Anti-activin a antibodies and uses thereof

Abstract

To provide antibodies that bind to Activin A, and to provide methods of using the same.SOLUTION: Provided is an isolated antibody or antigen-binding fragment thereof that specifically binds Activin A with a binding dissociation equilibrium constant (KD) of less than about 5 pM as measured in a surface plasmon resonance assay at 25°C. The antibody or antigen-binding fragment thereof may specifically bind Activin A with a KD of less than about 4 pM as measured in a surface plasmon resonance assay at 25°C, and may specifically bind Activin A with a binding association equilibrium constant (Ka) of less than about 500 nM.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibodies and antigen-binding fragments thereof specific for activin A, and and methods of using antibodies specific for bin A, including in combination with myostatin inhibitors. Regarding. [Background technology]

[0002] Activins are members of the transforming growth factor-beta (TGF-β) superfamily. Activins belong to the family of activins and exert a wide range of biological effects on cell proliferation, differentiation, and apoptosis. Activins are homo- or heterodimers of inhibin βA, inhibin βB, inhibin βC, and inhibin βE, the different combinations of which create the various members of the activin protein family. For example, activin A is a homodimer of inhibin βA, activin B is a homodimer of inhibin βB, whereas activin AB is a homodimer of inhibin βA and It is a heterodimer with inhibin βB, and activin AC is a heterodimer with inhibin βA and inhibin βC [Tsuchida, K. et al., Cell Commun Signal 7:15 (2009)].

[0003] Activin A binds to and activates receptor complexes on the cell surface known as activin type II receptors (types IIA and IIB, also known as ActRIIA and ActRIIB, respectively). Activation of these receptors results in phosphorylation of the activin type I receptors (e.g., Alk4 or 7), followed by phosphorylation of SMAD2 and 3 proteins, formation of a SMAD complex (with SMAD4), and translocation of the SMAD complex to the cell nucleus, where SMAD2 and SMAD3 upregulate various genes. It functions to regulate the transcription of [Sozzani, S. and Musso, T., Blood 117(19):5013~5015 (2011)]. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 5,500,362 [Patent Document 2] U.S. Patent No. 5,821,337 [Patent Document 3] WO05 / 103081 [Patent Document 4] U.S. Patent No. 8,586,713 [Patent Document 5] US2011-0293630A1 [Patent Document 6] US2007 / 0280945A1 [Patent Document 7] US8,309,082 [Patent Document 8] US2010 / 0272734A1 [Non-patent literature]

[0005] [Non-Patent Document 1] Tsuchida, K. et al., Cell Commun Signal 7:15 (2009) [Non-Patent Document 2] Sozzani, S. and Musso, T., Blood 117(19):5013~5015 (2011) [Non-Patent Document 3] McPherron AC et al. (1997). Nature 387(6628):83~90 [Non-Patent Document 4] Lee SJ et al. (2005) Proc Natl Acad Sci USA 102(50):18117~18122 [Non-Patent Document 5] Reddy et al., J Immunol 164:1925-1933 (2000) [Non-Patent Document 6] Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)

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[0006] GDF8 (myostatin), activin B, activin AB, inhibin A, inhibin B, GDF3 A number of other ligands, including GDF11, Nodal, BMP2, BMP4, BMP7, BMP9, and BMP10, bind to and activate ActRIIB. Blocking the interaction of ActRIIB with its ligands For example, GDF8 plays a central role in the development and maintenance of skeletal muscle and acts as a negative regulator of muscle mass [McPherron AC et al. (1997). Nature 387(6628):83-90]. Administration of ActRIIB-Fc (i.e., the extracellular portion of the type IIB receptor ActRIIB stabilized by fusion to the IgG Fc domain) increases skeletal muscle mass. A significant increase in muscle mass occurs, improving measures of muscle mass and strength in mice [Lee SJ et al. (2005) Proc Natl Acad Sci USA 102(50):18117-18122]. In Mstn (myostatin)-deficient mice, the efficacy of ActRIIB-Fc is attenuated but not eliminated, suggesting that in addition to myostatin, other ActRIIB ligands may also function as negative regulators of muscle growth. Therefore, there is a need for further ActRIIB signaling inhibitors that can provide clinical benefit. It is being considered. [Means for solving the problem]

[0007] The present invention relates to inhibin βA and dimers containing inhibin βA, e.g., activin A, The present invention provides an antibody that binds to activin AB, etc. The antibody of the present invention is, in particular, an antibody that binds to activin A. by inhibiting activin A-mediated signal transduction. These antibodies are useful for producing more beneficial clinical outcomes, for example, for treating diseases and disorders caused by or related to the activity and / or signaling of activin A. The antibodies of the present invention also have utility for use in conjunction with inhibitors of other ligands of the ActRIIA and ActRIIB receptors, for example GDF8 inhibitors.

[0008] The antibodies of the invention may be full length (e.g., IgG1 or IgG4 antibodies) or may contain only the antigen-binding site (e.g., Fab, F(ab')2 or scFv fragments) and may be modified to affect functionality, e.g., to remove residual effector functions [Reddy et al., J Immunol 164:1925-1933 (2000)].

[0009] The present invention provides a method for the preparation of a medicament having a binding of less than about 500 nM as measured by surface plasmon resonance assay at 25° C. Binding association equilibrium constant (K a ) and a dissociation average of less than about 5 pM Equilibrium constant (K D An isolated antibody or an antigen-binding flag thereof that specifically binds to activin A at In some embodiments of the invention, the isolated antibody or antigen-binding fragment thereof has an affinity for less than about 4 pM as measured by a surface plasmon resonance assay at 25° C. K D In some embodiments of the present invention, the isolated The antibodies or antigen-binding fragments thereof have a binding association equilibrium constant (K a ) to activate It specifically binds to thybin A.

[0010] The present invention relates to a method for the treatment of activin A-related disorders, comprising administering to a subject a therapeutic agent that specifically binds to activin A and activates at least one activin A receptor. The present invention provides an isolated antibody or antigen-binding fragment thereof that blocks binding to thymine A. In some embodiments of the invention, the isolated antibody or antigen-binding fragment thereof has a binding affinity to at least one of the following antibodies: , IC less than about 80 pM 50In some embodiments of the invention, the isolated antibody or antigen-binding fragment thereof blocks binding of activin A to the activin A receptor with a ligand binding activity of less than about 60 pM as measured in an in vivo receptor / ligand binding bioassay at 25° C. Mitsui IC 50 The invention also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to activin A and blocks activation of at least one activin A receptor by activin A. In some embodiments of the invention, the isolated antibody or antigen-binding fragment thereof does not significantly block binding of activin A to activin type II receptors. In one embodiment, the isolated antibody or antigen-binding fragment thereof is activin IIA. The antibody or antigen-binding fragment thereof inhibits binding of activin A to an activin A receptor selected from the group consisting of activin type A receptor (ActRIIA), activin type IIB receptor (ActRIIB), and activin type I receptor. In some embodiments of the invention, the isolated antibody or antigen-binding fragment thereof inhibits activin A-mediated activation of SMAD complex signaling.

[0011] The present invention relates to a heavy chain polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 106, 114, 122, 130, 138, 154, 162, 170, 178, 186, 194, and 202. The present invention provides an antibody or antigen-binding fragment thereof comprising a heterologous variable region (HCVR), or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0012] The present invention also provides an antibody or antigen-binding fragment of an antibody comprising a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 146, and 210, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0013] The present invention also provides methods for identifying HCVR and LCVR (HCVR / LCVR) sequence pairs comprising HCVR and LCVR (HCVR / LCVR) sequence pairs selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210, Antibodies or antigen-binding fragments thereof are also provided.

[0014] The present invention also relates to an antibody or an antibody fragment thereof, comprising a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 112, 120, 128, 136, 144, 160, 168, 176, 184, 192, 200, and 208, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 152, and 216, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The original binding fragment is also provided.

[0015] In certain embodiments, the antibody or antigen-binding portion of the antibody is selected from the group consisting of SEQ ID NOs: 8 / 16, 24 / 32 , 40 / 48, 56 / 64, 72 / 80, 88 / 96, 104 / 96, 112 / 96, 120 / 96, 128 / 96, 136 / 96, 144 / 152, 160 / 152, 168 / 152, 176 / 152, 184 / 152, 192 / 152, 200 / 152, and 208 / 216.

[0016] The present invention also relates to a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 108, 116, 124, 132, 140, 156, 164, 172, 180, 188, 196, and 204, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134, 142, 158, 166, 174, 182, 190, 198, and 206, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 148, and 212, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; light chain CDR1 (LCDR1) domain having a substantially similar sequence thereof having at least 99% sequence identity; and from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 150, and 214. The selected amino acid sequence, or at least 90%, at least 95%, at least 98% or Also provided are antibodies or fragments thereof further comprising a light chain CDR2 (LCDR2) domain having a substantially similar sequence thereto having at least 99% sequence identity.

[0017] Certain non-limiting exemplary antibodies and antigen-binding fragments of the invention include those of SEQ ID NOs: 4-6-8-12-14-16 (e.g., H4H10423P); 20-22-24-28-30-32 (e.g., H4H10424P); 36-38-40-44-46-48 (e.g., H4H10426P); 52-54-56-60-62-64 (e.g., H4H10429P); 68-70-72-76-78 -80 (e.g. H4H10430P); 84-86-88-92-94-96 (e.g. H4H10432P2); 100-102-104-92-94-96 (e.g. H4H10433P2); 108-110-112-92-94-96 (e.g. H4H10436P2); 116-118-120-92-94-96 (e.g. H4H10437P2); 124-126-128-92-94-96 (e.g. H4H10438P2);132-134-136-92-94-96 (e.g. H4H10440P2);140-142-144-148-150-152 (e.g. H4H10442P2);156-158-160-148-150-152 (H4H10445P2);164-166-168-148-150-152 (H4H10446P2);172-174-176-14 8-150-152 (H4H10447P2); 180-182-184-148-150-152 (H4H10448P2); 188-190-192-148-150-152 (H4H10452P2); 196-198-200-148-150-152 (H4H10468P2); and 204-206-208-212-214-216 (H2aM10965N), respectively. The present invention relates to a method for the preparation of a peptide comprising the steps of: nothing.

[0018] In a related embodiment, the invention provides heavy and light chain variable fragments selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210. The heavy and light chain CDR domains contained within the HCVR / LCVR sequences are specifically identified as activin A. Methods and techniques for identifying CDRs within the amino acid sequences of HCVRs and LCVRs are well known in the art and are described herein. can be used to identify CDRs within the amino acid sequences of a particular disclosed HCVR and / or LCVR. Exemplary routine methods that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is , based on sequence variability, whereas the Chothia definition is based on the location of structural loop regions. The AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., PNAS (USA) 86:9268-9272 (1989). Public Databases A sequence similar to that described above can also be used to identify CDR sequences within an antibody.

[0019] The present invention also provides nucleic acid molecules encoding anti-activin A antibodies or portions thereof. For example, the invention provides a nucleic acid molecule encoding any of the HCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0020] The present invention also provides nucleic acid molecules encoding any of the amino acid sequences of the LCVRs listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0021] The present invention also relates to a method for the preparation of a polypeptide encoding any one of the amino acid sequences of HCDR1 listed in Table 1. Also provided are nucleic acid molecules that encode the HCDR1 polypeptide, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of HCDR1 listed in Table 2, or These include substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0022] The present invention also relates to a method for the preparation of a polypeptide encoding any one of the amino acid sequences of HCDR2 listed in Table 1. Also provided are nucleic acid molecules that encode the HCDR2 sequences, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of HCDR2 listed in Table 2, or These include substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0023] The present invention also relates to a method for the preparation of a polypeptide encoding any one of the amino acid sequences of HCDR3 listed in Table 1. Also provided are nucleic acid molecules that encode the HCDR3, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of HCDR3 listed in Table 2, or These include substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0024] The present invention also relates to a method for the production of a peptide encoding any one of the amino acid sequences of LCDR1 listed in Table 1. Also provided are nucleic acid molecules that encode the LCDR1 polypeptide, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or These include substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0025] The present invention also relates to a method for the production of a peptide encoding any one of the LCDR2 amino acid sequences listed in Table 1. Also provided are nucleic acid molecules that encode the LCDR2 polypeptides, and in certain embodiments, the nucleic acid molecules are selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or These include substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0026] The present invention also relates to a method for the production of a peptide encoding any one of the amino acid sequences of LCDR3 listed in Table 1. Also provided are nucleic acid molecules that encode the LCDR3 polypeptides, and in certain embodiments, the nucleic acid molecules are selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or These include substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0027] The present invention also relates to a nucleic acid molecule encoding an HCVR, which comprises a set of three CDRs (i.e. The set of amino acid sequences of HCDR1-HCDR2-HCDR3 is shown in Table Also provided are nucleic acid molecules as defined in any of the exemplary anti-activin A antibodies listed in Table 1.

[0028] The present invention also relates to a nucleic acid molecule encoding an LCVR, which comprises a set of three CDRs (i.e. The set of amino acid sequences of LCDR1-LCDR2-LCDR3 is shown in Table Also provided are nucleic acid molecules as defined in any of the exemplary anti-activin A antibodies listed in Table 1.

[0029] The invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises any of the amino acid sequences of the HCVRs listed in Table 1, and the LCVR comprises any of the amino acid sequences of the LCVRs listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same anti-activin A antibody listed in Table 1.

[0030] The present invention also relates to a method for expressing a polypeptide comprising a heavy or light chain variable region of an anti-activin A antibody. Also provided are recombinant expression vectors that can be used to express the nucleic acid molecules of interest. For example, the invention encompasses recombinant expression vectors that include any of the nucleic acid molecules described above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences as set forth in Table 1. Also included within the scope of the invention are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions permissive for the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

[0031] The present invention encompasses anti-activin A antibodies having altered carbohydrate content. In some applications, modifications to remove undesirable glycosylation sites may be useful. In some applications, modifications to alter glycosylation patterns may be useful, for example, antibodies may be modified to delete fucose moieties present in oligosaccharide chains, for example to increase antibody-dependent cellular cytotoxicity (ADCC) function [see Shield et al., J Biol Chem 277:26733 (2002)]. In other applications, modifications to alter complement-dependent cytotoxicity (CDC) may be useful. Galactosylation modifications may be made. In some applications, the antibodies may have altered glycosylation patterns to minimize effector function. For example, the antibodies may be modified to obtain more glycosylated or sialylated antibodies.

[0032] In another aspect, the present invention relates to a recombinant human antibody or a method thereof that specifically binds to activin A. and a pharma- ceutical composition comprising a fragment of and a pharma- ceutical acceptable carrier. In a related aspect, the invention features a composition that is a combination of an anti-activin A antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that may be advantageously combined with an anti-activin A antibody. Examples of agents that may be advantageously combined with an anti-activin A antibody include, but are not limited to, Examples of suitable agents include, but are not limited to, other agents that inhibit activin A activity (e.g., other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that do not directly bind to activin A but interfere with, block, or attenuate activin A-mediated signal transduction. In one embodiment, the second therapeutic agent inhibits, interferes with, blocks, and / or attenuates the activity of another ligand of the ActRIIA and / or ActRIIB receptor (e.g., GDF8, activin B, activin AB, inhibin A, inhibin B, GDF3, GDF11, Nodal, BMP2, BMP4, and / or BMP7). In one embodiment, the second therapeutic agent is an anti-GDF8 antagonist (e.g., a human anti-GDF8 antibody or antigen-binding fragment thereof). Exemplary anti-GDF8 agents for use with the anti-activin A antibodies of the invention include human anti-GDF8 Antibodies [e.g., amino acid sequences of HCVR / LCVR or CDRs as described in US2011-0293630A1 and an anti-GDF8 antibody comprising any one of the sequences of SEQ ID NO: 217 (e.g., H4H1657N2, which is an HCVR sequence comprising SEQ ID NO: 217). A heavy chain complementarity determining region (HCDR) (e.g., the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 218, 219, and 220, respectively) and a light chain complementarity determining region (LCDR) of LCVR comprising SEQ ID NO: 221 (e.g., and CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NOs: 222, 223, and 224). Additional combination therapies and co-formulations comprising the anti-activin A antibodies of the invention are disclosed elsewhere herein.

[0033] In a further embodiment of the invention, an activin A specific binding domain and a GDF8 specific binding domain are provided. In one embodiment of this aspect of the invention, the antigen binding molecule is a bispecific antibody comprising a first variable domain that specifically binds activin A and a second variable domain that specifically binds GDF8.

[0034] In yet another aspect, the present invention relates to an anti-activin A antibody or an antigen-binding portion of an antibody of the present invention. A therapeutic method for inhibiting activin A activity using a polypeptide of the present invention, comprising administering to said subject a therapeutically effective amount of said polypeptide. The disorder to be treated is characterized by the ablation, inhibition or amelioration of activin A activity or signaling. The anti-activin A antibody or antibody fragment of the present invention binds to activin A and activin type II receptors (e.g., For example, it may function to block the interaction of activin A with the activin type I receptor; to block the interaction of activin A with both type II and type I receptors; or to otherwise inhibit the signaling activity of activin A.

[0035] The invention also encompasses the use of an anti-activin A antibody or an antigen-binding portion of an antibody of the invention in the manufacture of a medicament for treating a disease or disorder related to or caused by activin A activity in a patient. The invention also provides a method for increasing muscle mass or strength in a subject by administering to the subject an activin A antibody or antigen-binding fragment thereof. The invention also provides an activin A specific binding protein, administering to a subject a protein and a GDF8-specific binding protein, or an activin A-specific binding protein Also provided is a method for increasing muscle mass or strength in a subject by administering to the subject an antigen-binding molecule comprising the domain and a GDF8-specific binding domain.

[0036] The present invention also encompasses methods for treating, preventing, and / or alleviating a disease or disorder characterized by loss of muscle mass or strength by administering an activin A specific binding protein (e.g., an anti-activin A antibody) to a subject in need thereof. The methods of the invention include treating, preventing, and / or alleviating a disease or disorder characterized by loss of muscle mass or strength by administering an activin A specific binding protein and a GDF8 specific binding protein (e.g., an anti-activin A antibody and an anti-GDF8 antibody) to a subject in need thereof. The method of the present invention also includes a method for producing a activin A-specific binding domain and a GDF8-specific binding domain. The present invention also includes treating, preventing and / or alleviating a disease or disorder characterized by a decrease in muscle mass or strength by administering to a subject in need thereof an antigen-binding molecule comprising the domain. Muscle mass or strength that can be treated, prevented and / or ameliorated using the methods of the present invention. Diseases or disorders characterized by a decrease in the level of glucose in the blood include sarcopenia, cachexia [e.g., idiopathic cachexia or cachexia secondary to another condition (e.g., cancer, chronic renal failure, or chronic obstructive pulmonary disease)], muscle injury, muscle wasting and / or atrophy (e.g., due to disuse, immobilization, bed rest, injury, , caused by or associated with medical procedures, surgical interventions (e.g., hip fractures, hip replacements, and knee replacements) and the need for artificial ventilation], cancer, obesity, diabetes Disease, arthritis, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, and metabolic syndrome (e.g., diabetes, obesity, , malnutrition, organ atrophy, chronic obstructive pulmonary disease, and anorexia).

[0037] The present invention also provides a method for administering to a subject in need thereof a protein that specifically binds activin A (i.e., an inhibin βA dimer), such as an anti-activin A antibody or an antigen-binding fragment thereof. A molecule containing inhibin βA (e.g., a bispecific molecule containing inhibin βA) is Also provided are methods for treating, preventing and / or alleviating a disease or disorder caused by, promoted by, exacerbated by, or aggravated by the activity of a cytochrome P450 antagonist, such as activin A, activin AB, or the like. In one embodiment of the present invention, the method of the present invention includes administering an anti-activin A antibody to a subject in need thereof. and / or a method for treating, preventing and / or alleviating renal fibrosis by administering to a subject In certain embodiments of the invention, the methods of the invention include administering an anti-activin A antibody to a subject in need thereof. A method for treating chronic kidney disease [e.g., hypertension, diabetes, glomerulonephritis, due to genetic disorders (such as polycystic kidney disease), kidney malformations, autoimmune diseases (e.g., lupus), or as a result of obstruction (e.g., kidney stones, tumors, prostate enlargement), or repeated urinary tract infections The present invention also includes methods for treating, preventing and / or alleviating renal fibrosis caused by Additional embodiments include the treatment of sepsis by administering an anti-activin A antibody to a subject in need thereof. , chronic heart failure, chronic obstructive pulmonary disease, benign or malignant pheochromocytoma, uterine fibroids / leiomyomas , pre-eclampsia, keloids, hypertrophic scars, or pulmonary hypertension. Additional aspects of the invention include methods of administering an anti-activin A antibody to a subject in need thereof. The present invention relates to a method for treating a disease caused by, promoted by, or aggravated by activin A activity, comprising administering the drug to a patient having a disease to which the drug is administered. The present invention also encompasses methods for treating, preventing and / or alleviating cachexia, which is caused or exacerbated by the The present embodiment provides a method for treating activin A infection by administering an anti-activin A antibody to a subject in need thereof. The present invention relates to a method for treating weight loss caused by, promoting, exacerbating, or worsening hypercalcaemia caused by insulin A activity. The present invention includes methods for treating, preventing and / or ameliorating the symptoms of the disease.

[0038] Other embodiments will become apparent from review of the detailed description set forth below. [Brief description of the drawings]

[0039] [Figure 1]1 is a matrix showing the results of an antibody cross-competition assay in which a first anti-activin A antibody ("antibody sample") is applied to an anti-human FC-coated sensor chip, followed by exposure to a solution of a second anti-activin A antibody (1 μM) that has been pre-bound to activin A. Binding responses (values ​​between 0.22 and 1.84) are shown for each antibody combination tested. Binding responses shown in black type in white boxes indicate no competition for activin A binding, suggesting separate binding regions. [Diagram 2] Panel A shows the effect of 21 days of anti-GDF8 antibody treatment (H4H1657N2, 10 mg / kg or 30 mg / kg) on ​​mean peak tetanic force compared to isotype control antibody. Data was analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test. * is significance of p<0.05 vs. isotype control (n=6, unpaired Student's t-test); ns=not statistically significant compared to isotype 30 mg / kg. Panel B shows the increase in peak tetanic force of tibialis anterior (TA) muscle in H4H1657N2 treated mice (10 mg / kg) versus isotype control antibody treated mice (n=6) for 3 weeks when stimulated with currents over a range of frequencies (40 to 100 Hz). Data are presented as mean peak force ± SEM. [Diagram 3] Panel A shows the experimental design to evaluate the effect of H4H1657N2 during recovery from hindlimb suspension-induced muscle atrophy. Panel B shows the percentage change in muscle mass of TA and gastrocnemius (GA) in mice treated with H4H1657N2 and mice treated with isotype control antibody (HLS+7Rec) after 7 days of recovery from hindlimb suspension compared to mice without recovery period (HLS) and control mice (non-HLS control) after 7 days of hindlimb suspension. Values ​​are expressed as mean percentage change relative to control non-HLS values ​​± SEM. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test. *=significant at p<0.05 vs. non-HLS group. #=significant at p<0.05 vs. HLS group. [Figure 4] Panel A shows the effect of anti-activin A antibody H4H10446P2 administration on body weight (compared to isotype control) in mice overexpressing activin A. Data were analyzed using a two-way analysis of variance (repeated measures ANOVA + Bonferroni multiple comparison test) followed by Tukey's test. * = p<0.05 vs. isotype control; # = p<0.05 vs. activin A + isotype control. Panel B shows the effect of anti-activin A antibody H4H10446P2 on muscle mass of the tibialis anterior (TA) and gastrocnemius (GA) (compared to isotype control) in mice overexpressing activin A. Data were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's test. * = p<0.05 vs. vector + isotype control; # = p<0.05 vs. activin A + isotype control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Before describing the invention, it is to be understood that the invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary, and the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention will be limited only by the appended claims.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary by 1% or less from the recited value. For example, the expression "about 100" means As used herein, it includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0042] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0043] Antigen-specific binding proteins The present invention relates to compositions comprising antigen-specific binding proteins. More specifically, the present invention provides compositions comprising activin A-specific binding proteins.

[0044] The expression "antigen-specific binding protein" as used herein means a protein that contains at least one domain that specifically binds to a particular antigen. Exemplary categories of fusion proteins include antibodies, antigen-binding sites of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, and proteins that contain the ligand-binding portion of a receptor that specifically binds to a particular antigen.

[0045] The present invention relates to an antigen-specific binding protein that specifically binds to activin A, i.e., Activin A-specific binding proteins. Activin is a beta subunit, i.e., inhibin βA, inhibin βB, inhibin βC, and / or inhibin βE. The βA subunit has the amino acid sequence of SEQ ID NO: 226, and the βB subunit has the amino acid sequence of SEQ ID NO: 228. Activin A is a homodimer of two βA subunits, activin B is a homodimer of two βB subunits, activin AB is a heterodimer of one βA subunit and one βB subunit, and activin AC is a heterodimer of one βA subunit and one βC subunit. The activin A-specific binding protein can be an antigen-specific binding protein that specifically binds to the βA subunit. The βA subunit is found in activin A, activin AB, and activin AC molecules, and is therefore referred to as an "activin A-specific binding protein." The antibody "activin A" specifically binds to activin AB and activin AC. The activin A specific binding protein may be an actin specific binding protein (as it interacts with the βA subunit). Thus, according to one embodiment of the present invention, the activin A specific binding protein specifically binds to activin A; or activin A and activin AB; or activin A and activin AC; or activin A, activin AB and activin AC, but not to other ActRIIB ligands, such as activin B, GDF3, GDF8, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, BMP2, BMP4, BMP7, BMP9, BMP11, BMP2, BMP4 ... , Nodal, etc. Thus, in one embodiment of the present invention, the activin A specific binding protein specifically binds to activin A, but does not bind to activin B or activin C. In another embodiment, the activin A specific binding protein does not significantly bind to It can also bind to activin B (to cross-react with the βB subunit, i.e., inhibin βB). In another embodiment, the activin A-specific binding protein is a binding protein that specifically binds to activin A but does not bind to any other ligand of ActRIIB. In another embodiment, the activin A-specific binding protein is a binding protein that specifically binds to activin A but does not bind to any other ligand of ActRIIB. In another embodiment, the binding protein specifically binds to bin A but does not bind to any bone morphogenetic protein (BMP) (e.g., BMP2, BMP4, BMP6, BMP9, BMP10). The activin A-specific binding protein specifically binds to activin A but not to any other members of the transforming growth factor beta (TGFβ) superfamily. It is a synthetic protein.

[0046] The present invention also encompasses antigen-specific binding proteins that specifically bind to GDF8, i.e., "GDF8-specific binding proteins." The terms "GDF8" ("growth differentiation factor-8" and "myostatin"), The protein having the amino acid sequence of SEQ ID NO: 225 (mature protein) is According to the present invention, a GDF8-specific binding protein is a protein that specifically binds to GDF8. However, other ActRIIB ligands, such as GDF3, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, It does not bind to activin A, activin B, activin AB, Nodal, etc.

[0047] In the context of the present invention, molecules such as ActRIIB-Fc (e.g., "ACE-031") that contain the ligand-binding portion of the ActRIIB receptor can bind to multiple ligands in addition to GDF8, activin A, and activin AB. Because such molecules bind to activin A specific binding proteins or GDF8 specific binding proteins, such molecules are not considered to be "activin A specific binding proteins" or "GDF8 specific binding proteins."

[0048] The references herein to proteins, polypeptides and protein fragments are All are intended to refer to the human version of the respective protein, polypeptide or protein fragment, unless expressly stated to be from a non-human species.

[0049] An antigen-binding molecule having two different antigen-specific binding domains The present invention also encompasses antigen-binding molecules that contain two different antigen-specific binding domains. In particular, the present invention relates to a method for producing an activin A specific binding domain and a GDF8 specific binding domain. The term "antigen-specific binding domain" as used herein includes (i) an antigen-binding fragment molecule of an antibody, (ii) a molecule that specifically interacts with a particular antigen. and / or (iii) a polypeptide that comprises or consists of a ligand-binding portion of a receptor that specifically binds to a particular antigen. For example, the invention includes a polypeptide having one arm that comprises a first heavy chain variable region / light chain variable region (HCVR / LCVR) pair that specifically binds to activin A and another arm that comprises a second HCVR / LCVR pair that specifically binds to GDF8. The present invention also includes bispecific antibodies having the above structure.

[0050] Specific binding The term "specifically binds" or cognate terms as used herein means that an antigen-specific binding protein or antigen-specific binding domain binds to an antigen at or above 500 pM under normal test conditions. A dissociation constant (K D ) and does not bind to other unrelated antigens. "Unrelated antigens" are antigens that have less than 95% amino acid identity with each other. It is a protein, peptide, or polypeptide that specifically binds to two molecules. Methods for determining whether an antigen is a cytoplasmic ligand are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antigen-specific binding protein or an antigen-specific binding domain, as used in the context of the present invention, has a molecular weight of less than about 500 pM, less than about 400 pM, less than about 300 pM, or less than about 500 pM as measured by a surface plasmon resonance assay. K of less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM. D and binds to a specific antigen (e.g., activin A and / or AB, or GDF8) or a portion thereof. This includes molecules that

[0051] As used herein, an antigen-specific binding protein or antigen-specific binding domain "does not bind" to a specified molecule (e.g., "does not bind GDF11," "does not bind BMP9," "does not bind BMP10," etc.) means that when tested for binding to that molecule in a surface plasmon resonance assay at 25° C., the protein or binding domain has a K of greater than 50.0 nM in such an assay or an equivalent assay. D or no bond can be presented at all.

[0052] The term "surface plasmon resonance" as used herein refers to, for example, the surface plasmon resonance (SPPR) assay using the BIAcore™ system. The stem (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ) was used. This refers to an optical phenomenon that allows for the analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix using a fluorophore.

[0053] The term “K D " as used herein refers to the equilibrium dissociation constant of a particular protein-protein interaction (e.g., an antibody-antigen interaction). Unless otherwise specified, the K D Values ​​are determined by surface plasmon resonance assay at 25 °C. D Points to a value.

[0054] Antibodies and Antigen-Binding Fragments of Antibodies As indicated above, the antigen-specific binding protein may comprise or consist of an antibody or an antigen-binding fragment of an antibody. In the case of an antigen-binding molecule comprising specific binding domains, one or both of the antigen-specific binding domains may comprise or consist of an antigen-binding fragment of an antibody.

[0055] As used herein, an "antibody that binds activin" or an "anti-activin A antibody" refers to an antibody that binds activin. " includes antibodies and antigen-binding fragments thereof that bind to soluble fragments of activin A protein and may also bind to activin heterodimers containing the βA subunit of activin.

[0056] The term "antibody" as used herein refers to an antibody specific to a particular antigen (e.g., activin A). The term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that heterologously binds to or interacts with an antigen. The term "antibody" refers to an immune system that contains four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. globulin molecules, as well as multimers thereof (e.g., IgM). Regions (referred to herein as HCVR or V H The heavy chain constant region is made up of three domains: H 1. C H 2 and C H Each light chain comprises a light chain variable region (herein referred to as LCVR or is V L The light chain constant region comprises one domain (C L 1) Including Mm. V H and V LThe regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more highly conserved regions, termed framework regions (FRs). . Each V H and V L are in the following order from the amino terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. It is composed of three CDRs and four FRs arranged at the carboxy terminus. In one embodiment, the FR of the anti-activin A antibody (or an antigen-binding portion thereof) is a human germline sequence. They may be identical or may be naturally or artificially modified. The amino acid consensus sequence is defined based on a side-by-side analysis of two or more CDRs. It is possible.

[0057] The term "antibody" as used herein also encompasses antigen-binding fragments of full-length antibody molecules. The terms "antigen-binding site" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, encompass any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be isolated by any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optionally constant domains of an antibody. Such DNA can be derived, for example, from a full-length antibody molecule using methods known in the art. The nucleic acid sequence may be readily available and / or may be readily available, for example, from commercial sources, DNA libraries (e.g., phage-antibody libraries, etc.), or may be synthesized. DNA may be sequenced and manipulated chemically or using molecular biology techniques to identify, for example, one or more arranging one or more variable and / or constant domains into a suitable configuration; Or codons can be introduced, cysteine ​​residues can be created, amino acids can be modified, added or deleted, and the like.

[0058] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) isolated complementarity determining fragments, such as hypervariable regions of antibodies (e.g., CDR3 peptides). The term "antibody" includes a minimal recognition unit consisting of amino acid residues that mimic the CDR, or constrained FR3-CDR3-FR4 peptide. Other engineered molecules include domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bispecific antibodies, and the like. Also included are specific antibodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains. Included within the expression "antigen-binding fragment" as used herein.

[0059] An antigen-binding fragment of an antibody typically contains at least one variable domain. Variable domains may be of any size or amino acid composition and are generally adjacent to or in frame with one or more framework sequences. It is expected that the amino acid sequence of each of the above CDRs will be the same as that of the corresponding CDRs of the above CDRs. H Domain and V L In the antigen-binding fragment of the V H and V L The domains may be arranged relative to each other in any suitable configuration. For example, the variable region may be a dimer and the V H -V H , V H -V L Or V L -V L dual amount Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V H Or V L It may contain domains.

[0060] In certain embodiments, the antigen-binding fragment of an antibody comprises at least one constant The domain may comprise at least one variable domain covalently linked to the domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by all or part of a hinge or linker region. A hinge region provides flexibility between adjacent variable and / or constant domains in a single polypeptide molecule. At least two (e.g., 5, 10, 15, 20, 40) The antigen of the antibody of the present invention may be composed of 1 to 60 amino acids. The binding fragments may be linked to each other and / or to one or more monomeric V H Or V L The domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, with the domains non-covalently linked (e.g., by disulfide bonds).

[0061] As with full-length antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Antigen-binding fragments of multispecific antibodies will typically contain at least two different variable domains, where Each variable domain can specifically bind to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be suitable for use in the context of an antigen-binding fragment of an antibody of the present invention, using routine techniques available in the art.

[0062] The antibodies of the present invention may function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the present invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to the lysis of antigen-expressing cells by non-specific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, by recognizing bound antibodies on target cells. CDC and ADCC are well known in the art and refer to cell-mediated responses that result in the lysis of targeted cells. The antibody activity can be measured using available assays [see, e.g., U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes et al., PNAS USA 95:652-656 (1998)]. The constant region is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype may be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0063] In certain embodiments of the invention, the anti-activin A antibodies of the invention are human antibodies. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The disclosed human antibodies may, for example, contain human germline immunoglobulin sequences in the CDRs, particularly CDR3. Amino acid residues not encoded by the sequence (e.g., random or site-directed in vitro Mutations introduced by mutagenesis or by somatic mutation in vivo However, the term "human antibody", as used herein, refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences. The term "antibody" is not intended to encompass modified antibodies.

[0064] The antibodies of the invention may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, created, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes [see, e.g., Taylor et al., Nucl Acids Res 20:6287-6295 (1992)], or antibodies isolated from a human antibody library (described further below) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al., Nucl Acids Res 20:6287-6295 (1992)). Any other splicing of human immunoglobulin gene sequences into other DNA sequences is The term "recombinant human antibodies" is intended to encompass antibodies that have been prepared, expressed, engineered, or isolated by any means. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be produced by in vitro mutagenesis (or, for human Ig sequences, by the use of recombinant human antibodies). When transgenic animals are used, they are subjected to in vivo somatic mutagenesis ( , V of recombinant antibody H and V L The amino acid sequence of the region is human germline V H and V L These are sequences which, while derived from and related to the sequences, may not naturally exist in the germline repertoire of human antibodies in vivo.

[0065] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form In this state, the dimers are not linked via interchain disulfide bonds, and a molecule of about 75 to 80 kDa composed of covalently coupled light and heavy chains is formed (half antibody). These forms have been extremely difficult to separate, even after affinity purification.

[0066] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, isotype-related structural differences in the hinge region of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge causes the occurrence of the second form to be more pronounced than that observed with the human IgG1 hinge. (Angal et al., Molecular Immunology 30:105 1993). The present invention provides a method for improving the production of desired antibody forms by modifying the hinge region of the antibody. , C H 2 or C H The present invention has one or more mutations in the three regions that may be desirable, for example, in production. The present invention also includes antibodies that

[0067] The antibody of the present invention may be an isolated antibody. "Isolated antibody," as used herein, means an antibody that has been identified, separated and / or recovered from at least one component of its natural environment, e.g., from at least one component of an organism or organism in which the antibody is naturally occurring. An antibody that has been isolated or removed from the tissue or cell in which it is naturally produced is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in its natural location within a recombinant cell. An isolated antibody has not been subjected to at least one purification or isolation step. According to one embodiment, the isolated antibody is an antibody isolated from other cellular material and / or It may be substantially free of chemicals.

[0068] The present invention encompasses neutralizing and / or blocking anti-activin A antibodies. A "neutralizing" or "blocking" antibody, as used herein, is an antibody whose binding to activin A is: (i) an antibody that inhibits activin A; Activin A and activin A receptors (e.g., activin type IIA receptor, activin type IIB receptor) (ii) disrupt the formation of an activin-activin receptor complex; and / or (iii) disrupt at least one biological function of activin A. The term activin A neutralizing or blocking antibody is intended to refer to an antibody that inhibits activin A. The inhibition caused by the body does not necessarily have to be complete, as long as it is detectable using a suitable assay. Exemplary assays are described in the Examples.

[0069] The anti-activin A antibodies disclosed herein are similar to those described herein in that they are identical in structure to the corresponding germline mutants from which they were derived. The heavy and light chain variable domains may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains when compared to the sequences disclosed herein. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention provides One or more amino acids within one or more framework and / or CDR regions of an antibody to the corresponding residue in the derived germline sequence, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such as The sequence variations are collectively referred to herein as "germline mutations" and are disclosed herein. Starting from the sequences of the heavy and light chain variable regions disclosed herein, one skilled in the art can easily create one or more antibodies and antigen-binding fragments thereof that are derived from any of the amino acid sequences disclosed herein. Large numbers of antibodies and antigen-binding fragments can be readily produced that contain multiple individual germline mutations or combinations thereof. H and / or V L Domestic In other embodiments, all of the framework and / or CDR residues within the FR1 and / or CDR2 residues are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found within CDR1, CDR2, or CDR3. Alternatively, one or more of the CDR residues may be a different germline sequence (i.e., the sequence from which the antibody was originally derived). The corresponding residues in the germline sequence are mutated to those in the corresponding germline sequence (which differs from the germline sequence). The disclosed antibody may comprise any of two or more germline mutations within the framework and / or CDR regions. For example, certain individual residues may be mutated to the corresponding residue in a particular germline sequence, but certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once antibodies and antigen-binding fragments containing cellular mutations are obtained, they can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are included within the scope of the present invention.

[0070] The present invention also provides HCVRs, LCVRs, or polypeptides disclosed herein that have one or more conservative substitutions. and / or any variant of the amino acid sequence of the CDR. For example, the present invention provides HCVR, LCVR, and / or CDR amino acid sequences that contain, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. The present invention encompasses an anti-activin A antibody having the sequence

[0071] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes are also known as conformational epitopes. Conformational epitopes may be either conformational or linear epitopes. Conformational epitopes are produced by spatially adjacent amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include saccharide moieties, phosphoryl groups, or sulfonyl groups on the antigen.

[0072] The term "substantial identity" or "substantially identical," when referred to a nucleic acid or fragment thereof, when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, can be determined by any of the well-known techniques, such as FASTA, BLAST, or Gap, as discussed below. At least about 95%, more preferably at least about 95%, as measured by a sequence identity algorithm. "Substantial identity" refers to nucleotide sequence identity in at least about 96%, 97%, 98% or 99% of the nucleotide bases. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or a substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0073] The term "substantial similarity" or "substantially similar," as applied to polypeptides, means that two peptide sequences, when optimally aligned, for example by the GAP or BESTFIT programs using default gap weights, have at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. "Conservative amino acid substitutions" refer to amino acid residues that have side chains (R groups) with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions are substitutions in which a amino acid residue having the same structure as the amino acid residue is substituted with another amino acid residue having the same structure as the ... In cases where the amino acid sequences of amino acids differ from each other, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, WR, Methods Mol Biol 24:307-331 (1994). Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamate; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: (5) amino acid side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamic acid. Instead, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix as described in Gonnet et al., Science 256:1443-1445 (1992). A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0074] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications such as conservative amino acid substitutions. For example, GCG software can be used to match homologous polypeptides from different species. The GCG GCG Library contains programs such as Gap and Bestfit that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides such as ribozymes, or between wild-type proteins and their mutant proteins. See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., For example, FASTA2 and FASTA3) search for the best overlap region between the query and search sequences. The alignment and percent sequence identity of the sequences are provided in [e.g., Pearson, WR, Methods Mo [See Biol 132:185-219 (2000)]. Another preferred algorithm for comparing a database with the sequences of the present invention is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al., J Mol Biol 215:403-410 (1990) and Altschul et al., Nucleic Acids Res 25:3389-402 (1997).

[0075] Biological properties of antibodies The present invention encompasses anti-activin A antibodies and antigen-binding fragments thereof that bind with high affinity to activin A. For example, the present invention encompasses anti-activin A antibodies and antigen-binding fragments thereof that bind with high affinity to activin A, for example, anti-activin A antibodies and antigen-binding fragments thereof as defined herein in Example 3. A K of less than about 30 nM as measured by surface plasmon resonance using such an assay format. D Antibodies and antigen-binding fragments of antibodies that bind to activin A at 25° C. or 37° C. In certain embodiments, the antibodies or antigen-binding fragments of the invention are used in assay formats such as those defined in Example 3 herein or substantially similar assay formats. When measured by surface plasmon resonance using a 3-aminobutyric acid (3-aminobutyric acid) assay, the 3-aminobutyric acid (3-aminobutyric acid) is less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than about 250 pM, less than about 240 pM, less than about 230 pM, less than about 220 pM, less than about 210 pM, less than about 200 pM, less than about 190 pM, less than about 180 pM, less than about 170 pM, less than about 160 pM, less than about 150 pM, less than about 140 pM, less than about 130 pM, less than about 120 pM, K less than about 110 pM, less than about 100 pM, less than about 95 pM, less than about 90 pM, less than about 85 pM, less than about 80 pM, less than about 75 pM, less than about 70 pM, less than about 65 pM, less than about 60 pM, less than about 55 pM, less than about 50 pM, less than about 45 pM, less than about 40 pM, less than about 35 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM, or less than about 3 pM D It binds to activin A.

[0076] The present invention also encompasses anti-activin A antibodies and antigen-binding fragments thereof that inhibit activin A-mediated intracellular signaling. For example, the present invention relates to antibodies that inhibit activin A, such as those described herein in Example 5, or those that inhibit activin A, such as those described herein in Example 6, or those that inhibit activin A, such as those described herein in Example 7, or those that inhibit activin A, such as those described herein in Example 8, or those that inhibit activin A, such as those described herein in Example 9, or those that inhibit activin A, such as those described herein in Example 10, or those that inhibit activin A, such as those described herein in Example 11, or those that inhibit activin A, such as those described herein in Example 12, or those that inhibit activin A, such as those described herein in Example 13, or those that inhibit activin A, such as those described herein in Example 14, or those that inhibit activin A, such as those described herein in Example 15, or those that inhibit activin A, such as those described herein in Example 16, or those that inhibit activin A, such as those described herein in Example 17, or those that inhibit activin A, such as those described herein in Example 18, or those that inhibit activin A, such as those described herein in Example 19, or those that inhibit activin A, such as those described herein in Example 20, or those that inhibit activin A, such as those described herein in Example 21, or those that inhibit activin A, such as those described herein in Example 22, or those that inhibit activin A, such as those described herein in Example 23, or those that inhibit activin A, such as those described herein in Example 24, or those that inhibit activin A, such as those described herein in Example 25, or those that inhibit activin A, such as those described herein in Example 26, or those that inhibit activin A, such as those described herein in Example 27, or those that inhibit activin A, such as those described herein in Example IC of less than approximately 4 nM as measured by cell-based blocking bioassay 50 Value, A In certain embodiments, the present invention includes an anti-activin A antibody that inhibits activation of the SMAD complex signaling pathway through binding of activin A to the activin type I or type II receptor. Such antibodies or antigen-binding fragments may, for example, be used as defined herein in Example 5. When measured in a cell-based blocking bioassay using such an assay format or a substantially similar assay, the antibody has an activity of less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than about 250 pM, less than about 240 pM, less than about 230 pM, less than about 220 pM, less than about 210 pM, less than about 200 pM, less than about 190 pM, less than about 180 pM, less than about 170 pM, less than about 160 pM, less than about 150 pM, less than about 140 pM, less than about 130 pM, less than about 120 pM, less than about 110 pM, less than about 100 pM, less than about 95 pM, less than about 90 pM, less than about 85 pM, IC of less than about 80 pM, less than about 75 pM, less than 70 pM, less than about 65 pM, less than about 60 pM, less than about 55 pM, less than about 50 pM, less than about 49 pM, less than about 48 pM, less than about 47 pM, less than about 46 pM, less than about 45 pM, less than about 44 pM, less than about 43 pM, less than about 42 pM, less than about 41 pM, less than about 40 pM, or less than about 39 pM 50 In certain embodiments, the antibodies or antigen-binding fragments of the invention inhibit activation of the SMAD complex signaling pathway via binding of activin A to the activin type I or type II receptors at a concentration of 0.01 to 0.15 mM. IC of less than about 50 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, or less than about 1 nM as measured by a cell-based blocking bioassay using the assay 50 Values ​​of activin B In certain embodiments, the antibodies or antigen-binding fragments of the invention are assayed in an assay format, such as, for example, as defined herein in Example 5, or substantially similar assay formats, to inhibit the signaling activity of activin B by interfering with binding to the activin type I or type II receptors. When measured in a cell-based blocking bioassay using a similar assay, the antibody titers less than about 500 pM, less than about 450 pM, less than about 440 pM, less than about 430 pM, less than about 420 pM, less than about 410 pM, less than about 400 pM, less than about 390 pM, less than about 380 pM, less than about 370 pM, less than about 360 pM, less than about 350 pM, less than about 340 pM, less than about 320 pM, less than about 310 pM, less than about 300 pM, less than about 290 pM, less than about 280 pM, less than about 270 pM, IC of less than about 260 pM, less than about 250 pM, less than about 240 pM, less than about 230 pM, less than about 220 pM, less than about 210 pM, less than about 200 pM, less than about 190 pM, less than about 180 pM, less than about 170 pM, less than about 160 pM, less than about 150 pM, or less than about 140 pM 50 In certain embodiments, the antibody or antigen-binding fragment of the invention inhibits activation of the SMAD complex signaling pathway via binding of activin AB to the activin type I or type II receptor at a value of 0.1 to 0.25 μg / mL, e.g., as defined in Example 5 herein. IC50 of less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 750 pM, less than about 700 pM, less than about 650 pM, less than about 600 pM, or less than about 580 pM when measured in a cell-based blocking bioassay using an assay format as described above or a substantially similar assay. 50 Activin AC activates the SMAD complex signaling pathway through binding to activin type I or type II receptors at high levels. It inhibits sexualization.

[0077] The antibodies of the present invention have one or more of the above-mentioned biological properties, or any combination thereof. Other biological properties of the antibodies of the present invention will be apparent to those of skill in the art upon consideration of the present disclosure, including the Examples provided herein.

[0078] Anti-activin A antibodies, including Fc variants According to certain embodiments of the present invention, there are provided anti-activin A antibodies comprising an Fc domain that comprises one or more mutations that enhance or reduce antibody binding to the FcRn receptor at acidic pH, e.g., as compared to neutral pH. For example, the present invention provides an anti-activin A antibody comprising an Fc domain that comprises one or more mutations that enhance or reduce antibody binding to the FcRn receptor at acidic pH, as compared to neutral pH. H 2 or C H Included are anti-activin A antibodies that contain mutations in the three regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes with a pH ranging from about 5.5 to about 6.0). Such mutations may result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); positions 250 and 428 (e.g., L or F); positions 252 (e.g., L / Y / F / W or T), 254 (e.g., , S or T) and at 256 (e.g., S / R / Q / E / D or T); or at 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or at 250 and / or 428; or at 307 or 308 (e.g., 308F, V308F) and at 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and and 434 (e.g., 434Y); 252, 254, and 256 (e.g., 252Y, 254T, and 256E); 250Q and 428L (e.g., T250Q and M428L); and 307 and / or 308 (e.g., 308F or 308P). In yet another embodiment, the modification includes a 265A (e.g., D265A) and / or a 297A (e.g., N297A) modification.

[0079] For example, the present invention provides 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., For example, one or more pairs or groups of mutations selected from the group consisting of P257I and Q311I; 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). The present invention also encompasses anti-activin A antibodies comprising an Fc domain comprising the above-mentioned Fc domain mutations, and All possible combinations of other mutations within the variable domains of the antibodies disclosed herein are considered within the scope of the invention.

[0080] The present invention also relates to an anti-activin A antibody comprising a chimeric heavy chain constant (CH) region, The region is a segment derived from the CH region of more than one immunoglobulin isotype. For example, the antibody of the present invention includes human IgG1, human IgG2, or a part or the whole of the CH2 domain derived from a human IgG4 molecule, In some embodiments, the antibodies of the invention may comprise a chimeric hinge region comprising a CH3 domain derived from a human IgG4 molecule in combination with a portion or all of a CH3 domain derived from a human IgG4 molecule. For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. Antibodies comprising a chimeric CH region as described herein may, in certain embodiments, exhibit altered Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, US Pat. No. 6,333,351, issued Feb. 1, 2013). (See U.S. Provisional Application No. 61 / 759,578, filed on May 18, 2003).

[0081] Epitope mapping and related techniques The present invention encompasses anti-activin A antibodies that interact with one or more amino acids found within activin A (e.g., within the activin type II receptor binding site). The pitopes may be any of three or more (e.g., , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more Alternatively, the epitope may consist of a single continuous sequence of (many) amino acids. The fragments are made up of multiple non-contiguous amino acids (or amino acid sequences) located within the activin A dimer. It may be so.

[0082] Does the antibody "interact with one or more amino acids" within a polypeptide or protein? Various techniques known to those of skill in the art can be used to determine whether a particular antibody is a cytosine-binding protein. Exemplary techniques include, for example, conventional cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine stearate assays, and the like. These include canning mutation analysis, peptide blot analysis [Reineke, Methods Mol Biol 248:443-463 (2004)], and peptide cleavage analysis. Methods such as peptide extraction and chemical modification of the antigen may be employed [Tomer, Protein Science 9:487-496 (2000)]. Another method that can be used to identify the amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves labeling the protein of interest with deuterium, followed by binding of an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water and the antibody is then reacted with the protein / antibody complex to obtain a deuterium-labeled protein. Hydrogen-deuterium exchange was performed on all residues except for the residues protected by After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry to reveal deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, e.g., Ehring, Analytical Biochemistry 267(2):252-259 (1999); Engen and Smith, Anal. Chem. 73:256A-265A (2001).

[0083] The present invention relates to antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P2, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, H2aM10965N, etc.). The present invention further includes anti-activin A antibodies comprising the components described herein. and anti-activin A antibodies that compete for binding to activin A with any of the specific exemplary antibodies (e.g., H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P2, H4H10447P2, H4H10448P2, H4H10452P2, H4H10468P2, H2aM10965N, etc.). For example, the present invention encompasses one of "bin 1" as defined in Example 4 herein. one or more antibodies (e.g., H4H10423P, H4H10446P2, H4H10468P2 and H4H10442P2) and The present invention also encompasses anti-activin A antibodies that cross-compete for binding to activin A. The present invention also encompasses one or more antibodies of "bin 2" as defined in Example 4 herein (e.g., H4H1 Also encompassed are anti-activin A antibodies that cross-compete for binding to activin A with the following antibodies: H4H10432P2, H4H10436P2, and H4H10440P2.

[0084] The antibody binds to the same epitope as a reference anti-activin A antibody or Whether or not a compound competes for binding with a vasopressin A antibody is known in the art and is exemplified herein. For example, to determine whether a test antibody binds to the same epitope as a reference anti-activin A antibody of the invention, The reference antibody is allowed to bind to activin A (or a heterodimer containing the βA subunit). The ability of the test antibody to bind to activin A is then evaluated. If the test antibody is able to bind to activin A after saturation binding with the reference anti-activin A antibody, then the test antibody is considered to be capable of binding to activin A after saturation binding with the reference anti-activin A antibody. It can be concluded that the antibody binds to a different epitope than the antibody against activin A. If the test antibody is unable to bind to activin A after saturation binding with the reference anti-activin A antibody, then the test antibody binds to the same epitope as that bound by the reference anti-activin A antibody of the invention. Further routine experiments (e.g., peptide mutations and and binding analysis) to confirm that the observed lack of binding of the test antibody actually binds to the same endonucleases as the reference antibody. It can be ascertained whether the lack of binding is due to binding to a pitope or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, BIAcore, flow cytometry or any other quantitative or quantitative method available in the art. This can be done using qualitative antibody binding assays. According to certain embodiments of the invention, the antibody is bound to a 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess, for example, as measured in a competitive binding assay. Two antibodies bind to the same (or overlapping) epitope if one of the antibodies inhibits binding of the other by at least 50%, but preferably by 75%, 90%, or even 99%. [See, e.g., Junghans et al., Cancer Res. 50:1495-1502 (1990)]. Two antibodies are considered to bind the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have "overlapping epitopes" if only the subset of amino acid mutations that reduce or eliminate binding of one antibody reduces or eliminates binding of the other. It is considered that.

[0085] To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-activin A antibody, the binding method described above is performed in two directions. In one direction, the reference antibody was incubated with activin A protein (or βA subunit) under saturating conditions. In the second orientation, the test antibody is bound to activin A under saturating conditions, followed by evaluating the binding of the reference antibody to activin A. In both orientations, if only the first (saturating) antibody can bind to activin A, it is concluded that the test antibody and the reference antibody compete for binding to activin A (see, for example, the assay format described in Example 4 herein, in which the test activin A antibody is captured on a sensor chip and then bathed in a solution containing a reference activin A antibody previously bound to activin A). As would be understood by one skilled in the art, an antibody that competes with the reference antibody for binding may not necessarily be able to bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0086] The anti-activin A antibodies of the invention bind to activin A receptors within or near the binding site for the activin type II receptor. Anti-activin A antibodies of the invention can bind to an epitope on activin A nearby the binding site for activin type I receptor to directly block the interaction of activin A with the activin type II receptor, or indirectly block the interaction of activin A with the activin type I receptor. The present invention can directly block the interaction of activin A with the activin type I receptor. In an embodiment, an anti-activin A antibody of the invention that binds to activin A at or near the activin type I receptor binding site is capable of inhibiting the interaction of activin A with the activin A type II receptor. Do not block use.

[0087] Preparation of human antibodies Methods for generating monoclonal antibodies, such as fully human monoclonal antibodies, are known in the art. To generate a human antibody that specifically binds to human activin A, Any such known method can be used in the context of the present invention.

[0088] VELOCIMMUNE™ technology, e.g., or for generating fully human monoclonal antibodies Using any other known method, a high affinity chimeric antibody against human activin A having a human variable region and a mouse constant region is first isolated. As shown in the experimental section below. Subsequently, antibodies are characterized and selected for desired properties such as affinity, selectivity, epitope, etc. Optionally, to generate a fully human anti-activin A antibody, the murine constant regions can be modified to The desired human constant region is replaced, such as wild-type or modified IgG1 or IgG4. The constant region selected may vary according to the specific use, but the high affinity antigen binding and target specificity properties reside in the variable region. In certain cases, fully human anti-activin A antibodies are isolated directly from antigen-positive B cells.

[0089] biological equivalent The anti-activin A antibodies and antibody fragments of the present invention are similar to the amino acid sequences of the described antibodies. Proteins with amino acid sequences distinct from those of human activin A but that retain the ability to bind to human activin A Such variant antibodies and antibody fragments contain one or more amino acid additions, deletions, or substitutions when compared to the parent sequence, but retain the biological activity of the described antibodies. Similarly, the anti-activin A antibody of the present invention exhibits essentially equivalent biological activity. The DNA sequences disclosed herein may contain one or more nucleotide additions, deletions, or substitutions when compared to the disclosed sequences, but are essentially biologically identical to the anti-activin A antibodies or antibody fragments of the invention. The present invention also encompasses sequences encoding substantially equivalent anti-activin A antibodies or antibody fragments. Examples of such variant amino acid and DNA sequences are discussed above.

[0090] Two antigen-binding proteins or antibodies are considered bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in the rate and extent of absorption, for example, when administered in the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies would be expected to be considered equivalents or pharmaceutical substitutes even if they are equivalent in their extent of absorption but not in their rate of absorption, and may also be considered bioequivalent because such differences in absorption rates are intentional, reflected in the label, are not essential to achieving effective body drug concentrations, for example, during chronic use, and are not considered medically significant for the particular drug product being studied.

[0091] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0092] In one embodiment, a patient can switch between a reference product and a biological product one or more times without experiencing a clinically significant change in immunogenicity or an increased risk of predicted adverse effects, such as a decrease in efficacy, when compared to continued therapy without switching. Two antigen-binding proteins are biologically equivalent if they are both identical in structure and function.

[0093] In one embodiment, the two antigen-binding proteins both have one or more uses. Such mechanisms are driven by one or more common mechanisms of action in relation to the conditions of use. If they act to a known extent, they are bioequivalent.

[0094] Bioequivalence can be demonstrated by in vivo and in vitro methods. Measures of bioequivalence include, for example, (a) the presence or absence of an antibody in blood, plasma, serum, or other biological fluids. (b) in vitro studies that have been shown to correlate with and are reasonably predictive of in vivo human bioavailability data; (c) in vivo studies in humans or other mammals where the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) appropriate controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0095] Biologically equivalent variants of the anti-activin A antibodies of the invention include, for example, variants of the residue or sequence Biologically equivalent antibodies can be constructed by making various substitutions in the activin A antibody or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other situations, biologically equivalent antibodies include anti-activin A antibody variants that contain amino acid changes that alter the glycosylation characteristics of the antibody, e.g., mutations that remove or transfer glycosylation. may include.

[0096] Species selectivity and species cross-reactivity The present invention, according to one embodiment, provides a method for the production of activin A antibodies that bind to human activin A but not to activin A from other species. The present invention also provides an anti-activin A antibody that does not bind to activin A. The present invention also provides an anti-activin A antibody that does not bind to activin A. Also included are human activin A from any species and anti-activin A antibodies that bind to activin A. For example, an anti-activin A antibody of the invention can bind to human activin A, and optionally may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus monkey, or chimpanzee activin A. According to certain exemplary embodiments of the invention, a heterodimer containing human activin A (e.g., activin A or βA subunit) and cynomolgus activin A is Anti-activin A antibodies are provided that specifically bind to activin A of monkeys (eg, Macaca fascicularis).

[0097] Immunoconjugates The present invention encompasses anti-activin A monoclonal antibodies conjugated to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope (an "immunoconjugate"). Cytotoxic agents include any agent that is detrimental to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immunoconjugates are known in the art (e.g., See, for example, WO05 / 103081.

[0098] multispecific antibodies The antibodies of the invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., J Immunol 147:60-69 (1991); Kufer et al., Trends Biotechnol 22:238-244 (2004). The anti-activin A antibodies of the invention may be linked to or expressed in conjunction with another functional molecule, such as another peptide or protein. For example, the antibody or a fragment thereof may be linked to one or more other molecular entities, such as another antibody or is operably linked (e.g., by chemical coupling, genetic fusion) to the antibody fragment. Bispecific or multispecific antibodies may be produced that have a second binding specificity (by covalent association, non-covalent association or otherwise). For example, the present invention provides The other arm of the immunoglobulin is specific for human activin A or a fragment thereof. is specific for a second therapeutic target or is conjugated to a therapeutic moiety. One embodiment of the present invention includes an immunoglobulin in which one arm is specific for human activin A or a fragment thereof and the other arm of the immunoglobulin is specific for GDF8. The present invention includes bispecific antibodies, which are heterologous.

[0099] An exemplary bispecific antibody format that can be used in the context of the present invention is H 3 domain and second IgCH The use of three domains, where the first and second IgC H The three domains are They differ from each other in at least one amino acid, compared to bispecific antibodies that have no amino acid difference. When compared, at least one amino acid difference reduces binding of the bispecific antibody to Protein A (see, e.g., U.S. Patent No. 8,586,713). In one embodiment, the first IgC H 3Dome The IgC binds to protein A and is then released into the blood. H The third domain contains a mutation that reduces or eliminates Protein A binding, such as the H95R modification (according to the IMGT exon numbering; H435R in EU numbering). H 3 may further comprise a Y96F modification (according to IMGT; Y436F in EU). H Additional modifications that may be found within 3 include, in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I in EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I in EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, V82I, and L105P (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, V422I in EU). , and L445P). Variations on the bispecific antibody formats described above are considered within the scope of the present invention.

[0100] Other exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or bispecific antibody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 (For a review of the aforementioned formats, see, e.g., Klein et al., mAbs 4:6, 1-11 (2012), and references cited therein). can also be constructed using, for example, peptide / nucleic acid conjugation, in which case In this case, unnatural amino acids with orthogonal chemical reactivity can be used to site-specifically synthesize antibody-oligonucleotides. Nucleotide conjugates are generated and then self-assembled into multimeric complexes with defined composition, valency and geometry. [See, e.g., Kazane et al., J. See Am Chem Soc. 135(1):340-346 (2013).

[0101] Therapeutic Formulations and Administration The present invention relates to a pharmaceutical composition comprising the anti-activin A antibody or an antigen-binding fragment thereof of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improved migration, delivery, tolerance, and the like. A large number of suitable formulations can be found in the formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, paper tablets, and the like. Excipients for parenteral formulations include ointments, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA, J Pharm Sci Technol 52:238-311 (1998).

[0102] The dose of antibody administered to a patient may vary depending on the age and size of the patient, the target disease, condition, route of administration, and the like. Preferred doses are typically based on body weight or total body mass. Conditions or diseases associated with activin A activity in adult patients When the antibodies of the present invention are used to treat diseases, the usual dose is about 0.01 to about 20 mg / kg body weight, It may be advantageous to administer the antibody of the present invention intravenously, more preferably at a single dose of about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. It is effective for administration of anti-activin A antibodies Appropriate dosages and schedules can be determined empirically. For example, a patient's progress can be monitored by periodic evaluation and dosages adjusted accordingly. Additionally, interspecies scaling of dosages can be performed using methods well known in the art [e.g. See, for example, Mordenti et al., Pharmaceut Res 8:1351 (1991)].

[0103] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibodies or other therapeutic proteins of the invention, receptor-mediated endocytosis, etc. See, e.g., Wu et al., J Biol Chem 262:4429-4432 (1987)]. The antibodies and other therapeutically active components of the invention can also be delivered by gene therapy techniques. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be delivered to epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa) by any convenient route, e.g., by infusion or bolus injection. It may be administered by absorption through a membrane or with other biologically active substances. Administration may be systemic or local.

[0104] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily adapted to deliver the pharmaceutical composition of the present invention. Such a pen delivery device The pen delivery device may be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have a replaceable cartridge. Instead, disposable pen delivery devices come pre-filled with the pharmaceutical composition that is held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is depleted, the entire device is discarded.

[0105] A number of reusable pen and autoinjector delivery devices are adapted for subcutaneous delivery of the pharmaceutical compositions of the present invention, including, but not limited to, the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25 (Disetronic Medical Systems, Bergdorf, Switzerland), to name just a few. pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices applicable to subcutaneous delivery of the pharmaceutical composition include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), to name just a few. (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park IL).

[0106] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In some embodiments, pumps may be used [see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987)]. In other embodiments, polymeric materials may be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet other embodiments, a controlled release system can be placed in proximity to the target of the composition, such that only a fraction of the systemic dose is required ( See, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138.) Other controlled release systems are discussed in the review by Langer, Science 249:1527-1533 (1990).

[0107] Injectable preparations include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared by dissolving, suspending or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous medium or oily medium customarily used for injections. Aqueous media for injections include, for example, physiological saline, isotonic solutions containing glucose and other auxiliary agents, and the like, which can be dissolved in a suitable solubilizing agent, for example, alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene glycol of hydrogenated castor oil)] Ethylene (50 mol) adduct)], etc. can be used in combination with oil-based media such as: For example, sesame oil, soybean oil, etc. are employed, which can be used in combination with a solubilizing agent, such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled into a suitable ampoule.

[0108] Advantageously, the above-mentioned pharmaceutical composition for oral or parenteral use is prepared in a dosage form with a unit dose suitable for the dose of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained is generally about 5 to about 500 mg per unit dose dosage form, particularly In the case of an injectable formulation, the antibody is preferably contained in an amount of about 5 to about 100 mg, and in the case of other dosage forms, the antibody is preferably contained in an amount of about 10 to about 250 mg.

[0109] Therapeutic Uses of Antibodies The antibodies of the present invention may be used to inhibit or inhibit activin A expression, signaling, or activity. or by blocking the interaction of activin A with activin A receptors (e.g., ActRIIA, ActRIIB, activin type I receptors, etc.) or by otherwise inhibiting activin A. It is particularly useful for the treatment, prevention and / or amelioration of any disease or disorder treatable by inhibiting the activity and / or signaling of thybin A. by specifically binding to activin A but not other ActRIIB ligands, or by specifically binding to activin A and GDF8 but not other ActRIIB ligands, the antibody may enhance muscle strength / force and / or muscle mass and / or muscle function in an individual. The present invention provides a method for treating a condition or affliction that can be cured, alleviated or ameliorated by increasing metabolism or favorably altering metabolism (processing of carbohydrates, lipids and proteins). For example, the present invention provides a method for treating a condition or affliction that can be cured, alleviated or ameliorated by administering to a subject an activin A specific binding protein. thereby increasing muscle strength / power and / or muscle mass and / or muscle function in a subject. The present invention also encompasses methods for administering activin A specific binding protein to a subject, or for treating a disease or disorder characterized by a loss of muscle mass or strength in a subject. and a GDF8-specific binding protein to the subject, thereby improving muscle strength / muscle function in the subject. Also encompassed are methods for increasing strength and / or muscle mass and / or muscle function, or for treating a disease or disorder characterized by a decrease in muscle mass or strength in a subject. Any of the activin A-specific binding proteins and / or GDF8-specific binding proteins disclosed or referenced herein can be used in the context of these aspects of the invention. For example, the therapeutic methods of the invention include administering an anti-activin A antibody and / or an anti-GDF8 antibody to a subject. It encompasses the process.

[0110] Thus, in the context of the methods of treatment described herein, the anti-activin A antibody: It may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (e.g., anti-GDF8 antibodies), further examples of which are described elsewhere herein. is explained in.

[0111] Administering an activin A specific binding protein and a GDF8 specific binding protein to a subject. In a method comprising the steps of: The substances may be administered to a subject at the same time or substantially the same time, e.g., in a single therapeutic dose, or in two separate doses administered simultaneously or within less than about 5 minutes of each other. Alternatively, the activin A-specific binding protein and the GDF8-specific binding protein may be administered sequentially. For example, separate therapeutic doses may be administered to a subject more than about 5 minutes apart from one another. good.

[0112] The present invention also provides an antibody comprising an activin A specific binding domain and a GDF8 specific binding domain. For increasing muscle strength / power and / or muscle mass and / or muscle function in a subject, or for increasing muscle mass or strength in a subject, by administering to the subject an original binding molecule. The present invention also encompasses a method for treating a disease or disorder characterized by a decrease in the level of activin A (HCVR / LCVR) in a patient. Any of the antigen binding molecules disclosed or referred to herein can be used in the context of this aspect of the invention. For example, the therapeutic method of the present invention includes a method for treating a disease or disorder characterized by a decrease in the level of activin A (HCVR / LCVR) in a patient. The method includes administering to a subject a bispecific antibody comprising the antibody.

[0113] The compositions of the invention may be administered to a subject with one or more additional therapeutic agents, such as, for example, growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and cytotoxic / cytostatic agents. The additional therapeutic agent may be an activin A-specific and GDF8-specific binding agent of the invention. It may be administered prior to, simultaneously with, or after administration of the protein.

[0114] Exemplary diseases, disorders and conditions that can be treated with the compositions of the present invention include, but are not limited to, sarcopenia, cachexia (idiopathic cachexia, or other conditions, such as cancer, chronic renal failure, pulmonary circulation problems, or cachexia secondary to chronic obstructive pulmonary disease), muscle injury, muscle trauma , muscle wasting and atrophy, e.g., due to disuse (e.g., of muscles), immobilization, bed rest, injury, medical treatment or surgical intervention (e.g., hip fracture, hip replacement, knee replacement, and other joint, Muscle atrophy or wasting caused by or associated with tendon or ligament injuries, such as rupture of the anterior cruciate ligament (ACL) and / or medial collateral ligament (MCL), muscular dystrophies (e.g. For example, myotonic, Duchenne, Becker, limb-girdle, facioscapulohumeral (FSHD), and Landau et al. Also known as Eugie-Dejerine's disease, congenital, oculopharyngeal, peripheral, Emery-Drift myopathy), glucocorticoid-induced myopathy, stroke rehabilitation (e.g., rehabilitation for hemiparesis due to stroke) or the need for mechanical ventilation. The compositions of the present invention are also useful for treating cancer, obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, and metabolic syndrome (including, but not limited to, diabetes, obesity, nutritional disorders, organ atrophy, chronic kidney disease, and osteoporosis). It may also be used to treat, prevent, or alleviate diseases such as chronic obstructive pulmonary disease, and anorexia. Additional conditions that can be prevented, treated and / or ameliorated using the compositions of the present invention include: The diseases, disorders, and conditions include sepsis, chronic heart failure, benign and malignant pheochromocytoma, uterine fibroids / leiomyomas, pre-eclampsia, keloids and hypertrophic scars, and pulmonary arterial hypertension. can be done.

[0115] Improved binding and activity specificity The present invention provides a method for increasing muscle strength / power and / or muscle mass and / or muscle function in a subject, or for increasing muscle mass or function in a subject, without causing the adverse effects associated with administration of a molecule that binds multiple (e.g., three or more) ActRIIB ligands. For treating diseases or disorders characterized by decreased intensity or by activin A activity The present invention includes methods for treating diseases or disorders caused, promoted, or exacerbated by activin A. In other words, methods using anti-activin A antibodies or antigen-binding proteins thereof (e.g., where the anti-activin A antibodies significantly bind only to activin A) can treat diseases or disorders without causing unwanted or adverse effects seen with molecules that bind to multiple ActRIIB ligands. For example, a clinical molecule called ACE-031 (Acceleron Pharma, Inc., Cambridge, MA) is a multimer consisting of the extracellular portion of ActRIIB fused to the Fc domain of IgG (this molecule is also referred to as "ActRIIB-Fc" herein). ActRIIB-Fc binds to other ActRIIB ligands, such as activin B, GDF8, GDF11, BMP9, BMP10, and TGFβ, in addition to activin A, and is known to cause various adverse effects when administered to human patients. Importantly, the present inventors have unexpectedly demonstrated that activin A and GDF8 can be specifically inhibited (e.g., by administering anti-activin A and anti-GDF8 antibodies). They have discovered that inhibiting ActRIIB-Fc (by inhibiting other ActRIIB ligands, such as activin B, GDF11, BMP9, BMP10, and TGFβ) results in increases in muscle mass at least equal to those observed with administration of ActRIIB-Fc, without causing the adverse effects associated with binding agents such as ActRIIB-Fc.

[0116] Combination Therapies and Formulations The present invention encompasses compositions and therapeutic formulations comprising any of the anti-activin A antibodies described herein in combination with one or more additional therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject in need thereof. The present invention also encompasses compositions and therapeutic formulations comprising any of the anti-activin A antibodies described herein in combination with one or more additional therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject in need thereof. For example, the anti-activin A antibodies of the present invention The antibodies may also be administered in combination with and / or formulated with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, metal chelators, IFN-gamma, and / or NSAIDs. The anti-activin A antibodies of the present invention may also be administered as part of a treatment regimen that also includes radiation treatment and / or conventional chemotherapy (e.g., in the context of a method for treating cancer or inhibiting tumor growth). Any of the additional therapeutically active components of the present invention may be used to treat any disease or disorder in which administration of an anti-activin A antibody would be beneficial, such as sarcopenia, cachexia, muscle damage, muscle wasting and muscle atrophy. or in combination with any of the anti-activin A antibodies of the invention to treat Any of the above additional therapeutically active ingredients may also be used as GDF8 inhibitors (e.g., anti-GDF8 The antibody) may be administered in combination with any of the anti-activin A antibodies of the invention.

[0117] The additional therapeutically active ingredient is administered to the subject prior to administration of the anti-activin A antibody of the invention. For example, the first component may be administered 1 week, 72 hours, 60 hours, or before the administration of the second component. A first component can be considered to be administered "before" a second component if it is administered within 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of an anti-activin A antibody of the invention. In other embodiments, an additional therapeutically active component is administered to a subject following administration of an anti-activin A antibody of the invention. For example, if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours after administration of the second component, the first component may be administered It can be considered to be administered "after" the second component. The therapeutically active ingredient is administered to a subject simultaneously with administration of the anti-activin A antibody of the present invention. "Concurrent" administration, for purposes of the present invention, may refer to, for example, administration in a single dosage form or in separate dosage forms. and an anti-activin A antibody, This includes administering to a subject an additional therapeutically active component. When administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-activin A antibody and the additional therapeutically active component may be administered intravenously, subcutaneously, intravitreally, etc.). Alternatively, Each dosage form may be administered via a different route (e.g., administering the anti-activin A antibody locally (e.g., For example, intravitreally) and the additional therapeutically active component may be administered systemically. In either case, for purposes of the present disclosure, administration of the components in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes, will all be considered "co-administration." For purposes of the present disclosure, "before," "at the same time as," or "after" the administration of the additional therapeutically active component (as these terms are defined herein above). Administering an anti-activin A antibody (as described above) is considered to be administering the anti-activin A antibody "in combination" with an additional therapeutically active component.

[0118] The present invention relates to an anti-activin A antibody of the invention, comprising an additional It encompasses pharmaceutical compositions formulated with one or more therapeutically active ingredients.

[0119] dosage In general, the active ingredient (e.g., an anti-activin A antibody, an anti-activin A antibody in combination with A therapeutically effective amount is an amount of an antigen-specific binding protein and / or a bispecific antibody that specifically binds activin A and GDF8 that can be administered to a subject, such as an anti-GDF8 antibody given in a therapeutically effective manner, that results in a detectable increase in one or more of the following parameters: body weight, muscle mass (e.g., tibialis anterior [TA] muscle mass, gastrocnemius [GA] muscle mass, quadriceps [Quad] muscle mass, etc.), muscle strength / power, and / or muscle function. It refers to the dose of an antigen-binding molecule. For example, a "therapeutically effective amount" of an activin A-specific binding protein and / or a GDF8-specific binding protein can be, for example, as exemplified in Example 7 herein. As used herein, for example, the term includes an amount of activin A-specific binding protein and / or GDF8-specific binding protein that, when administered to a test subject, results in an increase in TA or GA muscle mass of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or more compared to a control-treated subject.

[0120] The antibodies of the invention (e.g., anti-activin A antibodies, In the case of an anti-GDF8 antibody that specifically binds to activin A and GDF8, or a bispecific antibody that specifically binds to activin A and GDF8, the therapeutically effective amount is from about 0.05 mg to about 600 mg; for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 480mg, about 490m g, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 6 00mg, about 610mg, about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about 680mg, about 690mg, about 700mg, Approximately 710mg, approximately 720mg, approximately 730mg, approximately 740mg, approximately 750mg, approximately 760mg, approximately 770mg, approximately 780mg, approximately 790mg , about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg, about 950 mg, about 960 mg, about 970 mg, about 980 mg, about 990 mg, or about 1000 mg of each antibody.

[0121] The antibody of the invention contained in each dose (e.g., an anti-activin A antibody, an anti-activin A Anti-GDF8 antibodies given in combination with antibodies that specifically bind activin A and GDF8 The amount of bispecific antibody administered is expressed in milligrams of antibody per kilogram of patient weight (i.e. For example, the anti-activin A, anti-GDF8 and / or anti-activin B of the present invention can be expressed in mg / kg. The thybin A / anti-GDF8 bispecific antibody may be administered in an amount ranging from about 0.0001 to about 50 mg (e.g., 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg , 10.0 mg / kg, 10.5 mg / kg, 11.0 mg / kg, 11.5 mg / kg, 12.0 mg / kg, 12.5 mg / kg, 13.0 mg / kg, 13.5 mg / kg, 14.0 mg / kg, 14.5 mg / kg, 15.0 mg / kg, 15.5 mg / kg, 16.0 mg / kg, 16.5 mg / kg, 17.0 mg / kg, 17.5 mg / kg, 18.0 mg / kg, 18.5 mg / kg, 19.0 mg / kg, 19.5 mg / kg, 20.0 mg / kg, etc.

[0122] The compositions of the present invention contain equal amounts of activin A specific binding protein and GDF8 specific binding protein. Alternatively, the composition may contain an activin A specific binding protein. The amount of the substance may be less than or greater than the amount of GDF8-specific binding protein. It is expected that one of ordinary skill in the art will be able, using routine experimentation, to determine the appropriate amounts of the individual components in the compositions of the invention required to produce the desired therapeutic effect.

[0123] Dosing regimen According to certain embodiments of the invention, multiple doses of an active ingredient (e.g., an anti-activin A antibody, an anti-GDF8 antibody administered in combination with an anti-activin A antibody, a pharmaceutical composition comprising a combination of an anti-activin A antibody and any of the agents having additional therapeutic activity described herein, such as an anti-GDF8 antibody, or a bispecific antibody that specifically binds to activin A and GDF8) may be administered to a subject over a defined time course. The method according to this aspect of the invention comprises sequentially administering multiple doses of an active ingredient of the invention to a subject. "Sequential administration," as used herein, means administering each dose of the active ingredient to a subject at different times, e.g., on different days, separated by a predetermined interval (e.g., hours, days, weeks or months). The invention encompasses methods comprising sequentially administering to a patient a single initial dose of the active ingredient, followed by one or more second doses of the active ingredient, optionally followed by one or more third doses of the active ingredient.

[0124] The terms "first dose," "second dose," and "third dose" refer to the time sequence of administration of an active ingredient, e.g., an anti-activin A antibody of the invention, or a combination therapy of the invention, e.g., an anti-activin A antibody and an anti-GDF8 antibody. Thus, a "first dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), and a "second dose" is a dose administered after the first dose. The first, second and third doses all contain the same amount of active ingredient, e.g., an anti-activin A antibody. However, in certain embodiments, the active ingredient contained in the first, second and / or third dose, e.g. For example, the amounts of anti-activin A antibodies are varied (e.g., adjusted up or down as needed) over the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose" at the beginning of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered less frequently.

[0125] In certain exemplary embodiments of the invention, the second and / or third doses each comprise: 1 to 26 weeks after the previous dose (e.g., 1, 1 and 1 / 2, 2, 2 and 1 / 2, 3, 3 and 1 / 2, 4, 4 and 1 / 2, 5, 5 and 1 / 2, 6, 6 and 1 / 2, 7, 7 and 1 / 2, 8, 8 and 1 / 2, 9, 9 and 1 / 2, 10, 10 and 1 / 2, 11, 11 and 1 / 2) , 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more weeks later). The phrase "immediately preceding dose" as used herein means a dose of an active ingredient, e.g., an anti-activin A antibody, that is administered to a patient in a multiple dose sequence prior to administration of the immediately succeeding dose in the sequence, with no doses in between.

[0126] The method according to this aspect of the invention includes administering to a subject an active ingredient of the invention, such as an anti-activin A antibody. The method may include administering any number of second and / or third doses to the patient. For example, In certain embodiments, only one second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in certain embodiments, only one third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) doses are administered. A third (larger) dose is administered to the patient.

[0127] In embodiments including multiple second doses, each second dose is administered at the same frequency as the other second doses. For example, each second dose may be administered 1 to 2 weeks or 3 days after the immediately preceding dose. The second dose may be administered to the patient one to two months later. Similarly, in embodiments including multiple third doses, In some embodiments, each third dose may be administered at the same frequency as the other third doses. Each dose may be administered to the patient 2 to 12 weeks after the immediately preceding dose. In certain embodiments, the frequency at which the second and / or third doses are administered to the patient is determined based on the treatment level. The dosage can be varied over the course of the drug. The frequency of administration can also be adjusted by the physician during the course of treatment, depending on the needs of the individual patient after clinical testing.

[0128] The invention encompasses dosing regimens in which two to six loading doses are administered to a patient at a first frequency (e.g., once weekly, once every two weeks, once every three weeks, once monthly, once every two months, etc.), followed by two or more maintenance doses administered to the patient less frequently. For example, according to this aspect of the invention, if a loading dose is administered monthly, a maintenance dose may be administered to the patient once every six weeks, once every two months, once every three months, etc.

[0129] Diagnostic Uses of Antibodies The anti-activin A antibodies of the invention can also be used to detect and / or measure activin A or cells expressing activin A in a sample, e.g., for diagnostic purposes. For example, the anti-activin A antibodies or fragments thereof can be used to detect abnormal expression (e.g., overexpression) of activin A. The present invention can be used to diagnose conditions or diseases characterized by the expression of a gene that is a target of the present invention (such as expression, underexpression, lack of expression, etc.). An exemplary diagnostic assay for activin A can be, for example, by assaying a sample obtained from a patient. The method may include contacting the subject with an anti-activin A antibody of the present invention, wherein the anti-activin A antibody The antibody is labeled with a detectable label or reporter molecule. In this application, the unlabeled anti-activin A antibody is coupled to a secondary antibody that is itself detectably labeled. Detectable labels or reporter molecules may be radioisotopes, e.g. 3 H, 14 C. 32 P, 35 S, or 125 I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate, or rhodamine; or an enzyme, such as alkaline phosphatase , beta-galactosidase, horseradish peroxidase, or luciferase Specific examples of enzymes that can be used to detect or measure activin A in a sample include: Representative assays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

[0130] Samples that can be used in the activin A diagnostic assay of the present invention include those containing normal or pathological A patient who has a detectable amount of activin A protein or a fragment thereof under Examples of tissue or body fluid samples that can be obtained from a tissue source include those obtained from any tissue or body fluid sample that can be obtained from a tissue source. To establish a baseline or standard level, a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal activin A levels or activity) It is anticipated that the level of activin A in the blood will be measured. This baseline level of activin A will then be measured in blood samples obtained from individuals suspected of having an activin A-related disease or condition. The levels of activin A measured in the culture can be compared to levels of activin A measured in the culture. EXAMPLES

[0131] The following examples are provided to provide those skilled in the art with a full disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors perceive as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weights are average molecular weights, temperatures are given in degrees Celsius, and pressures are at or near atmospheric pressure.

[0132] Example 1 Generation of human antibodies against activin A VELOCIMMUNE® mice, which contain DNA encoding the heavy and kappa light chain variable regions of human immunoglobulin, were administered an immunogen containing activin A protein (inhibin βA dimer) directly with an adjuvant to stimulate an immune response. Antibody immune responses were monitored by activin A-specific immunoassays. Once the desired immune response was achieved, Spleen cells were collected and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. To identify cell lines producing activin A-specific antibodies, Hybridoma cell lines were then screened and selected. Using this technique, several anti-activin A chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained. An exemplary antibody obtained in this manner is H2aM10965N. A fully human anti-activin A antibody as described herein was generated by cloning the human variable domains from the antibody into human constant domains.

[0133] Anti-activin A antibodies have also been used to treat myeloma cells, as described in US2007 / 0280945A1. Using this method, several complete human IgG antibodies were isolated directly from antigen-positive B cells without fusion with a human IgG antibody. Anti-activin A antibodies (i.e., antibodies having human variable domains and human constant domains) Exemplary antibodies generated in this manner are designated as follows: H4H10423P, H4H10429P, H4H10430P, H4H10432P2, H4H10440P2, H4H10442P2, H4H10436P2, and H4H10446P2.

[0134] Certain biological properties of exemplary anti-activin A antibodies produced according to the methods of this Example The properties are explained in detail in the examples set forth below.

[0135] Example 2 Amino acid sequences of the heavy and light chain variable regions Table 1 shows the amino acid sequences of the heavy and light chain variable regions of selected anti-activin A antibodies. The string pairs are listed along with their corresponding antibody identifiers, and Table 2 lists the corresponding nucleic acid sequence identifiers.

[0136] [Table 1]

[0137] [Table 2]

[0138] As used herein, antibodies are typically described according to the following nomenclature: an Fc prefix (e.g., "H1M," "H2aM," "H4H") followed by a numerical identifier (e.g., "10423," "10424," or "10426," as shown in Tables 1 and 2), followed by a "P," "P2," or "N" suffix. Thus, according to this nomenclature, antibodies are referred to herein, e.g., as It can be stated as "H4H10423P", "H4H10432P2", "H2aM10965N", etc. The prefixes H1M, H2M and H4H in the antibody names used in this specification indicate the specific Fc The region isotype is shown. For example, the "H2aM" antibody has the Fc of mouse IgG2a, whereas Thus, the "H4H" antibody has a human IgG4 Fc. As will be understood by those skilled in the art, An antibody having a given Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a mouse IgG2a Fc can be converted to an antibody having a human IgG4, etc.). In each case, the variable domain (including the CDRs) has the number of amino acids shown in Table 1. Although indicated by identifiers, are expected to remain the same and the nature of the binding is expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0139] Control constructs used in the following examples In the following examples, an anti-activin A control molecule is included for comparison purposes. The control antibody, designated Control 1, is a human anti-activin A antibody having the heavy and light chain variable domain sequences of "A1" as described in US 8,309,082. Control 2 is an anti-human activin receptor type IIB antibody (anti-ActR2B mAb) disclosed as MOR8159 in US Patent Application Publication No. 2012 / 0237521 A1. Control 3 is a mouse anti-activin A monoclonal antibody from R&D Systems, Minneapolis, MN (catalog number MAB3381). Control 4 is an activin receptor type IIB-Fc A fusion molecule [soluble activin RIIB receptor extracellular domain produced using a C-terminal human IgG1 Fc fusion protein (E23-P133 of NP_001097 followed by a Gly-Ser linker followed by a C-terminal human IgG1 Fc fusion)], the sequence of which is provided as SEQ ID NO: 227.

[0140] Example 3 Antibody binding to human activin A as determined by surface plasmon resonance. Binding of antigens to selected and purified anti-human activin A monoclonal antibodies Affinity and kinetic constants were measured using a real-time surface plasmon resonance biosensor (Biacore T200 or Biacore 4000, GE Healthcare Life Sciences, Piscataway, NJ) at 25°C and 37°C. Assays were used to determine whether the Fc domains were human Fc (prefixed with H4H) or mouse Fc (prefixed with H2aM). Antibodies expressed as IgG were captured onto their respective anti-Fc sensor surfaces (mAb capture format). Goat anti-mouse IgG polyclonal antibody (GE Healthcare, no. BR-1008-38) or mouse anti-human IgG monoclonal antibody, generated via direct amine coupling to a Biacore CM5 sensor chip, were captured onto the surface of the Biacore CM5 sensor chip (mAb capture format). Anti-activin A antibodies were captured on either a monoclonal antibody (GE Healthcare, no. BR-1008-39) surface. Kinetic experiments were performed using either HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) or PBS-P (10 mM sodium phosphate, 2.7 mM KCl, 137 mM NaCl, 0.02% NaN3, 0.05% surfactant P20, pH 7.4) as both running and sample buffer. Various concentrations (50 to 100 mM) of PBS-P were applied across the captured antibody surface. Antigen-antibody association rates were measured by injecting either activin A (R&D Systems, no. 338-AC-050 / CF), activin B (R&D Systems, no. 659-AB-025 / CF), activin AB (R&D Systems, no. 1006-AB-005), activin AC (R&D Systems, no. 4879-AC / CF), or inhibin E (Novus Biologicals, no. H00083729-P01) at 4-fold dilutions ranging from 0.1 to 0.2 nM. Antibody-antigen association was monitored for 240 s and dissociation in buffer was monitored for 600 s simultaneously. Kinetic association and dissociation rate constants were determined by processing and fitting the data using Scrubber software version 2.0c. The binding equilibrium dissociation constant (K D ) and dissociation half-life (t 1 / 2 ) was calculated from the kinetic rate constants as follows: K D (M)=k d / k a and t 1 / 2 (min)=[ln2 / (60*k d Tables 3 to 10 show the kinetic binding parameters for the different anti-activin A monoclonal antibodies. (NB = conditions used No binding was observed below 0.01%; NT = not tested).

[0141] [Table 3]

[0142] [Table 4]

[0143] [Table 5]

[0144] [Table 6]

[0145] [Table 7]

[0146] [Table 8]

[0147] [Table 9]

[0148] [Table 10]

[0149] As shown in Table 3 and Table 4, the anti-activin A antibody of the present invention has a 25 K in the range of less than 3.18 pM (i.e., ≤3.18E-12) to 745 pM (i.e., 7.45E-10) at °C D Value and K ranging from less than 2.18 pM (i.e., ≤2.18E-12) to 1.77 nM (1.77E-09) at 37 °C. D As shown in Tables 5 and 6, several anti-activin A antibodies were The activin A antibodies (i.e., H4H10432P2, H4H10442P2, H4H10430P2, H4H10446P2, and H4H10468P2) demonstrated no measurable binding to activin B at 25° C. or 37° C. has a K ranging from approximately 18.5 pM (i.e., 1.85E-11) to 33.1 nM (i.e., 3.31E-08) at 25 °C. D values ​​[Table 7], and approximately 44.3 pM (i.e., 4.43E-11) to 24.2 nM (i.e., Several antibodies demonstrated measurable binding to activin AB with K values ​​ranging from approximately 99.7 pM (i.e., 9.97E-11) to 11.8 nM (i.e., 1.18E-08) at 25° C. [Table 9] and approximately 462 pM (i.e., 4.62E-10) to 22.5 nM (i.e., 2.25E-08) at 37° C. [Table 10]. D demonstrated measurable binding to activin AC at values Furthermore, the anti-activin A antibodies of the invention that were tested did not demonstrate measurable binding to inhibin E (data not shown).

[0150] Example 4 Antibody Binding to TGF-beta Family Members as Determined by Surface Plasmon Resonance Activin A mAb was assayed for binding cross-reactivity to a panel of TGF-beta family members. For binding experiments, a Biacore 4000 instrument was used. Antibodies H4H10429P, H4H10430P, H4H10436P2, H4H10442P2, H4H10446P2; control 4 [ActR2B soluble ectodomain protein produced with a C-terminal human IgG1 Fc tag (ActR2B-hFc; SEQ ID NO: 227)]; and isotype control antibodies were captured on a Biacore CM4 biosensor chip that was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, catalog no. BR-1008-39). All Biacore binding studies were performed in HBS-T running buffer (0.01 M HEPES, pH 7.4, 0.5 M NaCl, 3 mM EDTA, 0.5 mg / ml bovine serum albumin, 0.05% v / v surfactant P20). Human TGF-beta family member ligands were purchased from R&D Systems (Activin A, no. 338-AC; Activin B, no. 659-AB; Activin AB, no. 1066-AB; Activin AC, no. 4879-AC; BPM2, no. 355-BM; hBMP4, no. 314-BP; hBMP6, no. 507-BP; hBMP7, no. 354-BP; hBMP9, no. 3209-BP; hBMP10, no. 2926-BP; hGDF8, no. 788-G8; hGDF11, no. 1958-GD). All binding assays were performed at 37°C. Between 60 and 200 co-immunopreserved antibodies or soluble receptors were used. Capture levels in the range of 10 ...

[0151] [Table 11]

[0152] The observed binding response of the captured activin A antibody to TGF-beta family ligands injected at 200 nM was compared to the binding response of a negative control antibody (isotype control mAb). However, it provided a measure of background levels of non-specific binding and was further compared to the binding response of ActR2B-hFc, which was observed to bind to the entire panel of TGF-beta family members tested and therefore corresponds to a positive control ligand-binding protein. From this comparison, several antibodies (e.g., H4H10430, H4H10442) , H4H20446) were found to bind to activin A, activin AB, and activin AC, but did not appreciably bind to activin B or BMP or GDF ligands. It was also found that some antibodies bound to additional TGF-beta family ligands with broader cross-reactivity. For example, H4H10429P bound to activin A, activin B, and activin C. It appreciably bound to activin AB, activin AC, and also to BMP9 and BMP10. showed clear binding to activin A, activin B, activin AB, activin AC, BMP2, BMP4, BMP7, BMP9, and BMP10. These data suggest that activin A ligands can mediate mitochondrial function. It is shown that after immunization of mice, antibodies with different binding specificities for TGF-beta family ligands can be obtained.

[0153] Example 5 Cross-competitive analysis of anti-activin A antibodies Nine antibodies (H4H10446P2, H4H10468P2, H4H10442P2, H4H10423P, H4H10430P, H4H10429P A cross-competition assay was performed to assess the ability of a panel of antibodies (H4H10432P2, H4H10436P2 and H4H10440P2) to compete with each other for binding to human activin A. The assay included two isotype control antibodies and two control activin A antibodies, control 1 (a human anti-activin A antibody with heavy and light chain variable domain sequences of "A1" as described in US 8,309,082) and control 2 isotype control antibodies (H4H10432P2, H4H10436P2 and H4H10440P2) to compete with each other for binding to human activin A. control 3 (MAB3381, available from R&D Systems, Inc., Minneapolis, MN) The cells were incubated with Octet HBST buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20, 0.1 mg / mL All assays were run for 25 min at 37 °C in 100 mL of BSA (100% BSA) using a microtiter plate shaking speed of 1000 rpm. Briefly, anti-activin A antibodies were captured on an Octet sensor chip (Fortebio, no. 18-0015) coated with anti-human Fc antibodies in an amount that resulted in a binding response of approximately 1.8 nM by immersing the chip in a 10 μg / mL solution of each anti-activin A antibody for 5 min. The chip was then incubated in a 50 μg / mL solution of an irrelevant antibody for 5 min. All remaining anti-hFc binding sites on the plate were then blocked with 1 μM The wells contained a 50 nM solution of activin A pre-bound with a second anti-activin A antibody (R&D Systems, no. 338-AC / CF). Binding to a bin A antibody-coated sensor chip was monitored for 5 min at 1000 rpm. The response of mAb / activin A complexes binding to anti-activin A coated sensor chips was compared to determine the competitive / non-competitive behavior of various anti-activin A monoclonal antibodies. The results are shown in Figure 1.

[0154] In Figure 1, competitive binding responses are indicated by black or light grey shading, and the corresponding antibody pairs were It is proposed that they compete with each other for binding to typin A. Light grey with black font Colored boxes represent binding responses for self-competition between the same antibodies. Black boxes with white font represent antibodies competing for activin A binding in both directions, independent of binding order. Dark grey boxes with black font represent isotype controls (i.e., non- (binding) antibody readout, which is in contrast to the isotype control antibody anti-activin A antibody- Lack of binding to activin A complex (isotype control antibody bound to Octet sensor chip) (when binding to activin A was observed) or the lack of binding of the isotype control antibody to activin A (when binding to isotype (when a control antibody is used as the second antibody in wells containing activin A) White boxes with black font represent no competition between the antibodies, suggesting that the antibodies have distinct binding epitopes on activin A.

[0155] Four antibodies, H4H10446P2, H4H10468P2, H4H10442P2, and H4H10423P, compete with each other for binding to activin A in a bidirectional manner. In addition, these four antibodies do not compete with control 1 or control 3 for binding. Three of these four activin A antibodies, H4H10446P2, H4H10468P2, and H4H10442P2, do not cross-compete with any other activin A antibodies. One of the four antibodies (H4H10423P) also competes with H4H10430P for binding to activin A in a bidirectional manner. Five antibodies, H4H10430P, H4H10429, H4H10432P2, H4H10436P2, and H4H10440P2, compete bidirectionally with each other, as well as with Control 1 and Control 3, for binding to activin A. Four of these five antibodies (i.e., H4H10429, H4H10432P2, H4H10436P2, and H4H10440P2) do not cross-compete with any other activin A antibodies, whereas H4H10423P also cross-competes with H4H10430P, as described above.

[0156] From the results of this Example, it is apparent that the anti-activin A antibody of the present invention has the following characteristics based on the epitope binding characteristics: It can be shown that the nucleotide sequences can be classified into two distinct "bins". Bin 1 is H4H10423P , H4H10446P2, H4H10468P2 and H4H10442P2. Bin 2 includes H4H10429, H4H1430P, H4H10432P2, H4H10436P2 and H4H10440P2. Furthermore, one antibody from each bin, i.e., H4H10423P and H4H1430P, cross-compete with each other. The results of this example suggest that bin 1 antibodies bind to a region on activin A that is distinct from bin 2 antibodies.

[0157] Example 6 Inhibition of activin A-mediated receptor activation and SMAD complex signaling with anti-activin A antibodies To further characterize the anti-activin A antibodies of the present invention, activin type IIA and type IIB receptors are Activation of the activin receptor (ActRIIA and ActRIIB, respectively) and subsequent phosphorylation of the activin type I receptor Bioassays were developed to detect oxidation and activation. The interaction of ActRIIA and ActRIIB with activin leads to induction of diverse cellular processes such as regulation of cancer cell proliferation, metastasis, and differentiation of embryonic stem cells [Tsuchida, K. et al., Cell Commun Signal 7:15(2009)]. Phosphorylation and activation of type I receptor leads to phosphorylation of SMAD2 and 3 proteins, which results in the formation of activated SMAD complexes that lead to transcriptional regulation of genes.

[0158] To detect activation of the SMAD complex signaling pathway via activin binding to the activin type II receptor, the human A204 rhabdomyosarcoma cell line (ATCC, no. HTB-82) was transfected with a Smad2 / 3-luciferase reporter plasmid (CAGAx12-Luc; Dennler, 1998) to generate the A204 / CAGAx12-Luc cell line. A204 / CAGAx12-Luc cells were maintained in McCoy's 5A (Irvine Scientific, no. 9090) supplemented with 10% fetal bovine serum (FBS), penicillin / streptomycin / glutamine, and 250 μg / mL G418. For the bioassay, A204 / CAGAx12-Luc cells were seeded into 96-well assay plates at 10,000 cells / well in low serum medium, 0.5% FBS and OPTIMEM (Invitrogen, no. 31985-070) and incubated overnight at 37°C and 5% CO2. To determine ligand dose response, Activin A (R&D Systems, #338-AC), Activin B (R&D Systems, #659-AB), Activin AB (R&D Systems, #1066-AB) and Activin AC (R&D Systems, #4879-AC / CF) were serially diluted 1:3 from 100 to 0.002 nM and added to cells starting with a no Activin control. Activin B, activin AB, and activin AC inhibited the A204 / CAGAx12-Luc cell line, respectively. EC values ​​of 99 pM, 47 pM, 19 pM, and 4.4 nM 50 It was observed that the activation was observed at the inhibition value. To determine the binding potential of activin A, activin B, activin AB, and activin AC, the antibodies were serially diluted 1:3 starting at 100 to 0.002 nM, activin B starting at 1000 to 0.02 nM, or activin AC starting at 300, including control samples containing either the appropriate isotype control antibody or no antibody, and added to cells at a constant concentration of 100 pM activin A, 50 pM activin B, 30 pM activin AB, or 4 nM activin AC. Control 4 (ActRIIB-hFc; SEQ ID NO: 227) was also used as a positive blocking control in this assay. After 5.5 h of incubation at 37° C. and 5% CO2, OneGlo substrate (Promega, no. E6051) was added and luciferase activity was then detected using a Victor X (Perkin Elmer) instrument. Nonlinear regression (4-parameter logistics) was performed with Prism 5 software (GraphPad). The results were analyzed using.

[0159] As shown in Table 12, the anti-activin A antibodies of the present invention inhibited 100 pM activin A with IC values ​​ranging from 39 pM to 3.5 nM. 50 value, while control 1 had an IC 50 Block by value A subset of the anti-activin A antibodies of the present invention were tested for blocking activin B, AB, and AC. Four of the nine antibodies tested blocked 50 pM activin B with IC values ​​ranging from 130 pM to 100 nM. 50 Five antibodies of the invention tested for blocking activin B only blocked at high antibody concentrations, while control 1 did not show any measurable blockade of activin B. Eight antibodies of the invention tested blocked 30 pM activin AB with IC values ​​ranging from 100 pM to 8.2 nM. 50 value, whereas control 4 had an IC 50 One antibody, H4H10423P, showed only weak blocking of activin AB. Seven of the eight antibodies tested demonstrated IC values ​​ranging from 580 pM to 6.5 nM for 4 nM activin AC. 50 value, whereas control 4 had an IC of 1.1 nM. 50 One antibody, H4H10423P, did not demonstrate any blockade of activin AC. Both mouse IgG (mIgG isotype control) and human IgG (hIgG isotype control) negative controls showed no significant inhibition of receptor agonists. did not block ligand activation.

[0160] [Table 12]

[0161] Bioassays using A204 / CAGAx12-Luc cells were also able to be stimulated by GDF8 (R&D Systems, Catalog No. 788-G8 / CF) and GDF11 (R&D Systems, Catalog No. 1958-GD-010 / CF). Functional inhibition of these ligands with activin A antibodies was also observed. To test this effect, the assay was performed using the conditions described above, but with GDF8 or GDF11 instead of the activating ligand, giving EC50 values ​​of 188 pM and 84 pM, respectively. IC4 of 298 pM and 214 pM, respectively. 50 These same constants were completely blocked by the value. Using concentrations of ligand, no inhibition of either GDF8 or GDF11 was observed by the activin A antibodies, H4H10446P2 and H4H10430P, when tested with up to 100 nM of antibody. On a separate day, the activin A antibodies H4H10429P and H4H10436P2 were tested for inhibition in this assay in the presence of a fixed concentration of 250 pM GDF8 or 250 pM GDF11, and no inhibition was observed after incubating cells with up to 150 nM of the activin A antibodies tested. In this assay, GDF8 and GDF11 alone displayed EC50 values ​​of 124 pM and 166 pM, respectively. These data demonstrate that the activin A antibodies H4H10446P2, H4H10430P, H4H10429P and H4H10436P2 do not functionally inhibit GDF8 or GDF11.

[0162] Example 7 Stimulation of skeletal muscle hypertrophy using activin A antibodies Skeletal muscle myostatin-specific antagonist, anti-GDF8 antibody H4H1657N2 (see US2011-0293630A1), or a combination of H4H1657N2 and a different anti-activin A antibody, induced Hypertrophy was assessed in CB17 SCID mice. The extent of hypertrophy was measured relative to treatment with an isotype-matched control antibody. These studies also included treatment with the extracellular domain of human ActRIIB (Control 4, SEQ ID NO: 227) produced with the C-terminal human IgG1 Fc domain. Control 4 was administered in vivo. It has been shown to induce muscle hypertrophy in the body and further to bind to and block the activity of multiple TGF-beta family member ligands [Souza, TA et al., Mol Endocrinol 22:2689-702 (2008); Lee, SJ et al., Proc Natl Acad Sci USA 102(50):18117-22 (2005)].

[0163] In eight studies, a total of eight anti-activin A antibodies of the present invention and one control were combined with H4H1657N2. H4H1657N2 alone or control2 (anti-activin RIIB antibody with VH / VL of antibody MOR08159 described in US2010 / 0272734A1) treated groups were tested together or alone. For this study, male CB17SCID mice (Taconic, no. CB17SC-M) approximately 10 weeks old were evenly divided into 6 groups of 5 mice according to body weight. Groups of mice were treated in each study as follows:

[0164] [Table 13A]

[0165] [Table 13B]

[0166] For studies 1-5, 7, and 8, antibody and control 4 were administered subcutaneously twice during week 1 of the experiment (days 0 and 3) at a dose of 10 mg / kg of each protein and once during week 2 (day 7) at a dose of 10 mg / kg of each protein. The last dose of antibody or control 4 during week 3 (day 14) was administered subcutaneously at 8 mg / kg for studies #1 and #2, or at 10 mg / kg for studies #3 to #8 [Table 13( On day 21, the mice were euthanized and the total body weight of each mouse was measured. administered antibodies as in previous studies 1-5, but extended treatment to day 28 with an additional injection on day 21. Tibialis anterior (TA) and gastrocnemius (GA) muscles were excised from each mouse and weighed. Tissue masses were normalized to starting body weight, and the mean percent change in mass relative to the mean mass of the isotype control antibody-treated group was calculated. Results summarized in Tables 14-21 are expressed as the mean percent increase relative to isotype control ± standard error.

[0167] [Table 14]

[0168] As shown in Table 14, in the first study, Control 4 induced significant hypertrophy in all muscles examined, with a 45.80±1.47% increase in TA mass and a 31.91±1.4% increase in GA mass when compared to mice treated with isotype control. Treatment with H4H1657N2 alone also induced hypertrophy in TA (19.54±2.67% increase) and GA (26.46±3.63% increase) muscle mass, but was less effective than Control 4. The combination of H4H1657N2+H4H10442P2 was comparable to Compared to mice treated with treatment with treatment 4, the mean TA (40.76 ± 2.59%) and GA (30.62 ± 2.32%) muscles H4H1657N2 / H4H10423P and H4H1657N2 / H4H10432P2 induced a similar increase in meat mass. Treatment with the combination of H4H16757N2 / H4H10442P or control 4 did not significantly increase However, it did not induce a significant increase in the average TA mass.

[0169] [Table 15]

[0170] As shown in Table 15, in the second study, Control 4 induced hypertrophy in all muscles examined, with a 43.47±2.37% increase in mean TA mass and a 29.24±2.22% increase in mean GA muscle mass when compared to mice treated with the isotype control. In this study, treatment with H4H1657N2 alone also induced hypertrophy in all muscles examined, with a 43.47±2.37% increase in mean TA mass and a 29.24±2.22% increase in mean GA muscle mass when compared to mice treated with the isotype control. Treatment with the H4H1657N2 / H4H10429P and H4H1657N2 / H4H10436P2 combinations induced increases in mean muscle mass in TA and GA (16.7±2.73% and 18.54±3.48%, respectively), although these mean increases were smaller than those observed for the control 4 treatment group. Treatment with the H4H1657N2 / H4H10429P and H4H1657N2 / H4H10436P2 combinations induced increases in mean TA (34.14±2.55% and 29.31±1.59%, respectively) and mean GA (26.24±3.11% and 26.55±2.41%, respectively). The increase was between that observed for either H4H1657N2 or control 4 alone. The H4H1657N2 / H4H10440P2 combination induced a 100% increase in IL-1 expression compared with the other two combinations or controls in this study. However, treatment with 4 did not induce an increase in the mean mass of TA or GA as large as that induced by 4-treatment.

[0171] [Table 16]

[0172] As shown in Table 16, in the third study, Control 4 induced hypertrophy in all muscles examined, with a 39.90±3.58% increase in mean TA muscle mass and a 34.01±2.87% increase in mean GA muscle mass when compared to mice treated with isotype control. Treatment with H4H1657N2 alone also induced increases in mean TA (14.19±3.19%) and GA muscle mass (15.73±0.58%) when compared to mice treated with isotype control, but these mean increases The increase was smaller than that observed for the control 4 treatment group. Treatment with the combination induced increases in mean muscle mass in TA (40.01 ± 3.67%) and GA (31.29 ± 2.60%) similar to those in mice treated with control 4. Treatment with H4H1657N2 induced an increase in mean muscle mass in TA (28.30±3.27%) and GA (21.55±2.30%), which was observed for treatment with H4H1657N2 alone and the H4H1657N2 / H4H10446P2 combination. It was among the

[0173] [Table 17]

[0174] As shown in Table 17, in the fourth study, patients were treated with an isotype control. H4H1657N2 induced hypertrophy in the muscles examined, with the average TA muscle In this study, treatment with H4H10430P alone resulted in an increase of 21.05±2.64% in mean GA muscle mass and 22.85±2.28% in mean GA muscle mass. Treatment with the combination of H4H1657N2 and H4H10430P at 10 mg / kg and 2 mg / kg, respectively, increased muscle mass slightly, but the value was not statistically significant. Treatment with the combination of H4H1657N2 and H4H10430P at 10 mg / kg and 10 mg / kg, respectively, induced increases in the mean muscle mass of TA (40.20 ± 2.48%) and GA (22.46 ± 5.03%), which was greater than that observed with H4H1657N2 or H4H10430P alone in the TA muscle. Treatment with the combination of H4H1657N2 and H4H10430P at 10 mg / kg and 25 mg / kg, respectively, induced an increase in the mean muscle mass of TA (44.92 ± 5.70%) and GA (30.22 ± 2.97%), which was greater than that observed with H4H1657N2 or H4H10430P alone. This was greater than that observed for either drug alone.

[0175] [Table 18]

[0176] As shown in Table 18, in the fifth study, patients treated with an isotype control H4H1657N2 induced hypertrophy in the muscles examined, with the average TA muscle There was a 25.4±1.35% increase in mean GA muscle mass and a 22.82±1.97% increase in mean GA muscle mass. In this study, treatment with H4H10446P2 alone induced a low level of muscle hypertrophy, with a 3.70±1.67% increase in mean TA muscle mass and a 2.70±1.06% increase in mean GA muscle mass when compared to mice treated with an isotype control. Treatment with a combination of H4H1657N2 and H4H10446P2 at 10 mg / kg and 2 mg / kg, respectively, induced increases in mean muscle mass of TA (51.29±4.20%) and GA (39.24±3.08%), which were greater than those observed for H4H1657N2 or H4H10446P2 alone. Treatment with the combination of H4H1657N2 and H4H10446P2, each at a dose of 10 mg / kg, induced increases in mean muscle mass in TA (49.64±4.08%) and GA (35.56±3.39%) that were greater than those observed with H4H1657N2 or H4H10446P2 alone. Treatment with the combination of H4H1657N2 and H4H10446P2 at 10 mg / kg and 25 mg / kg induced increases in mean muscle mass in TA (49.79±5.46%) and GA (35.14±3.49%) that were greater than those observed with H4H1657N2 or H4H10446P2 alone.

[0177] [Table 19]

[0178] As shown in Table 19, in the sixth study, control 4 had a 100% CI of 0.01 to 0.01 in all muscles examined. It induced hypertrophy in the muscle, with an increase of 47.34±2.63% in mean TA mass and 32.17±3.81% in mean GA muscle mass when compared to mice treated with isotype control. Treatment with H4H1657N2 alone also significantly increased mean TA mass compared to mice treated with isotype control in this study. In this study, the addition of 17.21±2.97% and 21.57±1.90% increased the mean muscle mass of the TA and GA, respectively, but these mean increases were smaller than those observed for the control 4 treatment group. In this study, treatment with control 1 alone induced low levels of muscle hypertrophy and Compared to control-treated mice, the mean TA muscle mass was increased by 4.54 ± 2.25% and the mean GA muscle mass by 1.01 ± 0.02%. There was a 2.72 + 1.30% increase in meat mass. Treatment with the combination of TA and control 1 significantly reduced mean muscle mass in the TA (30.06±5.51%) and GA (30.72±3.64%) rats. H4H1657N2 induced an increase in the amount of IL-1 that was greater than that observed with H4H1657N2 or control 1 alone.

[0179] [Table 20]

[0180] As shown in Table 20, in the seventh study, Control 4 induced hypertrophy in all muscles examined, with a 34.43±5.92% increase in mean TA mass and a 14.86±3.65% increase in mean GA muscle mass, when compared to mice treated with the isotype control. In this study, treatment with Control 2 alone induced hypertrophy in the muscles examined, with a 36.75±3.88% increase in mean TA mass and a 14.86±3.65% increase in mean GA muscle mass, when compared to mice treated with the isotype control. There was a 26.41±3.16% increase in meat mass. Treatment with the combination of H4H1657N2 and H4H10430P at 10 mg / kg and 10 mg / kg, respectively, induced an increase in mean muscle mass of TA (33.13±2.02%) and GA (22.82±1.34%), which was greater than that observed with ActRIIB-Fc alone and Control 2 alone. The combination of H4H1657N2 and H4H10446P2 at 10 mg / kg and 10 mg / kg, respectively, Treatment with sucrose induced an increase in mean muscle mass in TA (41.28 ± 2.76%) and GA (29.21 ± 2.62%).

[0181] [Table 21]

[0182] As shown in Table 21, in the eighth study, Control 4 induced hypertrophy in all muscles examined, with a 53.74±5.31% increase in mean TA mass and a 39.39±4.56% increase in mean GA muscle mass when compared to mice treated with the isotype control. In this study, treatment with H4H1657N2 alone also induced hypertrophy in all muscles examined, with a 53.74±5.31% increase in mean TA mass and a 39.39±4.56% increase in mean GA muscle mass when compared to mice treated with the isotype control. In this study, the addition of 18.44±2.30% and 21.17±1.72% increased the mean muscle mass of the TA and GA, respectively, but these increases were smaller than those observed for the control 4 treatment group. In the study, treatment with control 2 alone induced hypertrophy in the examined muscles, with a 39.90 ± 1.69% increase in mean TA mass and a 39.90 ± 1.69% increase in mean GA mass when compared to mice treated with isotype control. There was a 25.87±2.72% increase in muscle mass. Treatment with a combination of H4H1657N2 and H4H10423P at 10 mg / kg and 25 mg / kg, respectively, induced an increase in mean muscle mass in TA (36.33±3.67%) and GA (28.18±3.11%) when compared to mice treated with isotype control. Treatment with a combination of H4H1657N2 and H4H10430P at 10 mg / kg and 25 mg / kg, respectively, induced an increase in mean muscle mass in TA (43.83±1.56%) and GA (31.24±1.90%), which was greater than the control alone and between the increases observed for Control 2 alone.

[0183] These studies demonstrated that administering anti-activin A antibodies together with myostatin inhibitors was These results show that at the doses and injection frequencies tested, it is possible to further increase skeletal muscle hypertrophy to a significantly greater extent than treatment with a myostatin inhibitor alone.

[0184] Example 8 Blocking Activin A Binding Using Activin A Antibodies Activin A and its receptors ActRIIB and ActRIIA, as well as their endogenous antagonists Selected anti-activin A antibodies that block the interaction with the target, follistatin The potency of was determined using a Biacore 3000 instrument. For this experiment, control 4 [human ActRIIB expressed with a C-terminal human Fc tag (SEQ ID NO: 227)], human ActRIIB expressed with a C-terminal human Fc tag, Human ActRIIA (hActRIIA-Fc; R&D Systems, no. 340-R2-100) or follistatin-288 (R&D Systems, no. 5836-FS-025) were amine-coupled to the sensor surface of a Biacore CM5. Activin A (R&D Systems, #338-AC) at a fixed concentration of 5 nM was incubated for 1 hour at room temperature either alone or mixed with activin A antibody, hActRIIA-Fc, hActRIIB-Fc, or isotype control antibody at a final concentration of 60 nM (12-fold molar excess of activin A). The antibody-activin A mixture was then injected over the amine-coupled control 4, hActRIIA-Fc, or follistatin-288 surfaces at a flow rate of 20 μL / min. At 150 seconds after the start of the injection, The binding signal (RU) was measured and the specific binding signal was determined by subtracting the RU value measured for the negative control reference surface from this signal. The percentage of free activin A bound to the receptor or antagonist surface was determined by antibody The binding intensity was calculated as the ratio of the observed specific binding signal divided by the specific binding signal from 5 nM activin A in the absence of .

[0185] [Table 22]

[0186] As shown in Table 22, six of the seven anti-activin A antibodies of the present invention tested, as well as both Control 1 and Control 3, inhibited the binding of Actin A to follistatin-288. One antibody of the invention, H4H10423P, did not prevent activin A binding to follistatin-288. Control 4 and hActRIIA-Fc blocked activin A binding to follistatin-288 at higher concentrations.

[0187] [Table 23]

[0188] As shown in Table 23, four of the seven anti-activin A antibodies of the present invention tested, as well as both Control 1 and Control 3, blocked binding of hActRIIA-Fc to activin A. Three antibodies of the invention, H4H10442P2, H4H10446P2, and H4H10423P, did not prevent activin A from binding to hActRIIA-Fc. Control 4 and hActRIIA-Fc blocked activin A from binding to hActRIIA-Fc.

[0189] [Table 24]

[0190] As shown in Table 24, four of the seven anti-activin A antibodies of the invention tested, as well as both Control 1 and Control 3, blocked binding of activin A to hActRIIB-Fc. Two antibodies of the present invention, H4H10442P2 and H4H10446P2, inhibit the binding of activin A to hActRIIB-Fc. One antibody of the invention, H4H10423P, demonstrated the ability to partially block activin A binding to hActRIIB-Fc at the higher concentrations of the antibody tested. Both hActRIIB-Fc and hActRIIA-Fc blocked activin A binding to hActRIIB-Fc.

[0191] Example 9 Effects of H4H1657N2 on muscle mass and athletic performance The effects of anti-GDF8 antibody, H4H1657N2, on muscle mass and exercise capacity were evaluated in aged male C57BL / 6 mice (19 months of age).

[0192] Mice were randomly divided into four groups (n = 6-8 / group), sedentary or active, and were administered either H4H1657N2 or gave a subcutaneous dose (10 mg / kg) of isotype control antibody twice weekly (6 injections) for 21 days. Mice in the exercise group were subjected to an exercise regimen that included one training session per day of 20 min on an Exer 6M treadmill (Columbus Instruments, Columbus, OH) at 10 m / min and 5° incline, 5 days per week for 3 consecutive weeks. At the end of the 3-week treatment, endurance was measured in all four groups using a treadmill exhaustion test. Data were analyzed using two-way ANOVA followed by Tukey's HSD test. Muscle mass was reported as normalized mass (i.e., muscle (Mass was normalized to body weight measured at the start of the experiment). Table 25 shows the results for quadriceps muscle. Results are presented as the mean % change (± standard error) for each group compared to the isotype control antibody group. .

[0193] [Table 25]

[0194] As seen in Table 25, H4H1657N2 treatment resulted in a significant increase in quadriceps muscle mass. (p<0.01 significance vs. isotype control in both H4H1657N2 groups). Increases in leg muscle mass (TA, GA) were observed with exercise compared to the isotype control antibody ( The isotype was seen in aged mice (17.4% and 12.5%) and inactive (14.1% and 11.6%, respectively). A slight increase in muscle mass was observed between exercised and inactive aged mice given the control antibody, but this was not statistically significant (Table 25).

[0195] The effect of H4H1657N2 treatment on exercise endurance was also examined in 19-month-old male C57BL / 6 mice [Table 26].

[0196] [Table 26]

[0197] In exercised aged mice, H4H1657N2 increased the activity of the GABA receptor agonist (GAA) in response to exercise compared to isotype control. It also induced a significant increase in endurance as measured by running time (50.2 min vs. 73.2 min) and distance (0.93 km vs. 1.33 km) in a dorm (Table 26). However, in inactive mice, H4H1657N2 did not significantly increase endurance compared to the isotype control group. Ta.

[0198] As in the muscle mass studies, H4H1657N2 significantly increased treadmill running time and distance. These results suggest that H4H1657N2, in combination with exercise training, induced a significant increase in endurance, as measured by The results show that when

[0199] Example 10 Effects of H4H1657N2 on skeletal muscle mass and isometric strength in mice The ability of H4H1657N2 to induce skeletal muscle hypertrophy was evaluated in vivo in 9-week-old male C57BL / 6 mice. did.

[0200] Repeated subcutaneous doses of H4H1657N2 or isotype control antibody were administered at either 10 or 30 mg / kg twice weekly (n=6) for 3 weeks. H4H1657N2 treatment for 21 days resulted in weight gains of 4.7±2.3% (ns) and 7.1±1.5% (ns), respectively, compared to mice receiving isotype control administered at an equivalent dose. Individual muscle masses were increased compared to isotype control (10 mg / kg and 30 mg / kg): tibialis anterior (19.4±4.9%** and 20.6±1.5%**), calf (14.7±2.8%**), and femoral (13.3±2.4%**). abdominal muscles: (14.9±2.9%** and 25.3±1.9%***), and quadriceps muscles (17.7±3.6%* and 26.2±3.8%**). (All statistics by one-way ANOVA with Tukey's post-hoc test [*p<.05; **p<.01; ***p<.001; ns=not statistically different]).

[0201] Increased tibialis anterior (TA) muscle mass was accompanied by increased ex vivo isometric strength, indicating the ability to maintain both muscle function and mass. Repeated subcutaneous doses of type-control antibody (10 mg / kg twice weekly for 3 weeks, n=6 / group) were administered. Mice treated for this purpose were individually anesthetized and maintained under isoflurane gas while the TA muscle was excised and placed in a bath of oxygenated lactated Ringer's solution constantly maintained at 25°C. The upper end of the TA was tightly tied to a post immersed in the bath while the distal tendon was simultaneously tied to a 305C lever arm (Cambridge Systems). The optimal length was determined by slightly stretching the TA and testing the force generated by 1Hz stimulation at the lowest voltage. The TA muscle was stimulated to repeatedly stretch until a drop in force occurred and then allowed to relax to the previous position. The voltage was then gradually increased in a series of 1Hz stimulations. The length and voltage were gradually increased to achieve maximal force output. Once the optimal length and voltage were determined, the TA muscle was stimulated for 400 ms at increasing frequencies (40-100 Hz) to determine maximal tetanic force. Between each tetanic stimulation, the TA muscle was given a 2 min rest period.

[0202] TA muscles from mice treated for 21 days with isotype control antibody at doses of 10 mg / kg and 30 mg / kg The meat produced mean peak tetanic forces of 892.6 ± 37 and 906.1 ± 37.8, respectively. TA muscles from mice treated with H4H1657N2 produced mean peak tetanic forces of 1041.3 ± 31.7 and 1003.3 ± 35.7 mN, respectively. These force values ​​represent a 16.7%* (10 mg / kg) and 10.7%ns (30 mg / kg) increase in mean peak tetanic force compared to isotype controls (Figure 2A). The overall drug effect of H4H1657N2 treatment on peak tetanic force was significantly greater than that of isotype controls at both the 10 mg / kg and 30 mg / kg doses. The results were statistically different from the control group (10 mg / kg dose shown in Figure 2B). (Figure 2A: Tukey Statistical analysis by one-way ANOVA with Sidak's post-hoc test [*p<.05; ns=not statistically different]. Figure 2B: Statistical analysis by two-way ANOVA and Sidak's post-hoc test [p>0.0001]).

[0203] Example 11 H4H1657N2 improves recovery from hindlimb suspension (HLS)-induced atrophy The effect of H4H1657N2 on skeletal muscle mass during recovery from 7-day hindlimb suspension (HLS)-induced atrophy was assessed in 1-year-old C57BL / 6 male mice.

[0204] On day 0, 18 mice were suspended by their tails so that both hind legs did not touch the ground for a duration of 7 days. The mice were placed in special cages with free access to food and water. One additional group of 6 mice was kept in a normal cage and served as a control (non-HLS control). On day 7, the suspended mice were lowered and randomized into three groups (n=6 each) according to the percentage of body weight lost during HLS. On day 7, muscle mass from the non-HLS control group and one HLS group (HLS group) was obtained to assess the percentage of atrophy in response to HLS. Two groups were randomized to separate groups (n=6 each) according to the percentage of body weight lost during HLS. The remaining HLS groups (n=6 each) were housed in normal cages for 8 days (i.e., from day 7 to day 15 of the experiment). 1) were allowed to recover and then administered 10 mg / kg on days 7 and 10 (i.e., 0 and 3 days after recovery). Mice were treated subcutaneously with either H4H1657N2 or isotype control at 100 mg / kg (HLS+7Rec+H4H1657N2 and HLS+7rec+isotype control, respectively). On day 15 (i.e., after 8 days of recovery), muscle mass was obtained to assess the percentage of recovery after HLS-induced atrophy.

[0205] As seen in Figure 3B, 7 days of HLS resulted in a significant loss of mass in both the tibialis anterior (TA) and gastrocnemius (GA) muscles (HLS group) when compared to the non-HLS control group (-13.7%* and -14.8%*, respectively). After 8 days of recovery, the HLS+7rec+isotype control group showed a significant loss of mass compared to the non-HLS control group. Both groups maintained a loss of TA and GA muscle mass (-6.3% and -7.5%), whereas the HLS+7Rec+H4H1657N2 group showed an increase in mass (4.7% and 5%) when compared to the non-HLS group.

[0206] Comparing the two recovery groups (i.e., HLS+7Rec+H4H1657N2 vs. HLS+7rec+isotype control), the effect of H4H1657N2 on TA and GA mass was similar to that seen with the isotype control antibody. However, muscle mass in the HLS+7rec+isotype control group was not statistically different. was not statistically different from the HLS or non-HLS control groups, whereas the HLS+7Rec+H4H1657N2 group had statistically greater TA and GA mass when compared to the HLS group (all statistics by one-way ANOVA with Tukey's post-hoc test [*, p<0.05 vs. non-HLS; ##, p<0.01 vs. HLS).

[0207] Example 12 Inhibition of BMP receptor type I and type II activation by anti-activin A antibody and ActRIIB-Fc Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily and have BMP receptors type I and type II. They are involved in the regulation of many physiological processes by activating receptor complexes on the cell surface composed of SMAD proteins and SMAD-dependent transcriptional activation of genes responsive to the ligand.

[0208] W-20-17 cells, a mouse bone marrow stromal cell line that has been shown to respond to BMP2, We developed a bioassay to detect the modulation of BMP signaling in cells. The luciferase reporter (i.e., BMP-responsive element (BRE(2X)-luciferase-IRES-GFP)) was induced The cells were engineered to express GFP at a constant rate and sorted for high GFP expression. The cell line was designated W-20-17 / BRE-luc and was maintained in 10% FBS, DMEM, Pen / Strep, and 200 μg / ml G418. These cells were used to measure BMP activation and inhibition of this activation by anti-activin A antibody and ActRIIB-hFc (Control 4, SEQ ID NO: 227).

[0209] Using the W-20-17 / BRE-luc cell line, we investigated the BMP signaling pathway of four anti-activin A antibodies and ActRIIB-hFc. The ability of the IL-16 receptor agonist to inhibit signal transmission was evaluated. For the bioassay, W-20-17 / BRE-luc cells were used. were seeded at 10,000 cells / well onto 96-well assay plates and incubated at 37°C and 5% CO2 overnight. The following day, BMP2, BMP4, BMP6, BMP9 or BMP10 were serially diluted 1:3 and added to the cells at 100 nM to 0.002 nM (including a no BMP control for dose response). For inhibition of BMP by Bin A antibodies or ActRIIB-hFc, antibodies or ActRIIB-hFc were serially diluted 1:3 from 1000 nM to 0.02 nM [including no antibody, control antibody, or a negative control for ActRIIB-hFc (i.e., an unrelated protein tagged with hFc, "control protein")] and assayed against the indicated As described above, together with 100 pM BMP2, 100 pM BMP4, 10 nM BMP6, 800 pM BMP9 or 4 nM BMP10 The cells were incubated at 37°C and 5% CO2 using a Victor X (Perkin Elmer). Luciferase activity was detected 5.5 hours after incubation, and the results were analyzed using nonlinear regression (4-parameter logistics) with Prism 5 software (GraphPad).

[0210] As shown in Table 27 below, H4H10446P2 and H4H10430P did not inhibit any of the BMPs tested, whereas other activin A antibodies tested against them (H4H10429 and H4H10436P2) and ActRIIB-hFc all showed some inhibition of some BMPs. H4H10429P had IC of 8.1 nM and 3.5 nM, respectively. 50 The values ​​showed inhibition of BMP9 and BMP10, but not BMP2 or BMP4. and BMP6. H4H10436P2 showed weak inhibition of BMP2 and BMP4 and an IC of >100 nM at the highest antibody concentration. 50 showed inhibition of BMP10 at 100-fold increase in β-blockers, but did not show any inhibition of BMP6 or BMP9. ActRIIB-hFc had IC values ​​of 2 nM and 1 nM. 50 The control molecules [i.e., an isotype control antibody (control mAb) and an unrelated protein tagged with hFc (control protein)] both showed inhibition of BMPs at 1000 and 10000 ng / mL, but did not inhibit BMP2, BMP4, or BMP6. No inhibition was observed, whereas BMP2, BMP4, BMP6, BMP9, or BMP10 alone (i.e., without antibody or hFc-tagged protein) had ECs of 34 pM, 63 pM, 4.5 nM, 260 pM, and 2.5 nM, respectively. 50 W-20-17 / BRE-luc cells were activated with

[0211] [Table 27]

[0212] Example 13 Treatment with anti-activin A antibody H4H10446P2 reduces renal fibrosis in vivo The effect of the specific anti-activin A antibody of the present invention, H4H10446P2, on renal fibrosis was determined in a unilateral ureteral ligation (UUO) mouse model of renal fibrosis. The UUO model preserves the function of the right kidney while The left ureter was completely ligated while the patient was in a ketamine / In mice anesthetized with xylazine, the left ureter was accessed via a flank incision and inserted with a 5-0 silk suture. UUO was performed by placing two ligatures around the proximal third of the ureter, 5 mm apart, using a Sham surgery was performed in a similar manner, but without any ligature on the ureter. In this model, severe fibrosis developed in the kidney within 14 days of UUO, and hydroxyproline This was assessed by measuring kidney collagen by a direct measurement of hydroxyproline content in samples, a method called hydroxyproline quantification. Hydroxyproline is a unique component of collagen and accounts for approximately 14.4% of the amino acid composition of collagen in most mammalian tissues. [Cochrane et al., J Am Soc Nephrol 16:3623-30 (2005)]. To measure collagen content by the hydroxyproline method, the frozen kidney samples were first dried overnight using a vacuum chamber. The dried kidney tissue samples were then homogenized in ice-cold NaCl / NaHCO3 solution. The sample was then hydrolyzed using 6M HCl, after which it was dried using a vacuum centrifuge. The hydroxypropyl phosphate group in the rehydrated sample was then rehydrated using 0.1 M HCl. The amine was oxidized with 300 mM chloramine T (Sigma, no. 857319) and then subjected to Ehrlich Reagent: 3.5 M p-dimethylaminobenzaldehyde in 60% perchloric acid (Sigma, no. 311413). Color was developed by adding 558 nm hydroxyproline standard solution (FW: 149.19, Sigma, No. 39070). Finally, the absorbance of the sample was measured at 558 nm using a spectrophotometer. The hydroxyproline content of the kidney was determined by comparing it with a standard (Sigma, no. H5534). The measured hydroxyproline value was then multiplied by a factor of 6.94 to determine collagen value. Dry kidney mass was reduced 14 days after administration as a result of parenchymal damage. Male C57BL / 6 mice (Taconic farms, Inc.) aged 16 weeks were subjected to sham (n=10) or UUO (n=20) surgery. Mice that underwent UUO surgery were then divided into two groups. Each UUO group received either H4H10446P2 (40 mg / kg, n=10) or any known or an isotype control antibody (40 mg / kg, n = 10) that does not bind to mouse proteins Starting the day before surgery, mice received subcutaneous injections of 1, 3, 6, 8, 10, and 13 days after surgery. Sham-operated mice received vehicle (sterile PBS) during this time using the same schedule as the UUO group. All mice were sacrificed on day 14 after surgery. Kidney mass was measured, and kidneys were flash frozen using liquid nitrogen and kept at -80°C until collagen content was measured. Hydrochloride was added to the 100% ethanol solution. Renal collagen content was measured using the xyproline method and then expressed as either total kidney collagen (μg) or kidney collagen normalized to kidney mass (μg / mg dry mass). Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. In Table 28 below, results are summarized for total kidney collagen, normalized kidney collagen, and dry kidney mass for each treatment group, expressed as the mean±SEM.

[0213] [Table 28]

[0214] As shown in Table 28, both total kidney collagen and kidney collagen normalized to kidney mass were significantly increased in UUO mice compared to sham-operated mice. UUO mice treated with H4H10446P2 showed a significant reduction in both total kidney collagen and kidney collagen normalized to kidney mass (approximately 45% reduction in fibrotic collagen) compared to UUO mice treated with isotype control antibody, indicating that anti-activin A antibody reduces fibrosis in the kidney. Mice showed increased dry kidney mass compared to UUO mice treated with isotype control antibody. This indicates preservation of parenchyma in mice treated with anti-activin A antibodies.

[0215] Example 14 Effects of H4H10446P2 on body weight and muscle mass in mice overexpressing activin A Efficacy of H4H10446P2 in neutralizing high levels of activin A in mice To evaluate the effect of activin A on the expression of activin A in C57BL / 6 mice (10 weeks of age), we overexpressed activin A in these mice by hydrodynamic delivery (HDD) of a DNA construct encoding full-length activin A. Mice were randomly divided into three groups (n = 5-6 / group), one of which received an isotype control antibody. In the presence of α-aminobutyric acid, the mice were injected with a mixture of saline / 2.5 μg of DNA construct control, and two groups were injected with α-aminobutyric acid. A mixture of saline / 2.5 μg of DNA construct containing activin A in the presence of isotype control antibody or H4H10446P2 was injected. DNA construct was injected on day 0, and antibody was administered at 2.5 mg / kg (in two injections) on days 0 and 4 for 7 days. Muscle mass was normalized to mass and The results are reported as mean percent change (± standard error) in each group compared to the HDD delivery of the construct control + isotype control antibody group in Figure 4. Data were analyzed by two-way ANOVA followed by Tukey's HSD test.

[0216] As seen in Figure 4, 7 days after HDD, in mice treated with isotype control antibody Delivery of activin A in mice treated with H4H10446P2 resulted in a significant decrease in body weight (-10.81 ± 2.46%) and tibial and gastrocnemius muscle mass (-13.96 ± 1.85% and -10.34 ± 1.51%, respectively) (significance of p < 0.01 vs. isotype control). Delivery of activin A in mice treated with H4H10446P2 resulted in a significant attenuation in body weight (-1.49 ± 1.98%) and tibial and gastrocnemius muscle mass (-2.57 ± 1.26% and -1.77 ± 2.42%, respectively) at the end of 7 days of treatment.

[0217] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

Claims

1. A binding-dissociation equilibrium constant (K) of less than 5 pM as measured by surface plasmon resonance assay at 25 °C D ) an isolated antibody or antigen-binding fragment thereof that specifically binds to activin A 。

2. K<~4 pM as measured by surface plasmon resonance assay at 25 °C D 2. The isolated antibody or antigen-binding fragment thereof of claim 1, which specifically binds to activin A at

3. Binding association equilibrium constant (K a ) specifically binds to activin A. The isolated antibody or antigen-binding fragment thereof described above.

4. 3. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2, which blocks the binding of at least one activin A receptor to activin A.

5. Blocks activation of at least one activin A receptor by activin A, claims Item 3. An isolated antibody or antigen-binding fragment thereof according to item 1 or 2.

6. 6. The isolated antibody or antigen-binding fragment thereof of claim 5, which does not significantly block the binding of activin A to the activin type II receptor.

7. IC of less than approximately 80 pM as measured in an in vivo receptor / ligand binding bioassay at 25°C 50 5. The isolated antibody or antigen-binding fragment thereof of claim 4, which blocks binding of activin A to an activin A receptor at a value of 0.1-0.

25.

8. IC of less than approximately 60 pM as measured in an in vivo receptor / ligand binding bioassay at 25°C 50 8. The isolated antibody or antigen-binding fragment thereof of claim 7, which blocks binding of activin A to an activin A receptor at a value of 0.1 to 0.5 μg / mL.

9. A compound that inhibits the binding of activin A to an activin A receptor selected from the group consisting of activin type IIA receptor (ActRIIA), activin type IIB receptor (ActRIIB), and activin type I receptor.

9. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. The method of claim 1 to 8, which inhibits activin A-mediated activation of SMAD complex signaling. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 4.

11. Reference sequences comprising heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pairs selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210. The method according to any one of claims 1 to 8, which competes with a control antibody for binding to activin A. An isolated antibody or antigen-binding fragment thereof.

12. SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146 on activin A HCVR / LCVR sequence pairs selected from the group consisting of 194 / 146, and 202 / 210.

9. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which binds to the same epitope.

13. An isolated antibody or antigen-binding fragment thereof that specifically binds to activin A. (a) a complementarity determining region (CDR) of HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 106, 114, 122, 130, 138, 154, 162, 170, 178, 186, 194, and 202; and (b) a complementarity determining region (CDR) of HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 146, and 210. An isolated antibody or antigen-binding fragment thereof comprising a CDR of an LCVR having an amino acid sequence selected from the group consisting of: Fragment.

14. 14. The isolated antibody or antigen-binding fragment thereof of claim 13, comprising heavy and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210.

15. Sequence numbers: 4-6-8-12-14-16; 20-22-24-28-30-32; 36-38-40-44-46-48; 52-54-56-60-62-64; 68-70-72-76-78-80; 84-86-88-92-94-96; 100-102-104-92- 94-96;108-110-112-92-94-96;116-118-120-92-94-96;124-126-128-92-94-96;132-134-136-92-94-96;140-142-144-148-150-152;156-158-160 15. The isolated antibody or antigen-binding fragment thereof of claim 14, comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains selected from the group consisting of -148-150-152; 164-166-168-148-150-152; 172-174-176-148-150-152; 180-182-184-148-150-152; 188-190-192-148-150-152; 196-198-200-148-150-152; and 204-206-208-212-214-216, respectively.

16. An isolated antibody or antigen-binding fragment thereof that specifically binds to activin A. and (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 146, and 210.

17. 17. The monoclonal antibody of claim 16, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 90, 106 / 90, 114 / 90, 122 / 90, 130 / 90, 138 / 146, 154 / 146, 162 / 146, 170 / 146, 178 / 146, 186 / 146, 194 / 146, and 202 / 210. Isolated antibodies or antigen-binding fragments thereof.

18. The antibody or antigen-binding fragment according to any one of claims 1 to 17 and a pharmaceutical A pharmaceutical composition comprising a suitably acceptable carrier or diluent.

19. 20. A method for increasing muscle mass or strength in a subject, comprising administering to the subject a pharmaceutical composition according to claim 18.

20. The antibody or antigen-binding fragment according to any one of claims 1 to 17, and a pharma- ceutical acceptable carrier or diluent.

21. The pharmaceutical composition of claim 20, wherein the GDF8 antagonist is selected from the group consisting of a fusion protein that inhibits GDF8, an anti-GDF8 antibody, and an antigen-binding fragment of an anti-GDF8 antibody.

22. 20. The method of claim 19, further comprising administration of a GDF8 antagonist, wherein the GDF8 antagonist is an anti-GDF8 antibody or an antigen-binding fragment thereof.

23. The GDF8 antagonist comprises a heavy chain complementarity determining region (HCDR) of HCVR comprising SEQ ID NO: 217, and a sequence An anti-GDF8 antibody or its antigen-binding fragment comprising a light chain complementarity determining region (LCDR) of LCVR comprising sequence number 221. The method of claim 22, wherein the fragment is a

24. GDF8 antagonists a) three HCDRs comprising SEQ ID NO: 218, SEQ ID NO: 219, and SEQ ID NO: 220, and b) three LCDRs comprising SEQ ID NO: 222, SEQ ID NO: 223, and SEQ ID NO: 224 The method of claim 22, wherein the antibody is an anti-GDF8 antibody or an antigen-binding fragment thereof comprising:

25. 21. A method for increasing muscle mass or strength in a subject, comprising administering to the subject the pharmaceutical composition of claim 20.

26. A method for increasing muscle mass or strength in a subject, comprising administering to a subject an activin A-specific The method comprises administering to a subject an antigen-binding molecule comprising a GDF8-specific binding domain and a GDF8-specific binding domain.

27. The method of claim 26, wherein the activin A specific binding domain comprises HCVR and LCVR.

28. The method of claim 26, wherein the GDF8-specific binding domain comprises HCVR and LCVR.

29. HCVR, (a) a CDR of a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 106, 114, 122, 130, 138, 154, 162, 170, 178, 186, 194, and 202; and (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 146, and 210; CDR of LCVR with acid sequence 28. The method of claim 27, comprising:

30. 29. The method of claim 28, wherein the HCVR comprises three heavy chain complementarity determining regions (HCDRs) comprising SEQ ID NO: 218, SEQ ID NO: 219, and SEQ ID NO: 220, and the LCVR comprises three light chain complementarity determining regions (LCDRs) comprising SEQ ID NO: 222, SEQ ID NO: 223, and SEQ ID NO:

224.

31. The activin A specific binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR). The method of claim 26, wherein the GDF8-specific binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR).

32. The method of claim 26, wherein the antigen-binding molecule is a bispecific antibody.

33. A method for treating, preventing or alleviating a disease or disorder characterized by loss of muscle mass or strength, comprising administering to a subject in need thereof an activin A specific binding protein. A method comprising:

34. A method for treating, preventing or alleviating a disease or disorder characterized by loss of muscle mass or strength, comprising administering to a subject a specific binding protein for activin A and a specific binding protein for GDF8. administering to a subject in need thereof.

35. The disease or disorder characterized by a decrease in muscle mass or strength is sarcopenia, cachexia, muscle injury, muscle wasting / atrophy, cancer, obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophy, 35. The method of claim 34, wherein the disease is selected from the group consisting of osteoarthritis, amyotrophic lateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, and metabolic syndrome.

36. 36. The method of claim 35, wherein the cachexia is idiopathic or secondary to another condition.

37. 37. The method of claim 36, wherein the condition is cancer, chronic renal failure, or chronic obstructive pulmonary disease.

38. Muscle wasting / atrophy can occur due to disuse, immobilization, bed rest, injury, medical procedures, surgical interventions, and 36. The method of claim 35, wherein the condition is caused by or associated with a condition selected from the group consisting of: need for mechanical ventilation.

39. 39. The method of claim 38, wherein the surgical intervention is selected from the group consisting of hip fracture, hip replacement, and knee replacement.

40. 36. The method of claim 35, wherein the metabolic syndrome comprises a disease or disorder selected from the group consisting of diabetes, obesity, malnutrition, organ atrophy, chronic obstructive pulmonary disease, and anorexia.

41. A method for treating, preventing or alleviating a disease or disorder characterized by loss of muscle mass or strength, comprising administering to a subject an antigen-binding domain comprising an activin A-specific binding domain and a GDF8-specific binding domain. administering the combined molecule to a subject in need thereof.

42. Diseases caused, promoted, exacerbated, or aggravated by activin A activity or a method for treating, preventing or alleviating a disorder comprising administering to said patient an activin A antibody or an antigen thereof. administering the binding fragment to a subject in need thereof.

43. 43. The method of claim 42, wherein the disease or disorder is renal fibrosis.

44. 43. The method of claim 42, wherein the disease or disorder is cachexia.

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