 14, the amino acid sequence of the CDR-H2 is as shown in SEQ ID NO: 15, and the amino acid sequence of the CDR-H3 is as shown in SEQ ID NO: 16; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is as shown in SEQ ID NO: 11, the amino acid sequence of the CDR-L2 is as shown in SEQ ID NO: 12, and the amino acid sequence of the CDR-L3 is as shown in SEQ ID NO: 13; and The activin A antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to activin A, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) of the heavy chain variable region, wherein the amino acid sequence of the CDR-H1 is shown in SEQ ID NO:22, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:23, and the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:24; and the light chain variable region comprises three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3) of the light chain variable region, wherein the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:19, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:20, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:

21.

20. The use according to any one of claims 13, 15, 18 and 19, wherein the combination is administered parenterally.

21. A method of manufacturing a combination of components according to any one of claims 1 to 8, comprising co-formulating the LEPR antagonist, the GDF8 antagonist and the activin A antagonist with a pharmaceutically acceptable carrier.

22. A combination of components which is the product of the process of claim 21.

23. A method of making a device or container according to claim 10, comprising introducing the combined components into the container or device.

24. A device or container which is the product of the method of claim 23.

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