ACTRII-BINDING PROTEINS AND USES THEREOF

Abstract

The present disclosure provides ActRII-binding proteins, such as anti-ActRIIA antibodies and anti-ActRIIB antibodies, as well as compositions and methods for producing the ActRII-binding proteins. In certain embodiments, the ActRII-binding proteins inhibit or antagonize ActRII activity. Furthermore, the present disclosure provides compositions and methods for diagnosing and treating diseases and conditions associated with muscle wasting; fibrotic conditions; inflammatory, cardiovascular, pulmonary, musculoskeletal, neurological, ocular, skeletal, autoimmune, or metabolic diseases or conditions; wound healing; and cancer, as well as other ActRII-mediated diseases and conditions.

Description

[Technical field]

[0001] Related Applications This patent application claims priority to U.S. Provisional Patent Application No. 62 / 854,625, filed May 30, 2019, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 14, 2020, is named APH-00925_SL.txt and is 57,075 bytes in size. [Background technology]

[0003] The transforming growth factor-beta (TGF-beta) family includes a variety of growth factors known to exert biological effects on a wide variety of cell types in both vertebrates and invertebrates. Members of the TGF-beta family play important functions in pattern formation and tissue fate determination during embryonic development and can affect a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family includes proteins variously described as growth differentiation factors (GDFs), bone morphogenetic proteins (BMPs), activins, and inhibins.

[0004] TGF-beta family members signal through a mechanism that involves a multistep process in which they bind to type II serine / threonine kinase receptors expressed on the cell surface, the type II receptor forms a heterocomplex with the cognate type I receptor and activates the type I receptor by phosphorylation, the activated type I receptor phosphorylates and activates Smad proteins that transmit signals from the cytoplasm to the nucleus, where Smad oligomers bind to DNA and associate with transcription factors to control the expression of target genes.

[0005] Two related members of the type II TGF-beta receptor family, ActRIIB and ActRIIA, have been identified as type II receptors for activin A and activin B, as well as other TGF-beta family members such as BMP7, BMP9, BMP10, GDF1, GDF3, GDF8 (myostatin), GDF11, and Nodal (Yamashita et al., J. Cell Biol. 130:217-226 (1995); Lee et al., PNAS 98:9306-9311 (2001); Yeo et al., Mol. Cell 7:949-957 (2001); and Oh et al., Genes Dev. 16:2749-54 (2002)). ALK4 and ALK7 are the major type I TGF-beta receptor family member receptors for activin A and activin B, respectively.

[0006] Altered expression and activity of members of the TGF-beta ligand and receptor family have been proposed to be associated with a variety of disorders and conditions, including muscle, bone, neurological and metabolic disorders and conditions, as well as cancer. Additional ActRII antagonists and their uses would be useful in the diagnosis and treatment, prevention and / or amelioration of diseases or conditions associated with ActRII and / or ligands of ActRII. Summary of the Invention

[0007] The present disclosure provides activin receptor type II (ActRII) binding proteins and methods of using such ActRII binding proteins. In certain embodiments, the ActRII binding proteins are capable of inhibiting or preventing ActRII from binding to one or more cognate ActRII ligands and / or one or more cognate ActRI receptors. In some embodiments, the ActRII binding proteins are capable of inhibiting or preventing ActRII from binding to an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10). The present disclosure also provides methods of using ActRII binding proteins for the diagnosis, or treatment, prevention, and / or amelioration of diseases or conditions associated with increased ActRII expression and / or ActRII-mediated signaling. Such diseases or conditions include, but are not limited to, muscle disorders such as degenerative muscle diseases, muscular dystrophies, muscle atrophy, or muscle wasting disorders; fibrotic conditions; inflammatory, autoimmune, cardiovascular, pulmonary, musculoskeletal, skeletal, ophthalmic, neurological, or metabolic diseases or conditions; obesity; wound healing; and cancer.

[0008] In some embodiments, the ActRII binding protein specifically binds to ActRIIB. In further embodiments, the ActRII binding proteins provided specifically bind ActRIIB and (a) compete with an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10) for binding to ActRIIB; (b) reduce phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIB and ALK4 and / or ALK7 in the presence of an ActRIIB ligand (e.g., activin A and / or GDF8 (myostatin)); (c) reduce phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRIIB in the presence of an ActRIIB ligand (e.g., activin A and / or GDF8); and (d) bind to ActRIIB with a K of 1 nM or less and 1 pM or more. D (e.g., as determined by BIACORE® analysis). In some embodiments, the ActRIIB binding protein has two, three, or four of the above properties. In some embodiments, the ActRIIB binding protein has at least two or at least three of the above properties. In further embodiments, the ActRIIB binding protein competes for binding to ActRIIB with an antibody having an ActRIIB-binding VH and VL pair disclosed herein. In further embodiments, the ActRIIB binding protein is an anti-ActRIIB antibody or an ActRIIB-binding antibody fragment.

[0009] In some embodiments, the ActRII binding protein specifically binds to ActRIIB and ActRIIA. In further embodiments, the ActRII binding protein provided specifically binds to ActRIIB and ActRIIA and (a) competes with an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10) for binding to ActRIIB and / or ActRIIA; (b) competes with a ligand of ActRIIB and / or ActRIIA (e.g., activin A and / or GDF8 (myostatin)). (c) reduces phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRIIB and / or ActRIIA in the presence of a ligand for ActRIIB and / or ActRIIA (e.g., activin A and / or GDF8); and (d) reduces phosphorylation of ActRIIB with a K of 1 nM or less and 1 pM or more. D (e.g., as determined by BIACORE® analysis). In some embodiments, the ActRIIB and ActRIIA binding proteins have two, three, or four of the above properties. In some embodiments, the ActRIIB and ActRIIA binding proteins have at least two or at least three of the above properties. In further embodiments, the ActRIIB binding proteins compete for binding to ActRIIB and ActRIIA with an antibody having a VH and VL pair that binds to ActRIIB and ActRIIA disclosed herein. In further embodiments, the ActRIIB and ActRIIA binding proteins are anti-ActRIIB and ActRIIB antibodies or ActRIIB antibody fragments and ActRIIB-binding antibody fragments.

[0010] In some embodiments, the ActRII binding protein specifically binds to ActRIIA. In further embodiments, the ActRII binding proteins provided specifically bind to ActRIIA and (a) compete with an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10) for binding to ActRIIA; (b) reduce phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIA and ALK4 and / or ALK7 in the presence of an ActRIIA ligand (e.g., activin A and / or GDF8 (myostatin)); (c) reduce phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRIIA in the presence of an ActRIIA ligand (e.g., activin A and / or GDF8); and (d) bind to ActRIIA with a K of 1 nM or less and 1 pM or more. D (e.g., as determined by BIACORE® analysis). In some embodiments, the ActRIIA binding protein has two, three, or four of the above properties. In some embodiments, the ActRIIA binding protein has at least two or at least three of the above properties. In further embodiments, the ActRIIA binding protein competes for binding to ActRIIA with an antibody having an ActRIIA-binding VH and VL pair disclosed herein. In further embodiments, the ActRIIA binding protein is an anti-ActRIIA antibody or an ActRIIA-binding antibody fragment.

[0011] In some embodiments, the ActRII binding protein comprises a set of complementarity determining regions (CDRs), i.e., heavy chain variable region (VH)-CDR1, VH-CDR2, VH-CDR3, light chain variable region (VL)-CDR1, VL-CDR2 and VL-CDR3, and / or a set of antigen binding regions (ABRs), i.e., heavy chain variable region (VH)-ABR1, VH-ABR2, VH-ABR3, light chain variable region (VL)-ABR1, VL-ABR2 and VL-ABR3, and such CDRs and / or ABRs are present in the heavy chain variable region (VH) and light chain variable region (VL) pairs disclosed in Table 1. In some embodiments, the ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH and VL pair selected from the group consisting of: (a) a VH sequence of SEQ ID NO: 20, 49, or 77 and a VL sequence of SEQ ID NO: 30, 39, 59, 67, or 85, wherein the protein binds to ActRIIB, and (b) a VH sequence of SEQ ID NO: 2 and a VL having the amino acid sequence of SEQ ID NO: 12, wherein the protein binds to ActRIIB and activin receptor type IIA (ActRIIA).

[0012] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 20 and a VL having the amino acid sequence of SEQ ID NO: 30, and such a protein binds to ActRIIB.

[0013] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 20 and a VL having the amino acid sequence of SEQ ID NO: 39, and such a protein binds to ActRIIB.

[0014] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 49 and a VL having the amino acid sequence of SEQ ID NO: 59, and such a protein binds to ActRIIB.

[0015] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 20 and a VL having the amino acid sequence of SEQ ID NO: 67, and such a protein binds to ActRIIB.

[0016] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 77 and a VL having the amino acid sequence of SEQ ID NO: 85, and such a protein binds to ActRIIB.

[0017] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 2 and a VL having the amino acid sequence of SEQ ID NO: 12, and such a protein binds to ActRIIA and ActRIIB.

[0018] In a further embodiment, the ActRII binding protein specifically binds to ActRII and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (a) (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21, 50, or 78, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, 51, or 79, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 23, 52, or 80, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 31, 40, 60, 68, or 86, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 32, 41, 61, 69, or 87, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 8 or (b) (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 3, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 4, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 5, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 13, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 14, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 15, and such protein binds to ActRIIB and ActRIIA, or has a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0019] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 31, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 32, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 33, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0020] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 40, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 41, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 42, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0021] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 50, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 51, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 60, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 61, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 62, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0022] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 68, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 69, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 70, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0023] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 having the amino acid sequence of SEQ ID NO: 78, (ii) VH-CDR2 having the amino acid sequence of SEQ ID NO: 79, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 86, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 87, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 88, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0024] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and ActRIIA and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 3, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 4, (iii) (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 13, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 14, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 15, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0025] In a further embodiment, the ActRII binding protein specifically binds to ActRII and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (a) (i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 24, 53, or 81, (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 25, 54, 55, or 82, (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 26, 56, 57, or 83, (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 34, 43, 63, 71, or 89, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 35, 44, 64, 72, or 90, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: or (b) (i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 6, (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 7 or 8, (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 9 or 10, (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 16, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 17, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 18, and such protein binds to ActRIIB and ActRIIA, or has a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0026] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 24, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 25, and (iii) VH-ABR3 having the amino acid sequence of SEQ ID NO: 26. (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 34, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 35, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 36, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0027] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 24, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 25, and (iii) VH-ABR3 having the amino acid sequence of SEQ ID NO: 26. (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 43, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 44, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 45, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0028] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 53, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 54 or 55, and (iii) VH- (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 63, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 64, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 65, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0029] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 24, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 25, and (iii) VH-ABR3 having the amino acid sequence of SEQ ID NO: 26. (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 71, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 72, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 73, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0030] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 81, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 82, and (iii) VH- (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 89, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 90, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 91, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0031] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and ActRIIA and comprises a set of ABRs, i.e., VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, i.e., (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 6, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 7 or 8, and (iii) ) VH-ABR3 has the amino acid sequence of SEQ ID NO: 9 or 10, (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 16, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 17, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 18, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0032] In some embodiments, the ActRII binding protein specifically binds to ActRII and comprises a VH and VL pair selected from the group consisting of: (a) (i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20, 49, or 77, and (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, 39, 59, 67, or 85, wherein the protein binds to ActRIIB; and (b) (i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, and (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, wherein the protein binds to ActRIIB and ActRIIA.

[0033] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:30.

[0034] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:39.

[0035] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:49 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:59.

[0036] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 67.

[0037] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:77 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:85.

[0038] In some embodiments, the ActRII binding protein specifically binds to ActRIIB and ActRIIA and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12.

[0039] In further embodiments, the ActRIIB binding protein (a) competes with an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10) for binding to ActRII; (b) reduces phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRII in the presence of an ActRII ligand (e.g., activin A or GDF8); (c) reduces phosphorylation of ALK4 and / or ALK7 in cells expressing ActRII and ALK4 and / or ALK7 in the presence of an ActRII ligand; and (d) binds ActRII with a K of 1 nM or less and 1 pM or more. D(e.g., as determined by BIACORE® analysis). In some embodiments, the ActRII binding protein has two, three, or four of the above properties. In some embodiments, the ActRII binding protein has at least two or at least three of the above properties.

[0040] In additional embodiments, the ActRII binding protein competes for binding to ActRII with an antibody comprising a VH and VL sequence pair disclosed herein. In some embodiments, the ActRII binding protein binds to the same epitope as an ActRII binding protein disclosed herein.

[0041] In some embodiments, the ActRII binding protein is an antibody that specifically binds to ActRII. In additional embodiments, the antibody is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the ActRII binding protein is an ActRII binding antibody fragment. In some embodiments, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab')2, Fv, a diabody, a DART, and a single chain antibody molecule (e.g., BiTE).

[0042] Nucleic acids and sets of nucleic acids encoding ActRII binding proteins are also provided.Vectors and sets of vectors containing the nucleic acids and sets of nucleic acids, and host cells transformed with the nucleic acids and vectors are further provided.In some embodiments, the host cells are mammalian host cells, such as hybridomas or NS0 mouse myeloma cells, PER.C6® human cells, or Chinese hamster ovary (CHO) cells.Host cells, including mammalian host cells and hybridomas, that produce ActRII binding proteins are also provided.

[0043] Also provided is a method for producing ActRII binding protein.In some embodiments, the method comprises culturing a host cell capable of expressing ActRII binding protein under suitable conditions for expressing such protein, and optionally isolating the expressed ActRII binding protein.Also provided is the ActRII binding protein prepared and / or isolated using the method disclosed herein or other methods known in the art.

[0044] Further provided is a pharmaceutical composition comprising an ActRII binding protein and a pharma- ceutically acceptable carrier. In some embodiments, the present disclosure provides a method for treating and / or ameliorating a condition in a subject associated with elevated ActRII expression or ActRII-mediated signaling. In some embodiments, the method reduces ActRII-mediated signaling in the subject. Also provided is the use of an ActRII binding protein (e.g., an anti-ActRIIB and / or ActRIIA binding antibody) provided herein in the manufacture or preparation of a medicament. In some embodiments, the medicament is for treating and / or ameliorating a condition in a subject associated with elevated ActRII expression or ActRII-mediated signaling. In additional embodiments, the present disclosure provides the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating a disease or condition described herein.

[0045] Conditions that may be treated and / or ameliorated in a subject using the provided methods include, but are not limited to, muscle disorders such as degenerative muscle diseases, muscular dystrophies, muscle atrophy, or muscle wasting disorders; fibrotic conditions (e.g., fibrotic conditions of the liver, lung, blood vessels, and / or eye, such as myocardial fibrosis, and idiopathic pulmonary fibrosis (IPF)); metabolic diseases (e.g., type II diabetes insulin resistance, hyperglycemia, and obesity); inflammatory diseases or conditions, autoimmune diseases, cardiovascular diseases (e.g., congestive heart failure and hypertension); eye diseases such as age-related macular degeneration; pulmonary diseases, musculoskeletal diseases, skeletal diseases, such as osteoporosis; neurological diseases; neuromuscular diseases, degenerative diseases, wound healing; weight loss; and cancer (e.g., carcinoma, myeloma, bone loss-induced cancer, pituitary cancer, and gastrointestinal cancer).

[0046] In some embodiments, the disclosed method comprises administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of ActRII binding protein.In some embodiments, the ActRII binding protein is administered alone.In other embodiments, the ActRII binding protein is administered as a combination therapy.In further embodiments, the ActRII binding protein is administered as a combination therapy to a standard treatment / therapy.

[0047] Methods of inhibiting or reducing ActRII activity (e.g., ligand binding and / or signaling) are also provided. In some embodiments, the methods include contacting an ActRII binding protein with a cell expressing ActRII. In some examples, the methods include contacting an ActRII binding protein with a cell expressing ActRII in the presence of an ActRII ligand (e.g., activin A). In some embodiments, the methods are performed in vivo. In other embodiments, the methods are performed in vitro. In some embodiments, the ActRII activity that is inhibited or reduced is phosphorylation of ActRI. In further embodiments, the phosphorylated ActRI is ALK4 and / or ALK7. In additional embodiments, the ActRII activity that is inhibited or reduced is phosphorylation of Smad (e.g., Smad2 and / or Smad3). In some embodiments, the present disclosure provides a method of inhibiting or reducing ActRII activity in a subject, comprising administering an effective amount of an ActRII binding protein to a subject in need thereof. In some embodiments, a method for reducing ActRIIA activity in a subject is provided, comprising administering an effective amount of an ActRIIA binding protein to a subject in need thereof. In some embodiments, a method for reducing ActRIIB activity in a subject is provided, comprising administering an effective amount of an ActRIIB binding protein to a subject in need thereof.

[0048] Also provided are methods of blocking or reducing ActRII activity in pathological conditions associated with increased ActRII expression and / or ActRII signaling, or in pathological conditions that can be treated and / or ameliorated by reducing or inhibiting the activity of an ActRII ligand. In some cases, the method comprises administering an ActRII binding protein to a subject with increased expression of ActRII or an ActRII ligand. In some embodiments, the pathological condition is a muscle disorder. In further embodiments, the muscle disorder is a wasting dystrophy or a muscular dystrophy. In some embodiments, the pathological condition is a metabolic condition, such as obesity or type II diabetes. In some embodiments, the pathological condition is a fibrotic condition of the lung or liver. In additional embodiments, the pathological condition is a cancer. In further embodiments, the cancer is myelofibrosis, myeloma (e.g., multiple myeloma), pituitary cancer, breast cancer, gastrointestinal cancer, or carcinoma. In additional embodiments, the pathological condition is a bone loss-inducing cancer (e.g., prostate and breast cancer). In some embodiments, the disclosure provides methods of blocking or reducing ActRII activity in pathological conditions associated with cancer therapy-induced bone loss.

[0049] In some aspects, the present disclosure provides a method for treating and / or ameliorating a muscle disorder. In some cases, the method comprises administering an ActRII binding protein (e.g., an anti-ActRII antibody) to a subject with a muscle disorder. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or ameliorating a muscle disorder, such as a wasting dystrophy or a muscular dystrophy. In other embodiments, the subject is at risk of developing a muscle disorder, such as a wasting dystrophy or a muscular dystrophy.

[0050] In some aspects, the disclosure provides a method of treating and / or ameliorating a fibrotic condition. In some cases, the method includes administering an ActRII binding protein (e.g., in a pharmaceutical composition described herein) to a subject having a fibrotic condition. In other embodiments, the subject is at risk of developing a fibrotic condition. In some embodiments, the fibrotic condition is chronic. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or ameliorating a fibrotic condition.

[0051] In some aspects, the present disclosure provides a method for reducing fibrosis in a subject.In some cases, the method comprises administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as a full-length ActRII antibody or an ActRII binding antibody fragment, and variants and derivatives thereof) to a subject with fibrosis.In some embodiments, the fibrosis is liver fibrosis or pulmonary fibrosis.Further provided is the use of the ActRII binding protein as provided herein in the manufacture of a medicament for treating or improving fibrosis.

[0052] In another aspect, the present disclosure provides a method for suppressing the loss of liver or lung function caused by fibrosis in a subject. In some embodiments, the method comprises administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as a full-length ActRII antibody and an ActRII binding fragment thereof) to a subject in need thereof. In some embodiments, the method suppresses the loss of liver function in the subject. In some embodiments, the method suppresses the loss of lung function in the subject. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present disclosure provides isolated recombinant ActRII binding proteins. In certain embodiments, the ActRII binding proteins specifically bind to ActRIIB and / or ActRIIA. In further embodiments, the ActRII binding proteins are anti-ActRII antibodies. Nucleic acids encoding the ActRII binding proteins, vectors and host cells containing the nucleic acids, and methods of making and using the ActRII binding proteins are also provided. The provided ActRII binding proteins are used for the diagnosis, treatment, and / or amelioration of diseases and conditions associated with ActRII expression and / or signaling. Such uses include, but are not limited to, preventing and / or ameliorating muscle disorders such as degenerative muscle diseases, muscular dystrophies, muscle atrophy or muscle wasting disorders; fibrotic conditions (e.g., fibrotic conditions of the liver, lung, blood vessels and / or eye, such as myocardial fibrosis and idiopathic pulmonary fibrosis (IPF)); metabolic diseases (e.g., type II diabetes and obesity); inflammatory diseases or conditions, autoimmune diseases, cardiovascular diseases (e.g., congestive heart failure and hypertension); eye diseases such as age-related macular degeneration; pulmonary diseases, musculoskeletal diseases, skeletal diseases, neurological diseases such as osteoporosis; wound healing; weight loss; and cancer (e.g., carcinoma, myeloma, bone loss-induced cancer, pituitary cancer, and gastrointestinal cancer).

[0054] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure. The headings set forth herein are not intended to be limiting for the various aspects, but may be used generally by reference to the specification. Additionally, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0055] The terms "a," "an," and "the" include plural referents unless the context in which the term is used clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more," and "at least one" are used interchangeably herein. Furthermore, when "and / or" is used herein, it should be construed that each of the two or more specified features or components, either with or without the other, is specifically disclosed. Thus, the term "and / or" when used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" when used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0056] The term "comprise" is used generally in the sense of include, i.e., permitting the presence of one or more features or components. Wherever in this specification an embodiment is described using the word "comprising," other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0057] The terms "about" and "approximately" as used in connection with numerical values ​​throughout the specification and claims refer to intervals of accuracy familiar and acceptable to those skilled in the art. Typically, such intervals of accuracy are ±10%. Alternatively, particularly in biological systems, the terms "about" and "approximately" can refer to values ​​that are within an order of magnitude of a given value, preferably within 5-fold or less, more preferably within 2-fold or less.

[0058] Numeric ranges are inclusive of the numbers defining the range.

[0059] An ActRII binding protein refers to a protein that specifically binds to ActRII (i.e., ActRIIB and / or ActRIIA), preferably to the extracellular domain of ActRII.

[0060] The terms "ActRII activin receptor type II and "ActRII" are used interchangeably and refer to activin receptor type IIA (ActRIIA) and / or activin receptor type IIB (ActRIIB), unless the context in which the term is used clearly dictates otherwise.

[0061] The terms "activin receptor type IIA", "ActRIIA receptor", and "ActRIIA" are used interchangeably herein and refer to ActRIIA (also referred to in the literature as ACVR2A, ActRIIA, ActRII, and EC 2.7.11.30). A reference sequence for human ActRIIA is provided in reference SEQ ID NO: NP_001607.1. The provided ActRIIA binding proteins bind to the extracellular domain of ActRIIA, which corresponds to amino acids 20-138 of SEQ ID NO: 92.

[0062] The terms "activin receptor type IIB", "ActRIIB receptor", and "ActRIIB" are used interchangeably and refer to ActRIIB (also referred to in the literature as ACVR2B, ActRIIB, HTX4, ErbB3 receptor, and EC 2.7.11.30). A reference sequence for human ActRIIB is provided in NCBI reference sequence NP_001097. The provided ActRIIB binding proteins bind to the extracellular domain of ActRIIB corresponding to amino acids 19-130 of SEQ ID NO:93.

[0063] The term "compete" when used in the context of an ActRII binding protein (e.g., a neutralizing antibody) means competition between antigen-binding proteins as determined by an assay in which a test antigen-binding protein (e.g., an anti-ActRII antibody or an ActRII-binding fragment thereof) prevents or inhibits a reference antigen-binding protein (e.g., a ligand, or a reference antibody) from specifically binding to a common antigen (e.g., the extracellular domain of ActRIIA or ActRIIB, or a fragment thereof). Numerous types of competitive binding assays can be used, such as direct or indirect solid-phase radioimmunoassays (RIA) (see, e.g., Moldenhauer et al., Scand. J. Immunol. 32:77-82 (1990) and Morel et al., Molec. Immunol. 25:7-15 (1988)), direct or indirect solid-phase enzyme immunoassays (EIA), direct solid-phase biotin-avidin EIA (see, e.g., Cheung, et al., Virology 176:546-552 (1990) and Kirkland et al., J. Immunol. 137:3614-3619 (1986)), and sandwich competition assays (see, e.g., Stahli et al., Methods in Enzymology 92:242-253 (1983)). Typically, such assays involve the use of purified antigen bound to a solid surface or cells (carrying either of these), an unlabeled test antigen-binding protein, and a labeled reference antigen-binding protein.

[0064] Competitive inhibition can be measured by determining the amount of label bound to a solid surface or cell in the presence of a test antigen-binding protein. Usually, the test antigen-binding protein is present in excess. Antigen-binding proteins identified in a competitive assay (competitor antigen-binding proteins) include ActRII binding proteins that bind to the same epitope as the reference ActRII binding protein, as well as ActRII binding proteins that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference ActRII binding protein so that steric hindrance occurs. Usually, when a competing ActRII (e.g., ActRIIA or ActRIIB) binding protein is present in excess, the specific binding of the reference ActRII binding protein ActRII (e.g., ActRIIA or ActRIIB) is inhibited by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some cases, the competing antigen binding protein inhibits specific binding of the reference ActRII binding protein by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99%.

[0065] The term "epitope" when used in the context of an ActRII protein refers to an ActRII (e.g., human ActRIIA, human ActRIIB, mouse ActRIIA or mouse ActRIIA) protein determinant capable of binding to an ActRII binding protein (e.g., an antibody) of the present disclosure. Epitopes usually consist of chemically active surface groups of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. The epitope of ActRII bound by an ActRII binding protein can be readily determined using techniques known in the art.

[0066] Antigen binding proteins, such as anti-ActRII binding antibodies and ActRII binding fragments, variants, or derivatives thereof disclosed herein, can be described or specified in terms of the epitope(s) or portion(s) of the antigen they recognize or specifically bind, e.g., target polypeptide. For example, a portion of ActRII that specifically interacts with the antigen binding domain of an ActRII binding protein disclosed herein is an "epitope". Epitopes can be formed either from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitope determinants can include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. An epitope typically includes at least 3, 4, 5, 6, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 amino acids in a unique spatial conformation. Epitopes can be routinely determined using methods known in the art.

[0067] The terms "inhibit," "block," "reduce," "reduce," "suppress," "antagonize," and "neutralize" are used interchangeably and refer to any statistically significant decrease in activity (e.g., ActRII ligand binding and ActRII signaling), including complete blockage of activity. For example, "inhibition" or "suppression" can refer to about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% decrease in activity compared to a control.

[0068] In some embodiments, the term "reduce" may refer to the ability of an ActRII binding protein, such as an antibody or ActRII-binding fragment thereof, to statistically significantly (e.g., p-value of 0.05 or less) reduce phosphorylation of one or more Smads (e.g., Smad2 and / or Smad3) induced by contact of a cell expressing ActRII and a type I receptor with an ActRII ligand, such as activin A, compared to the degree of Smad phosphorylation in a cell not contacted with the ActRII binding protein. Cells expressing ActRII (e.g., ActRIIB and / or ActRIIA) can be naturally occurring cells or cell lines, or can be recombinantly produced by introducing a nucleic acid encoding ActRII (e.g., ActRIIB and / or ActRIIA) into a host cell. In some embodiments, the ActRII binding protein, e.g., an ActRII antibody or an ActRII binding fragment thereof, reduces ActRII ligand-mediated phosphorylation of one or more Smads (e.g., Smad2 and / or Smad3) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or about 100%, as determined by, e.g., Western blotting followed by probing with an anti-phosphotyrosine antibody or by ELISA using other standard techniques and conditions described herein or known in the art.

[0069] In some embodiments, the ActRIIA binding protein reduces ActRIIA ligand (e.g., activin A)-mediated phosphorylation of one or more Smads (e.g., Smad2 and / or Smad3) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or about 100%, as determined using techniques described herein or other known in the art, for example, by Western blotting followed by probing with an anti-phosphotyrosine antibody, or by ELISA (e.g., P-Smad ELISA) or Smad-dependent reporter gene assay.

[0070] In additional embodiments, the ActRIIB binding protein reduces ActRIIB ligand (e.g., activin A or GDF8)-mediated phosphorylation of one or more Smads (e.g., Smad2 and / or Smad3) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or about 100%, as determined by, for example, Western blotting followed by probing with an anti-phosphotyrosine antibody, or by ELISA (e.g., P-Smad ELISA) or Smad-dependent reporter gene assay using other standard techniques and conditions described herein or known in the art.

[0071] The terms "enhance," "promote," and "agonist" are used interchangeably and refer to any statistically significant increase in activity (e.g., ActRII ligand binding and / or ActRII signaling). For example, "enhance" or "promote" can refer to an increase in activity of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a control.

[0072] The terms "antibody" and "immunoglobulin" are used interchangeably herein and include whole (full-length) antibodies and antigen-binding fragments or single chains thereof. A typical antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CHI, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FWs). Each VH and VL is composed of three CDRs and four FWs, arranged in the following order from amino-terminus to carboxy-terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Exemplary antibodies include classical antibodies, scFvs, and combinations thereof, where, for example, the scFvs are covalently linked (e.g., via a peptide bond or chemical linker) to the N-terminus or C-terminus of either the heavy and / or light chains of the classical antibody, or are interposed within the heavy and / or light chains of the classical antibody.

[0073] The terms "antibody" and "immunoglobulin" encompass intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), derivatives and variants of single-chain Fv (scFv), multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigenic determining portion of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibody exhibits the desired binding activity. Antibodies can be any of the five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, based on the identity of the heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2). The different classes of immunoglobulins have distinct and well-known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated to other molecules, such as toxins, radioisotopes, etc. The term "IgG" refers to a polypeptide belonging to a class of antibodies substantially encoded by the recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3.

[0074] The terms "ActRII antibody," "antibody that binds ActRII," or "anti-ActRII antibody" refer to an antibody that is capable of binding to ActRII (e.g., ActRIIB and / or ActRIIA, respectively) with sufficient affinity such that the antibody is useful in targeting ActRIIB and / or ActRIIA as a therapeutic or diagnostic reagent.

[0075] "Specifically binds" when used in the context of an ActRII protein generally refers to the ability of a binding protein, such as an antibody, to bind to ActRII (e.g., ActRIIB and / or ActRIIA, preferably human ActRIIA and / or human ActRIIB, preferably the extracellular domain of ActRIIB and / or ActRIIA) with greater affinity than the binding protein binds to an unrelated control protein. In some embodiments, the control protein is hen egg white lysozyme. Preferably, the binding protein binds to ActRII with an affinity that is at least 100-fold, 500-fold, or 1000-fold greater than the affinity for the control protein. Preferably, the binding protein has a binding affinity for human ActRII of 1×10 or greater, as measured using binding assays known in the art. -7 M or less or 1×10 -8 In some embodiments, binding affinity is measured using a radioimmunoassay (RIA) or BIACORE® (e.g., using ActRII (e.g., ActRIIB and / or ActRIIA) as the analyte and an ActRII binding protein as the ligand, or vice versa).

[0076] In some embodiments, the extent of binding of an ActRII binding protein (e.g., an anti-ActRII antibody) to an unrelated, non-ActRII protein is less than about 10% of the binding of that ActRII binding protein to ActRII, as measured, for example, by radioimmunoassay (RIA), BIACORE® (using recombinant ActRII as the analyte and an ActRII binding protein as the ligand, or vice versa), equilibrium exclusion assay (KINEXA®), or other binding assays known in the art. In certain embodiments, the ActRII binding protein has a dissociation constant (K D) is 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 10 pM or less, 1 pM or less, or 0.1 pM or less.

[0077] The term "antigen-binding antibody fragment" (e.g., "ActRII-binding antibody fragment", "ActRIIA-binding antibody fragment" and "ActRIIB-binding antibody fragment") refers to a fragment containing all or a portion of the antigen-binding variable region (e.g., CDR3) of an intact antibody. It is known that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from one or more antibody fragments. In some embodiments, the disclosure provides an ActRII-binding antibody fragment, where the antibody fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, or a single-chain antibody molecule.

[0078] The Fc region includes polypeptides that include the antibody constant region other than the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains for IgA, IgD, and IgG, and the last three constant region immunoglobulin domains for IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes immunoglobulin domains Cγ2 and Cγ3, and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the heavy chain Fc region of human IgG is usually defined to include residues C226 or P230 at its carboxyl terminus, where numbering is done according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc may refer to this region alone or in the context of a whole antibody, an antibody fragment, or an Fc fusion protein. Polymorphisms have been observed at several different Fc positions, including but not limited to, positions 270, 272, 312, 315, 356, and 358 as numbered in the EU index, so there may be slight differences between the presented sequence and prior art sequences.

[0079] A "monoclonal antibody" refers to a homogeneous antibody population involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to a polyclonal antibody, which typically contains different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments that contain antibody portions (such as Fab, Fab', F(ab')2, and Fv), single chain (scFv) variants, and fusion proteins), and any other modified immunoglobulin molecule that contains an antigen recognition site. Monoclonal antibodies may be produced in any number of ways, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0080] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from more than one species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and / or ability to bind to an antigen, while the constant regions are homologous to the sequences of antibodies derived from that species to avoid eliciting an immune response in another species (usually human).

[0081] The term "humanized antibody" refers to an antibody derived from a non-human (e.g., murine) immunoglobulin that has been engineered to contain few, preferably minimal, non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the CDRs are replaced with residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that has the desired specificity, affinity, and / or ability to bind to the antigen (Jones, Nature 321:522-525 (1986); Riechmann, Nature 332:323-327 (1988); Verhoeyen, Science 239:1534-1536 (1988)). In some cases, Fv framework region (FW) residues of the human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and / or ability to bind to the antigen. Humanized antibodies can be further modified by substitution of additional residues, either in the Fv framework regions and / or within the replaced non-human residues, to refine and optimize the specificity, affinity, and / or potency of the antibody. Generally, a humanized antibody contains substantially all of at least one variable domain, typically two or three variable domains, that contain all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, but all or substantially all of the FR regions are of the consensus sequence of a human immunoglobulin. A humanized antibody can also contain at least a portion of an immunoglobulin constant region or domain, typically a human immunoglobulin constant region or domain. Examples of methods used to make humanized antibodies are described in U.S. Patent Nos. 5,225,539 and 5,639,641.

[0082] The term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, made using any technique known in the art. The term "human antibody" includes intact (full-length) antibodies, fragments thereof, and / or antibodies that contain at least one human heavy and / or light chain polypeptide, such as an antibody that contains a murine light chain polypeptide and a human heavy chain polypeptide.

[0083] An "antagonist," "blocking," or "neutralizing" binding protein is one that inhibits or reduces the activity of an antigen, such as ActRIIB and / or ActRIIA, to which it binds. In some embodiments, an antagonist ActRII binding protein reduces or inhibits binding of an ActRIIA ligand, such as activin A, to ActRIIA. In some embodiments, an antagonist ActRII binding protein reduces or inhibits binding of an ActRIIB ligand, such as activin A, to ActRIIB. In certain embodiments, an antagonist ActRII binding protein substantially or completely inhibits the activity of ActRII. In some embodiments, ActRII activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or 100%. In certain embodiments, the antagonist ActRII binding protein is an anti-ActRIIA antibody, such as a full-length antibody or an ActRIIA-binding antibody fragment. In further embodiments, the antagonist anti-ActRIIA antibody inhibits or reduces the activity of ActRIIA by at least 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. In additional embodiments, the antagonist ActRII binding protein is an anti-ActRIIB antibody, such as a full-length antibody or an ActRIIB-binding antibody fragment. In further embodiments, the antagonist anti-ActRIIB antibody inhibits or reduces the activity of ActRIIB by at least 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

[0084] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein, and can be used for the purposes of this disclosure.

[0085] "Potency" is a measure of a compound's pharmacological activity expressed in terms of the amount of compound required to produce an effect of a given strength. It refers to the amount of compound required to achieve a defined biological effect; the lower the dose required, the more potent the drug. Potency is usually expressed in terms of the IC unless otherwise stated. 50 The IC values ​​are expressed as nM. 50 is the median inhibitory concentration of an ActRII binding protein (e.g., an anti-ActRIIA or anti-ActRIIB antibody). In functional assays, the IC 50 IC is the concentration that reduces a biological response by 50% of its maximum. In ligand-receptor binding studies, IC 50 is the concentration that reduces ligand-receptor binding by 50% of the maximum specific binding level. 50 can be calculated by any number of means known in the art. The fold improvement in potency of an antibody or other binding protein provided herein can be at least 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, or at least 180-fold when compared to a reference anti-ActRII antibody or other ActRII binding protein.

[0086] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Specific high-affinity IgG antibodies directed to the surface of the target cells are absolutely required for such killing, since they "arm" the cytotoxic cells. Lysis of the target cells is extracellular, requires direct cell-cell contact, and does not involve complement. In addition to antibodies, it is contemplated that other proteins containing an Fc region, particularly Fc fusion proteins, capable of specific binding to target cells bearing ActRII, can effect cell-mediated cytotoxicity. For simplicity, cell-mediated cytotoxicity resulting from the activity of Fc fusion proteins is also referred to herein as ADCC activity.

[0087] An "isolated" ActRII binding protein (e.g., an ActRII antibody, including its ActRII binding fragments, variants, and derivatives), polynucleotide, vector, cell, or composition is a protein (e.g., an antibody), polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated proteins, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated protein, polynucleotide, vector, cell, or composition is substantially pure. Isolated proteins and isolated nucleic acids are free or substantially free from materials with which they are naturally associated, such as other polypeptides or nucleic acids found with them in their natural environment or in the preparation environment (e.g., cell culture) when they are prepared by recombinant DNA techniques performed in vitro or in vivo. Proteins and nucleic acids may be formulated with a diluent or adjuvant, and may even be isolated for practical purposes; for example, proteins are usually mixed with gelatin or other carriers when used to coat microtiter plates for use in immunoassays, or with a pharma- ceutically acceptable carrier or diluent when used diagnostically or therapeutically.

[0088] The terms "subject," "individual," "animal," "patient," and "mammal" refer to any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, non-human primates, domestic animals, livestock, rodents, etc., who are the intended recipient of a particular treatment.

[0089] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient is in a form such that it can be effective, and which does not contain any additional components at concentrations that are unacceptably toxic to the subject to which the composition is intended to be administered. Such compositions may be sterile.

[0090] An "effective amount" of a polypeptide, e.g., an antigen-binding protein, such as an antibody, as disclosed herein, is an amount sufficient to carry out a specifically described purpose. An "effective amount" can be determined empirically and in a routine manner in relation to the described purpose. The term "therapeutically effective amount" refers to an amount of a polypeptide, e.g., an antigen-binding protein, such as an antibody, or other drug, that is effective to "treat" a disease or condition in a subject (e.g., a mammal, such as a human), and that provides some improvement or benefit to a subject having such disease or condition. Thus, a "therapeutically effective" amount is an amount that provides some relief, alleviation, and / or reduction of at least one clinical symptom of an ActRII-mediated disease or condition. The clinical symptoms associated with diseases or conditions that can be treated by the methods of the present disclosure are well known. Moreover, the therapeutic effect need not be complete or curative, so long as some benefit is provided to the subject. In some embodiments, the term "therapeutically effective" refers to an amount of a therapeutic agent that is capable of reducing ActRII activity in a patient in need thereof. The actual amount administered, and the rate and time-course of administration, will vary depending on the nature and severity of what is being treated. Prescribing treatment, e.g., determining dosage, etc., is within the responsibility of general practitioners and other physicians. Appropriate doses of antibodies and antigen-binding fragments thereof are generally known, see Ledermann et al., Int. J. Cancer 47:659-664 (1991); Bagshawe et al., Ant. Immun. and Radiopharm. 4:915-922 (1991).

[0091] A "sufficient amount" or "amount sufficient" to achieve a particular result in a patient having an ActRII-mediated disease or condition refers to an amount of a therapeutic agent (e.g., an antigen binding protein, such as an antibody, as disclosed herein) that is effective (i.e., by administration of a therapeutically effective amount) to produce a desired effect, optionally a therapeutic effect. In some embodiments, such a particular result is a reduction in ActRII activity in a patient in need thereof.

[0092] The term "label" refers to a detectable compound or composition that is attached directly or indirectly to a moiety, such as an anti-ActRII antibody, to produce a "labeled" moiety. The label may itself be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0093] The terms "treating" or "treatment", "treat" or "improving" and "ameliorating" refer to both (a) therapeutic measures that cure, slow, alleviate, and / or halt the progression of a diagnosed pathological condition or disorder, and (b) prophylactic or preventative measures that prevent and / or slow the onset of the targeted disease or condition. Thus, subjects in need of treatment include those already with a disease or condition, those at risk of developing a disease or condition, and those in whom a disease or condition is to be prevented. In certain embodiments, a subject has been successfully "treated" according to the methods provided herein when the subject exhibits, for example, a total, partial, or transient improvement or disappearance of symptoms associated with the disease or condition. In some embodiments, the present disclosure provides methods for treating muscle disorders, such as muscle wasting due to disease or disuse. In additional embodiments, the disclosure provides a method for treating a disease or condition selected from a muscle disorder, such as a degenerative muscle disease, a muscular dystrophy, a muscle atrophy, or a muscle wasting disorder; a fibrotic condition (e.g., a fibrotic condition of the liver, lung, blood vessels, and / or eye, such as myocardial fibrosis, and idiopathic pulmonary fibrosis (IPF)); a metabolic disease (e.g., type II diabetes and obesity); an inflammatory disease or condition, an autoimmune disease, a cardiovascular disease (e.g., congestive heart failure, and hypertension); an eye disease, such as age-related macular degeneration; a pulmonary disease, a musculoskeletal disease, a skeletal disease, a neurological disease, such as osteoporosis; wound healing; weight loss; and cancer (e.g., carcinoma, myeloma, bone loss-inducing cancer, pituitary cancer, and gastrointestinal cancer). In a further embodiment, the disclosure provides a use of an ActRII binding protein as provided herein in the manufacture of a medicament for the treatment or amelioration of one or more of the above diseases or conditions.

[0094] As used herein, "in combination with" or "combination therapy" refers to any form of administration in which an additional therapy (e.g., second, third, fourth, etc.) is more effective in the body (e.g., multiple compounds are effective in a subject simultaneously, which may include synergistic effects of the compounds). Efficacy may not correlate with measurable concentrations of the agents in blood, serum, or plasma. For example, different therapeutic compounds can be administered in the same formulation or in separate formulations, simultaneously or sequentially, on different schedules. Thus, subjects receiving such treatment can benefit from the combined effects of the different therapies. One or more ActRII binding proteins of the present disclosure can be administered simultaneously with, prior to, or after one or more other additional agents and / or supportive therapies. In general, each therapeutic agent is administered at a dose and / or time schedule determined for that particular agent. The particular combination used in the regimen takes into account the compatibility of the antagonist of the present disclosure with the treatment and / or desired outcome.

[0095] The methods and techniques of the present disclosure are generally carried out according to known conventional methods, unless otherwise indicated, as described in various general and more detailed references cited and discussed throughout this disclosure.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), all of which are incorporated herein by reference.

[0096] The terms "cancer," "tumor," "cancerous," and "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas such as adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's lymphoma and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer (including hormone-induced breast cancer; see, for example, Innes et al., Br. J. Cancer 94:1057-1065 (2006)), colon cancer, colorectal cancer, endometrial cancer, myeloma (multiple myeloma, etc.), salivary gland cancer, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, various head and neck cancers, and cancers that originate from mucus, such as mucinous ovarian cancer, and bile duct cancer (liver). In certain embodiments, the cancer is myelofibrosis, myeloma (e.g., multiple myeloma), or pituitary cancer. In other embodiments, the cancer is breast cancer, gastrointestinal cancer, or carcinoma (e.g., basal cell carcinoma and squamous cell carcinoma). In additional embodiments, the cancer is a bone loss-inducing cancer.

[0097] The terms "polynucleotide" and "nucleic acid" are used interchangeably and are intended to encompass single and multiple nucleic acids, and refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA). In certain embodiments, polynucleotides contain normal phosphodiester bonds or non-normal bonds (e.g., amide bonds as found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA, cDNA, or RNA fragments. When applied to a nucleic acid or polynucleotide, the term "isolated" refers to a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment, e.g., a recombinant polynucleotide encoding an antigen-binding protein contained within a vector is considered isolated for purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or in solution purified (partially or substantially) from other polynucleotides. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically.In addition, a polynucleotide or nucleic acid can include regulatory elements, such as a promoter, enhancer, ribosomal binding site, or transcription termination signals.

[0098] The term "vector" refers to a construct capable of delivering, and in some embodiments, expressing, one or more gene(s) or sequence(s) of interest into a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0099] The term "host cell" refers to a cell or population of cells that contains or is capable of containing a recombinant nucleic acid. Host cells can be prokaryotic (e.g., E. coli) or eukaryotic. Host cells can be fungal cells, including yeasts such as Saccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomyces pombe. Host cells can also be any of a variety of animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS-7, NIH-3T3, NS0, PER.C6®, and hybridomas). In further embodiments, the host cell is a CHO-K, CHO-0 CHO-Lec10, CHO-Lec13, CHO-Lec1, CHO Pro - 5, and CHO dhfr - In certain embodiments, the host cell is a CHO cell selected from the group consisting of:

[0100] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. Such terms also include amino acid polymers that are modified naturally or by intervention, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within this definition are, for example, polypeptides that contain one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. In some embodiments, because the ActRII binding proteins provided are based on antibodies, it is understood that the ActRII binding proteins can occur as single chains or linked chains.

[0101] A "recombinant" polypeptide, protein, or antibody refers to a polypeptide, protein, or antibody produced by recombinant DNA technology. Recombinantly produced, host cell-expressed polypeptides, proteins, and antibodies are considered isolated for the purposes of this disclosure, as are naturally occurring or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0102] The present disclosure also includes fragments, variants, or derivatives of polypeptides, and any combination thereof. The term "fragment" when referring to polypeptides and proteins includes any polypeptide or protein that retains at least some of the properties of the reference polypeptide or protein. Polypeptide fragments include proteolytic fragments and deletion fragments.

[0103] The term "variant" refers to an antibody or polypeptide sequence that differs from that of a parent antibody or polypeptide due to at least one amino acid modification. Antibody or polypeptide variants include fragments, as well as antibodies or polypeptides in which the amino acid sequence has been altered due to amino acid substitution, deletion, or insertion. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be made using mutagenesis techniques known in the art. Variant polypeptides may include conservative or non-conservative amino acid substitutions, deletions, or additions.

[0104] The term "derivative" when applied to an antibody or polypeptide refers to an antibody or polypeptide that has been modified so as to exhibit additional characteristics not found in the native antibody or polypeptide. One example of a "derivative" antibody is a fusion or conjugate with a second polypeptide or another molecule (e.g., a polymer such as PEG, a chromophore, or a fluorophore) or atom (e.g., a radioisotope).

[0105] The term "amino acid substitution" refers to the replacement of an amino acid residue present in a parent sequence with another amino acid residue. The amino acid can be replaced in the parent sequence, for example, by chemical peptide synthesis or by known recombinant methods. Thus, a reference to a "substitution at position X" or a "substitution at position X" refers to the replacement of the amino acid residue present at position X with an alternative amino acid residue. In some embodiments, the substitution pattern can be represented according to the scheme AXY, where A is the one-letter code corresponding to the amino acid residue naturally present at position X, and Y is the replacement amino acid residue. In other embodiments, the substitution pattern can be represented according to the scheme XY, where Y is the one-letter code corresponding to the amino acid residue that replaces the amino acid residue naturally present at position X.

[0106] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been previously defined and include basic side chains (e.g., Lys, Arg, His), acidic side chains (e.g., Asp, Glu), uncharged polar side chains (e.g., Gly, Asp, Gln, Ser, Thr, Tyr, Cys), non-polar side chains (e.g., Ala, Val, Leu, Ile, Pro, Phe, Met, Trp), beta-branched side chains (e.g., Thr, Val, Ile), and aromatic side chains (e.g., Tyr, Phe, Trp, His). Thus, if an amino acid residue in a polypeptide is replaced with another amino acid residue from the same side chain family, the substitution is considered conservative. In other embodiments, a set of amino acid residues can be conservatively replaced with a set of structurally similar side chain family members that differ in order and / or composition.

[0107] Non-conservative substitutions include (a) a residue having a positively charged side chain (e.g., Arg, His, or Lys) is substituted for or substituted with a negatively charged residue (e.g., Glu or Asp); (b) a hydrophilic residue (e.g., Ser or Thr) is substituted for or substituted with a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (c) Cys or Pro is substituted for or substituted with any other residue; or (d) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) is substituted for or substituted with a residue having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

[0108] Other substitutions can be easily identified. For example, for the amino acid alanine, the substitution can be any one of D-Ala, Gly, beta-Ala, L-Cys, and D-Cys. For lysine, the substitution can be any one of D-Lys, Arg, D-Arg, homo-Arg, Met, D-Met, omithine, or D-ornithine. In general, substitutions in functionally important regions that may be expected to induce changes in the properties of the isolated polypeptide include (a) polar residues (e.g., Ser or Thr) substituted for (or substituted by) hydrophobic residues (e.g., Leu, Ile, Phe, or Ala); (b) Cys residues substituted for (or substituted by) any other residue; (c) residues with positively charged side chains (e.g., Lys, Arg, or His) substituted for (or substituted by) residues with negatively charged side chains (e.g., Glu or Asp); or (d) residues with bulky side chains (e.g., Phe) substituted for (or substituted by) those without such side chains (e.g., Gly). The likelihood that one of the aforementioned non-conservative substitutions may alter the functional properties of a protein also correlates with the location of the substitution with respect to the functionally important regions of the protein, such that some non-conservative substitutions may have little or no effect on biological properties.

[0109] The term "amino acid insertion" refers to the introduction of a new amino acid residue between two amino acid residues present in a parent sequence. The amino acid residue can be inserted into the parent sequence, for example, by chemical peptide synthesis or by recombinant methods known in the art. Thus, in the phrase "insertion between positions X and Y" or "insertion between positions X and Y according to Kabat", X and Y correspond to the position of the amino acid residue (e.g., insertion between positions 239 and 240 of the cysteine ​​amino acid residues), and such phrase refers to the insertion of an amino acid residue between positions X and Y, and also to the insertion of a codon encoding an amino acid residue in a nucleic acid sequence between the codon encoding the amino acid residue at position X and the codon encoding the amino acid residue at position Y.

[0110] The term "percent sequence identity" or "percent identity" between two sequences of polynucleotides or polypeptides refers to the number of identical matching positions shared by the sequences over the entire comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment between the two sequences. A matching position is any position where an identical nucleotide or amino acid is presented in both the target sequence and the reference sequence. Gaps are not counted in the target sequence, since gaps are neither nucleotides nor amino acids. Similarly, gaps are not counted in the reference sequence, since nucleotides or amino acids of the target sequence are counted, not nucleotides or amino acids from the reference sequence. The percentage of sequence identity can be calculated by determining the number of positions where identical amino acid residues or nucleic acid bases occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percent identity between two sequences can be accomplished using readily available software programs. Suitable software programs are available from a variety of sources and are available for both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST package of programs available from the US government's National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses the BLASTN or BLASTP algorithm to perform a comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS package of bioinformatics programs, also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0111] The structure for carrying the CDR or set of CDRs is generally that of an antibody heavy or light chain sequence or a substantial portion thereof, with the CDR or set of CDRs located at a position corresponding to the CDR or set of CDRs of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structure and position of immunoglobulin variable domains and their CDRs can be readily determined by those skilled in the art using programs and known variable domain residue numbering systems such as Chothia, Chothia+, and Kabat, which can be routinely determined by reference to Kabat (Kabat et al., Sequences of Proteins of Immunological Interest. 4th Edition. USDHHS. 1987, which is incorporated herein by reference in its entirety, and tools available on the Internet (e.g., at bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.html, and immuno.bme.nwu.edu).

[0112] The CDRs can also be carried by other scaffolds, such as fibronectin, cytochrome B, albumin (e.g., ALBUdAb (Domantis / GSK) and ALB-Kunitz (Dyax)), unstructured repeat sequences of three or six amino acids (e.g., PASylation® technology and XTEN® technology), and sequences containing elastin-like repeat domains (see, e.g., U.S. Patent Application No. 61 / 442,106, which is incorporated by reference in its entirety).

[0113] The CDR amino acid sequences substantially as set out herein may be retained as a CDR in a human variable domain or a substantial portion thereof. The HCDR3 sequences substantially as set out herein represent embodiments of the present disclosure, each of which may be retained as an HCDR3 in a human heavy chain variable domain or a substantial portion thereof.

[0114] The variable domains used in this disclosure can be derived from any germline or rearranged human variable domain, or can be synthetic variable domains based on consensus sequences of known human variable domains. CDR sequences (e.g., CDR3) can be introduced into a repertoire of variable domains lacking a CDR (e.g., CDR3) using recombinant DNA techniques.

[0115] For example, Marks et al. (Bio / Technology 10:779-783 (1992), incorporated herein by reference in its entirety) provide a method for generating a repertoire of antibody variable domains in which a consensus primer directed at or adjacent to the 5' end of the variable domain region is used in conjunction with a consensus primer to the third framework region of the human VH gene to obtain a repertoire of VH variable domains lacking CDR3. Marks et al. further describe how this repertoire can be combined with the CDR3 of a particular antibody. Using similar techniques, the CDR3-derived sequences of the present disclosure can be shuffled with a repertoire of VH or VL domains lacking CDR3, and the completed shuffled VH or VL domains can be combined with cognate VL or VH domains to obtain an antigen-binding protein. The repertoire can then be displayed in a suitable host system, such as any of the phage display systems described in International Publication No. WO 92 / 01047, or the plethora of subsequent publications, such as Kay et al., (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press, so that suitable antigen-binding proteins can be selected. Repertoires can be displayed in a variety of formats, such as from 10 distinct members or more, e.g., from 10 6 ~10 8 , or 10 10The host cell may be any of the members of the family. Other suitable host systems include yeast display, bacterial display, T7 display, and ribosome display. For a review of ribosome display, see Lowe et al., Curr. Pharm. Biotech. 517-527 (2004) and International Publication No. WO92 / 01047, both of which are incorporated herein by reference in their entireties. A similar shuffling or combinatorial technique has also been disclosed by Stemmer (Nature 370:389-391 (1994)), who describes the technique in the context of a β-lactamase gene, but observes that the technique can be used to generate antibodies.

[0116] The term "antigen-binding region" or "ABR" refers to the paratope, which is the residue within an antibody that recognizes and binds an antigen (Ag). The ABR or set of ABRs is generally of an antibody heavy or light chain sequence or a substantial portion thereof, and the ABR or set of ABRs is located at a position corresponding to the ABR or set of ABRs of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structure and location of immunoglobulin variable domains and their ABRs can be readily determined by one of skill in the art using programs and known variable domain residue numbering systems and tools available on the internet, which are incorporated herein by reference in their entirety (e.g., Kunik V., et al., Nucleic Acids Res. 2012 July; 40).

[0117] An ActRII binding protein (e.g., an anti-ActRIIA antibody and an anti-ActRIIB antibody) is said to "compete" with a reference molecule for binding to ActRII if it binds to ActRII to an extent that it blocks the binding of the reference molecule to ActRII (e.g., ActRIIB and / or ActRIIA, respectively) to some extent. The ability of proteins to compete for binding to ActRII and thus prevent, block, or "cross-block" each other from binding to ActRII can be determined by any standard competitive binding assay known in the art, including, for example, a competitive ELISA assay, surface plasmon resonance (SPR; BIACORE®, Biosensor, Piscataway, NJ) or according to the method described by Scatchard et al. (Ann. NY Acad. Sci. 51:660-672 (1949)). ActRII binding protein may be said to competitively inhibit the binding of a reference molecule to ActRII, for example, by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.According to some embodiments, ActRII binding protein competitively inhibits the binding of a reference molecule to ActRIIA by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.According to other embodiments, ActRII binding protein competitively inhibits the binding of a reference molecule to ActRIIB by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0118] ActRII-binding protein Proteins that specifically bind to ActRII are provided. The term "ActRII" refers to activin receptor type IIA (ActRIIA) and / or activin receptor type IIB (ActRIIB).

[0119] As used herein, the term "ActRIIA" refers to a family of activin receptor type IIA proteins from any species and variants derived from such ActRIIA proteins by mutagenesis or other modifications. References to ActRIIA herein are understood to be references to any one of the currently identified forms. Members of the ActRIIA family are generally transmembrane proteins, composed of an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic serine-threonine kinase domain. There are various naturally occurring isoforms of human ActRIIA. The sequence of the standard human ActRIIA isoform 1 precursor protein (NCBI reference sequence NP_001265508.1) is as follows: [ka] The signal peptide is shown in single underline and the extracellular domain is shown in bold font.

[0120] As used herein, the term "ActRIIB" refers to a family of activin receptor type IIB proteins from any species and variants derived from such ActRIIB proteins by mutagenesis or other modifications. References herein to ActRIIA are understood to be references to any one of the currently identified forms. Members of the ActRIIB family are generally transmembrane proteins, composed of an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic serine-threonine kinase domain. There are various naturally occurring isoforms of human ActRIIB. The sequence of the standard human ActRIIB isoform 1 precursor protein (NCBI reference sequence NP_001097.2) is as follows: [ka] The signal peptide is shown in single underline and the extracellular domain is shown in bold font.

[0121] In some embodiments, the ActRII binding protein binds to ActRII with an affinity that is at least 100-fold, 500-fold, or 1000-fold greater than the affinity of the ActRII binding protein for a control protein that is not a TGF-beta receptor family member. In certain embodiments, the ActRII binding protein binds to ActRII with a dissociation constant (K D In some embodiments, the ActRII binding protein has a K for human ActRII of less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM, or less than 0.1 pM. D is within the range of 1 μM or less and 0.1 pM or more, 100 μM or less and 0.1 pM or more, or 100 μM or less and 1 pM or more.

[0122] In some embodiments, BIACORE® analysis is used to determine the ability of an ActRII binding protein (e.g., an anti-ActRII antibody) to compete / block binding to an ActRII protein by a reference ActRII binding protein (e.g., an anti-ActRII antibody). In a further aspect, the BIACORE® instrument (e.g., a BIACORE® 3000) is operated according to the manufacturer's recommendations, and ActRII-Fc fusion protein is captured on a CM5 BIACORE® chip with pre-bound anti-niFc IgG to generate an ActRII-coated surface. Typically, 200-800 resonance units of ActRII-Fc (dimer) will be coupled to the chip (an amount that readily provides measurable levels of binding, but is easily saturable by the concentration of test reagents used).

[0123] Two ActRII binding proteins (A * and B. *A test mixture is prepared by mixing ActRII binding proteins (i.e., ActRII binding sites ... * and B. * The concentration of ActRII-Fc in the mixture should be high enough to easily saturate the binding sites for that ActRII-binding protein on the ActRII-Fc molecule captured on the BIACORE® chip. * and B. * are at the same molar concentration (based on binding), which would typically be 1.00-1.5 micromolar (based on binding sites). * Alone and ActRII-binding protein B * Separate solutions containing ActRII binding protein A alone are also prepared. * and ActRII binding protein B * should be at the same concentration in the same buffer as in the test mixture. The test mixture is passed over a BIACORE® chip coated with ActRII-Fc and the total amount bound is recorded. The chip is then treated to remove the bound ActRII binding protein without damaging the chip-bound ActRII-Fc. Typically, this is done by treating the chip with 30 mM HC1 for 60 seconds. The ActRII binding protein A is then removed. * A single solution is passed over the ActRII-Fc coated surface and the amount of binding is recorded. The chip is treated again to remove bound antibody without damaging the chip-bound ActRII-Fc. ActRII binding protein B is then * A single solution is passed over the ActRII-Fc coated surface and the amount of binding is recorded. ActRII binding protein A * and ActRII-binding protein B *The theoretical maximum binding of the mixture is then calculated, which is the sum of the binding of each ActRII binding protein when passed alone over the ActRII surface. If the actual recorded binding of the mixture is less than this theoretical maximum, then the two ActRII binding proteins compete / block each other. Thus, in general, an inhibitory ActRII binding protein is one that binds to ActRII in the BIACORE® inhibition assay described above such that the recorded binding during the assay and in the presence of a second ActRII binding protein is between 80% and 0.1% (e.g., less than 80% to 4%) of the theoretical maximum binding (as defined above) of the two ActRII binding proteins combined, specifically between 75% and 0.1% (e.g., between 75% and 4%) of the theoretical maximum binding, and more specifically between 70% and 0.1% (e.g., between 70% and 4%) of the theoretical maximum binding.

[0124] The BIACORE® assay described above is an exemplary assay used to determine whether two ActRII binding proteins, such as anti-ActRII antibodies, compete / block each other for binding to ActRII. In rare cases, a particular ActRII binding protein may not bind to ActRII-Fc that is bound to a CM5 BIACORE® chip via anti-Fc IgG (this can occur if the relevant binding site on ActRII is masked or destroyed by the linkage of ActRII to Fc). In such cases, blocking can be determined using a tagged version of ActRII, for example, C-terminal His-tagged ActRII. In this particular format, an anti-His antibody is bound to the BIACORE® chip, and then the His-tagged ActRII is passed over the chip surface and captured by the anti-His antibody. The cross-blocking analysis is essentially performed as described above, except that after each chip regeneration cycle, the anti-His antibody-coated surface is again loaded with fresh His-tagged ActRII. Additionally, various other known tag and tag binding protein combinations can be used in such blocking assays (e.g., HA tag and anti-HA antibody; FLAG tag and anti-FLAG antibody; biotin tag and streptavidin).Outlined below is an ELISA assay to determine whether an ActRII binding protein blocks or is capable of blocking binding of a reference ActRII binding protein to ActRII.

[0125] In some embodiments, ELISA is used to determine the ability of an ActRII binding protein (e.g., an anti-ActRII antibody) to compete with a reference ActRII binding protein (e.g., an anti-ActRII antibody or an ActRII ligand) for binding to an ActRII protein. The general principle of such an assay is to coat a reference ActRII binding protein (e.g., an anti-ActRII antibody) onto the wells of an ELISA plate. An excess of a potentially inhibitory second test ActRII binding protein is added in solution (i.e., not bound to the ELISA plate). A limited amount of ActRII (or alternatively ActRII-Fc) is then added to the wells. The coated reference ActRII binding protein and the test ActRII binding protein in solution compete for binding of a limited number of ActRII (or ActRII-Fc) molecules. The plate is washed to remove ActRII that is not bound by the coated reference ActRII binding protein, and also to remove the liquid-phase test ActRII binding protein and the complex formed between the liquid-phase test ActRII binding protein and ActRII.The amount of bound ActRII is then measured using a suitable ActRII detection reagent.A test ActRII binding protein in solution that can block the binding of the coated reference ActRII binding protein to ActRII can cause a decrease in the number of ActRII molecules that the coated reference ActRII binding protein can bind to compared to the number of ActRII molecules that the coated reference ActRII binding protein can bind in the absence of a second liquid-phase test ActRII binding protein.The background signal of the assay is defined as the signal obtained in wells that contain the coated reference ActRII binding protein, the liquid-phase test ActRII binding protein, ActRII buffer only (i.e., no ActRII) and the ActRII detection reagent.The positive control signal of the assay is defined as the signal obtained in wells containing coated reference ActRII binding protein, solution-phase test ActRII binding protein buffer only (i.e., no solution-phase test ActRII binding protein), ActRII and ActRII detection reagent. The ELISA assay is performed to obtain a positive control signal at least three times the background signal. As a control for method artifacts, cross-blocking assays can be performed in the format described above or vice versa, using the test ActRII binding protein as the coated antibody and the reference ActRII binding protein as the solution-phase antibody.

[0126] In some embodiments, a reporter gene assay is used to determine the ability of an ActRII binding protein (e.g., an anti-ActRII antibody) to neutralize ActRII (e.g., ActRIIB). In some embodiments, a reporter gene assay is performed using recombinant A204 cells to determine the ability of an ActRII binding protein (e.g., an anti-ActRII antibody) to neutralize ActRII (e.g., ActRIIB) activity. This assay is based on a human rhabdomyosarcoma cell line transfected with a pGL3(CAGA)12 plasmid containing a (CAGA)12 motif (see, e.g., Dennler et al., EMBO 17:3091-3100 (1998) and U.S. Patent No. 8,765,385, each of which is incorporated herein by reference in its entirety) and a ReniUa reporter plasmid (pRLCMV) to confirm transfection efficiency. Since the CAGA12 motif is present in a TGF-beta responsive gene (PAI-1 gene), this vector is of general use for factors that signal through Smad2 and Smad3. Regarding using this assay to measure the ActRIIB binding activity of candidate proteins, since the A204 cell line expresses mainly ActRIIA rather than ActRIIB, it is not possible to directly test antibodies for their potential to neutralize ActRIIB. Instead, this assay is designed to detect the ability of the tested ActRII protein binding candidate to neutralize the inhibitory effect of the soluble fusion protein ActRIIB-Fc on the activation of endogenous ActRIIA by ligands (such as activin A or GDF11) that can bind both ActRIIB and ActRIIA with high affinity. Thus, in this assay, if ActRIIB binding is neutralized, ligand-mediated ActRIIA activation occurs despite the presence of ActRIIB-Fc.

[0127] On the first day of the assay, A204 cells (ATCC HTB-82) were plated at 10 5On the second day, a solution containing 10 μg pGL3(CAGA)12, 1 μg pRLCMV, 30 μl Fugene 6 (Roche Diagnostics), and 970 μl OptiMEM (Invitrogen) is preincubated for 30 minutes and then added to McCoy's growth medium, which is then added to the plated cells (500 μl / well) and incubated overnight at room temperature. On the third day, the medium is removed and the cells are incubated for 6 hours at 37° C. with a mixture of ligands and inhibitors prepared as described below.

[0128] According to some embodiments, the neutralizing capacity of ActRII binding proteins, such as anti-ActRII antibodies, is evaluated, whereby serial dilutions of the test protein are made in a 48-well plate in a volume of 200 μl of assay buffer (McCoy's medium + 0.1% BSA). For assays evaluating the ability of candidate proteins to neutralize ActRIIB activity, an equal volume of assay buffer containing ActRIIB-Fc (200 μg / ml) is then added. The test solution is incubated at 37° C. for 30 minutes, then 400 μl of activin A (10 ng / ml) is added to all wells, and 350 μl of this mixture is added to each well of the 48-well plate of A204 cells. Each concentration of the test protein is tested in duplicate. For assays evaluating the ability of candidate proteins to neutralize ActRIIB activity, the final concentration of ActRIIB-Fc is 50 ng / ml (this is the IC of this activin A signaling inhibitor at a final concentration of 5 ng / ml of activin A). 50 After 6 hours of incubation with the test solutions, the cells are rinsed with phosphate-buffered saline containing 0.1% BSA, then lysed with passive lysis buffer (Promega E1941) and stored overnight at -70°C. On the fourth and final day, the plates are warmed to room temperature with gentle shaking. The cell lysates are transferred in duplicate to chemiluminescence plates (96 wells) and analyzed in a luminometer using reagents from the Dual-Luciferase Reporter Assay system (Promega E1980) to determine normalized luciferase activity.

[0129] The pharmacodynamic parameters that depend on ActRIIB signal transduction can be measured as the end point of the in vivo test of ActRIIB binding protein, in order to identify the binding protein that can neutralize ActRIIB and provide therapeutic benefit.ActRIIB neutralizing binding substances are defined as those that can produce statistically significant changes in such pharmacodynamic parameters compared to vehicle-treated animals.Such in vivo tests can be carried out in any suitable mammal (for example, mouse, rat, or monkey).

[0130] In some embodiments, the ActRII binding protein binds to ActRIIA with an affinity that is at least 100-fold, 500-fold, or 1000-fold greater than the affinity of the ActRII binding protein for a control protein that is not a TGF-beta receptor family member. In additional embodiments, the ActRII binding protein binds to ActRIIA with an affinity that is at least 100-fold, 500-fold, or 1000-fold greater than the affinity of the ActRII binding protein for a control protein that is not a TGF-beta receptor family member. In certain embodiments, the ActRIIA binding protein binds to ActRIIA and has a dissociation constant (K D In some embodiments, the ActRIIA binding protein has a K for human ActRIIA of less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM, or less than 0.1 pM. D is within the range of 1 μM or less and 0.1 pM or more, 100 μM or less and 0.1 pM or more, or 100 μM or less and 1 pM or more.

[0131] In some embodiments, the ActRII binding protein binds to ActRIIB with an affinity that is at least 100-fold, 500-fold, or 1000-fold greater than the affinity of the ActRII binding protein for a control protein that is not a TGF-beta family member. In additional embodiments, the ActRII binding protein binds to ActRIIB with an affinity that is at least 100-fold, 500-fold, or 1000-fold greater than the affinity of the ActRII binding protein for a control protein that is not a TGF-beta receptor family member. In certain embodiments, the ActRII binding protein binds to ActRIIB with a dissociation constant (K D In some embodiments, the ActRIIB binding protein has a K for human ActRIIB of less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM, or less than 0.1 pM. D is within the range of 1 μM or less and 0.1 pM or more, 100 μM or less and 0.1 pM or more, or 100 μM or less and 1 pM or more.

[0132] In some embodiments, the ActRII binding protein binds ActRIIB and ActRIIA with an affinity that is at least 100-fold, 500-fold, or 1000-fold greater than the affinity of the ActRII binding protein for a control protein that is not a TGF-beta family member. In additional embodiments, the ActRII binding protein binds ActRIIB and ActRIIA with an affinity that is at least 100-fold, 500-fold, or 1000-fold greater than the affinity of the ActRII binding protein for a control protein that is not a TGF-beta receptor family member. In certain embodiments, the ActRII binding protein binds ActRIIB and ActRIIA with a dissociation constant (K D In some embodiments, the ActRIIA- and ActRIIB binding proteins have a K Dis within the range of 1 μM or less and 0.1 pM or more, 100 μM or less and 0.1 pM or more, or 100 μM or less and 1 pM or more.

[0133] In some embodiments, the ActRII binding protein is an antibody that specifically binds to ActRII. In additional embodiments, the ActRII binding protein is a full-length anti-ActRIIA antibody or a full-length anti-ActRIIB antibody. In additional embodiments, the antibody is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody, or an ActRII-binding antibody fragment thereof. In additional embodiments, the antibody specifically binds to ActRIIB and / or ActRIIA.

[0134] In some embodiments, ActRII binding proteins (eg, anti-ActRII antibodies and ActRII-binding antibody fragments) are capable of binding to ActRII molecules across species.

[0135] The mature ActRIIA extracellular domain of human ActRIIA (amino acids 20-138 of SEQ ID NO:92) differs from that of the mouse ActRIIA orthologue (reference P27038) by only two conserved amino acid substitutions (i.e., K19R and V72I). In additional embodiments, the ActRII binding protein is capable of binding to human ActRIIA (hActRIIA) and mouse ActRIIA (murActRIIA). In certain embodiments, the ActRII binding protein is capable of binding to an anti-ActRIIA antibody (e.g., a full-length ActRIIA antibody and an ActRIIA-binding antibody fragment, as well as variants and derivatives thereof) that is capable of specifically binding to ActRIIA (e.g., hActRIIA or murActRIIA) and has a dissociation constant or K as determined by BIACORE® or KINEXA®. D 10 -8 Less than M, 10 -9 Less than M or 10 -10 In a further embodiment, the anti-ActRIIA antibody has a K for ActRIIA of less than 1 nM.D In further embodiments, the anti-ActRIIA antibody binds to ActRIIA with a K within one order of magnitude of 1 nM or within two orders of magnitude of 1 nM (e.g., as determined by BIACORE® analysis). D In some embodiments, the ActRIIA binding protein binds to human ActRIIA at a K D is within the range of 1 μM or less and 0.1 pM or more, 100 μM or less and 0.1 pM or more, or 100 μM or less and 1 pM or more.

[0136] The mature extracellular domain of human ActRIIB (amino acids 19-130 of SEQ ID NO:93) differs from the corresponding sequence of the mouse ActRIIB ortholog (NCBI Reference Sequence NP031423) by one amino acid substitution (i.e., A95P). In certain embodiments, the ActRII binding protein has a 10-fold increase in ribosomal activity as determined by BIACORE® or KINEXA®. -8 Less than M, 10 -9 Less than M or 10 -10 Dissociation constant or K less than M D In a further embodiment, the anti-ActRIIB antibody has a K of less than 1 nM as determined by BIACORE® or KINEXA® analysis. D In a further embodiment, the anti-ActRIIB antibody binds to ActRIIB with a K within one order of magnitude of 1 nM or within two orders of magnitude of 1 nM. D In some embodiments, the ActRIIB binding protein binds to ActRIIB at a K D is within the range of 1 μM or less and 0.1 pM or more, 100 μM or less and 0.1 pM or more, or 1 nM or less and 1 pM or more.

[0137] In some embodiments, the anti-ActRII antibody is an ActRII-binding antibody fragment. In some embodiments, the ActRII-binding antibody fragment is a Fab, Fab', F(ab')2, Fv fragment, diabody, or single chain antibody molecule. In additional embodiments, the ActRII antibody is an Fd, single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv)2, scFv-Fc or bis-scFv.

[0138] In additional embodiments, the ActRII binding protein is an antibody comprising a VH and a VL. In some embodiments, the anti-ActRII antibody further comprises a heavy chain constant region or a fragment thereof. In some embodiments, the antibody comprises a heavy chain immunoglobulin constant region selected from the group consisting of: (a) a human IgA constant region or a fragment thereof, (b) a human IgD constant region or a fragment thereof, (c) a human IgE constant domain or a fragment thereof, (d) a human IgG1 constant region or a fragment thereof, (e) a human IgG2 constant region or a fragment thereof, (f) a human IgG3 constant region or a fragment thereof, (g) a human IgG4 constant region or a fragment thereof, and (h) a human IgM constant region or a fragment thereof. In certain embodiments, the ActRII binding protein comprises a heavy chain constant region or a fragment thereof, e.g., a human IgG constant region or a fragment thereof. In further embodiments, the ActRII binding protein comprises a heavy chain immunoglobulin constant domain that has been altered or mutated to alter effector function and / or half-life.

[0139] In certain embodiments, the ActRII binding protein is an antibody that comprises an IgG1 heavy chain constant region that contains a mutation that reduces effector function (see, e.g., Idusogie et al., J. Immunol. 166:2571-2575 (2001); Sazinsky et al., PNAS USA 105:20167-20172 (2008); Davis et al., J. Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23:403-411 (1993); Alegre et al., Transplantation 57:1537-1543 (1994); Xu et al., Cell Immunol. 200:16-26 (2000); Cole et al., J. Immunol. 20:16-26 (2000); each of which is incorporated by reference in its entirety. (see, for example, Hutchins et al., PNAS USA 92:11980-11984 (1995), Reddy et al., J. Immunol. 164:1925-1933 (2000); WO 97 / 11971, and WO 07 / 106585; U.S. Application Publication No. 2007 / 0148167 A1; McEarchern et al., Blood 109:1185-1192 (2007), Strohl, Curr. Op. Biotechnol. 20:685-691 (2009), and Kumagai et al., J. Clin. Pharmacol. 47:1489-1497 (2007)).

[0140] In some embodiments, the heavy chain constant region or fragment thereof includes one or more amino acid substitutions relative to a wild-type IgG constant domain, such that the modified IgG has reduced ADCC compared to the half-life of an IgG having a wild-type IgG constant domain. Examples of Fc sequence engineering modifications contained within the provided antibodies that reduce ADCC include IgG1-K326W, E333S; IgG2-E333S; IgG1-N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2-EU sequence 118-260; IgG4-EU sequence 118-260; and IgG1-L234F, L235E, P331S, where the numbering of the positions is according to the EU index, as in Kabat.

[0141] In certain embodiments, the ActRII binding protein comprises a heavy chain immunoglobulin constant domain that has reduced or been mutated to reduce CDC activity. In certain embodiments, the ActRII binding protein is an antibody that comprises an IgG1 heavy chain constant region that contains a mutation that reduces CDC activity (see, e.g., WO 97 / 11971 and WO 07 / 106585, U.S. Patent Publication No. 2007 / 0148167 A1, McEarchern et al., Blood 109:1185-1192 (2007), Hayden-Ledbetter et al., Clin. Cancer 15:2739-2746 (2009), Lazar et al., PNAS USA 103:4005-4010 (2006), Bruckheimer et al., Neoplasia 103:4005-4010 (2006), each of which is incorporated herein by reference in its entirety). 11:509-517 (2009); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Sazinsky et al., PNAS USA 105:20167-20172 (2008). Examples of Fc sequence engineering modifications contained within anti-ActRII antibodies that reduce CDC include one or more modifications corresponding to IgG1-S239D, A330L, I332E; 118-260 of the IgG2 EU sequence; 261-447 of the IgG4-EU sequence; IgG2-H268Q, V309L, A330S, A331S; IgG1-C226S, C229S, E233P, L234V, L235A; IgG1-L234F, L235E, P331S; and IgG1-C226S, P230S.

[0142] In further embodiments, the heavy chain constant region or fragment thereof comprises one or more amino acid substitutions relative to a wild-type IgG constant domain, such that the modified IgG has an increased half-life compared to the half-life of an IgG having a wild-type IgG constant domain. For example, the IgG constant domain can contain one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, where the numbering of the amino acid positions is according to the EU index as set forth in Kabat. In certain embodiments, an IgG constant domain can contain one or more of the following: a substitution of the amino acid at Kabat position 252 with Tyr, Phe, Trp, or Thr; a substitution of the amino acid at Kabat position 254 with Thr; a substitution of the amino acid at Kabat position 256 with Ser, Arg, Gln, Glu, Asp, or Thr; a substitution of the amino acid at Kabat position 257 with Leu; a substitution of the amino acid at Kabat position 309 with Pro; a substitution of the amino acid at Kabat position 311 with Ser; a substitution of the amino acid at Kabat position 428 with Thr, Leu, Phe, or Ser; a substitution of the amino acid at Kabat position 433 with Arg, Ser, Iso, Pro, or Gln; or a substitution of the amino acid at Kabat position 434 with Trp, Met, Ser, His, Phe, or Tyr. Specifically, the IgG constant domain can contain amino acid substitutions relative to the wild-type human IgG constant domain, including substitution of the amino acid at Kabat position 252 with Tyr, substitution of the amino acid at Kabat position 254 with Thr, and substitution of the amino acid at Kabat position 256 with Glu.

[0143] In additional embodiments, the ActRII binding protein is an antibody comprising a light chain immunoglobulin constant region. In further embodiments, the antibody comprises a human Ig kappa constant region or a human Ig lambda constant region.

[0144] The present disclosure provides activin receptor type II (ActRII) binding proteins and methods of using such ActRII binding proteins. In certain embodiments, the ActRII binding proteins are capable of inhibiting or preventing ActRII from binding to one or more cognate ActRII ligands and / or one or more cognate ActRI receptors. In some embodiments, the ActRII binding proteins are capable of inhibiting or preventing ActRII from binding to an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10). The present disclosure also provides methods of using ActRII binding proteins for the diagnosis, or treatment, prevention, and / or amelioration of diseases or conditions associated with increased ActRII expression and / or ActRII-mediated signaling. Such diseases or conditions include, but are not limited to, muscle disorders such as degenerative muscle diseases, muscular dystrophies, muscle atrophy, or muscle wasting disorders; fibrotic conditions; inflammatory, autoimmune, cardiovascular, pulmonary, musculoskeletal, skeletal, ophthalmic, neurological, or metabolic diseases or conditions; obesity; wound healing; and cancer.

[0145] In some embodiments, the ActRII binding protein specifically binds to ActRIIB. In further embodiments, the ActRII binding proteins provided specifically bind ActRIIB and (a) compete with an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10) for binding to ActRIIB; (b) reduce phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIB and ALK4 and / or ALK7 in the presence of an ActRIIB ligand (e.g., activin A and / or GDF8 (myostatin)); (c) reduce phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRIIB in the presence of an ActRIIB ligand (e.g., activin A and / or GDF8); and (d) bind to ActRIIB with a K of 1 nM or less and 1 pM or more. D (e.g., as determined by BIACORE® analysis). In some embodiments, the ActRIIB binding protein has two, three, or four of the above properties. In some embodiments, the ActRIIB binding protein has at least two or at least three of the above properties. In further embodiments, the ActRIIB binding protein competes for binding to ActRIIB with an antibody having an ActRIIB-binding VH and VL pair disclosed herein. In further embodiments, the ActRIIB binding protein is an anti-ActRIIB antibody or an ActRIIB-binding antibody fragment.

[0146] In some embodiments, the ActRII binding protein specifically binds to ActRIIB and ActRIIA. In further embodiments, the ActRII binding protein provided specifically binds to ActRIIB and ActRIIA and (a) competes with an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10) for binding to ActRIIB and / or ActRIIA; (b) competes with a ligand of ActRIIB and / or ActRIIA (e.g., activin A and / or GDF8 (myostatin)). (c) reduces phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRIIB and / or ActRIIA in the presence of a ligand for ActRIIB and / or ActRIIA (e.g., activin A and / or GDF8); and (d) reduces phosphorylation of ActRIIB with a K of 1 nM or less and 1 pM or more. D (e.g., as determined by BIACORE® analysis). In some embodiments, the ActRIIB and ActRIIA binding proteins have two, three, or four of the above properties. In some embodiments, the ActRIIB and ActRIIA binding proteins have at least two or at least three of the above properties. In further embodiments, the ActRIIB binding proteins compete for binding to ActRIIB and ActRIIA with an antibody having a VH and VL pair that binds to ActRIIB and ActRIIA disclosed herein. In further embodiments, the ActRIIB and ActRIIA binding proteins are anti-ActRIIB and ActRIIB antibodies or ActRIIB antibody fragments and ActRIIB-binding antibody fragments.

[0147] In some embodiments, the ActRII binding protein specifically binds to ActRIIA. In further embodiments, the ActRII binding proteins provided specifically bind to ActRIIA and (a) compete with an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10) for binding to ActRIIA; (b) reduce phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIA and ALK4 and / or ALK7 in the presence of an ActRIIA ligand (e.g., activin A and / or GDF8 (myostatin)); (c) reduce phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRIIA in the presence of an ActRIIA ligand (e.g., activin A and / or GDF8); and (d) bind to ActRIIA with a K of 1 nM or less and 1 pM or more. D (e.g., as determined by BIACORE® analysis). In some embodiments, the ActRIIA binding protein has two, three, or four of the above properties. In some embodiments, the ActRIIA binding protein has at least two or at least three of the above properties. In further embodiments, the ActRIIA binding protein competes for binding to ActRIIA with an antibody having an ActRIIA-binding VH and VL pair disclosed herein. In further embodiments, the ActRIIA binding protein is an anti-ActRIIA antibody or an ActRIIA-binding antibody fragment.

[0148] In some embodiments, the ActRII binding protein comprises a set of complementarity determining regions (CDRs), i.e., heavy chain variable region (VH)-CDR1, VH-CDR2, VH-CDR3, light chain variable region (VL)-CDR1, VL-CDR2 and VL-CDR3, and / or a set of antigen binding regions (ABRs), i.e., heavy chain variable region (VH)-ABR1, VH-ABR2, VH-ABR3, light chain variable region (VL)-ABR1, VL-ABR2 and VL-ABR3, and such CDRs and / or ABRs are present in the heavy chain variable region (VH) and light chain variable region (VL) pairs disclosed in Table 1. In some embodiments, the ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH and VL pair selected from the group consisting of: (a) a VH sequence of SEQ ID NO: 20, 49, or 77 and a VL sequence of SEQ ID NO: 30, 39, 59, 67, or 85, wherein the protein binds to ActRIIB, and (b) a VH sequence of SEQ ID NO: 2 and a VL having the amino acid sequence of SEQ ID NO: 12, wherein the protein binds to ActRIIB and activin receptor type IIA (ActRIIA).

[0149] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 20 and a VL having the amino acid sequence of SEQ ID NO: 30, and such a protein binds to ActRIIB.

[0150] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 20 and a VL having the amino acid sequence of SEQ ID NO: 39, and such a protein binds to ActRIIB.

[0151] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 49 and a VL having the amino acid sequence of SEQ ID NO: 59, and such a protein binds to ActRIIB.

[0152] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 20 and a VL having the amino acid sequence of SEQ ID NO: 67, and such a protein binds to ActRIIB.

[0153] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 77 and a VL having the amino acid sequence of SEQ ID NO: 85, and such a protein binds to ActRIIB.

[0154] In some embodiments, an ActRII binding protein comprises a set of CDRs and / or ABRs present in a VH having the amino acid sequence of SEQ ID NO: 2 and a VL having the amino acid sequence of SEQ ID NO: 12, and such a protein binds to ActRIIA and ActRIIB.

[0155] In a further embodiment, the ActRII binding protein specifically binds to ActRII and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (a) (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21, 50, or 78, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, 51, or 79, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 23, 52, or 80, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 31, 40, 60, 68, or 86, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 32, 41, 61, 69, or 87, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 8 or (b) (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 3, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 4, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 5, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 13, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 14, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 15, and such protein binds to ActRIIB and ActRIIA, or has a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0156] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 31, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 32, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 33, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0157] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 40, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 41, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 42, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0158] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 50, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 51, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 60, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 61, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 62, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0159] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 68, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 69, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 70, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0160] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 having the amino acid sequence of SEQ ID NO: 78, (ii) VH-CDR2 having the amino acid sequence of SEQ ID NO: 79, and (iii) VH- (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 86, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 87, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 88, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0161] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and ActRIIA and comprises a set of CDRs, i.e., VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e., (i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 3, (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 4, (iii) (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 13, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 14, and (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 15, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0162] In a further embodiment, the ActRII binding protein specifically binds to ActRII and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (a) (i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 24, 53, or 81, (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 25, 54, 55, or 82, (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 26, 56, 57, or 83, (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 34, 43, 63, 71, or 89, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 35, 44, 64, 72, or 90, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: or (b) (i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 6, (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 7 or 8, (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 9 or 10, (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 16, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 17, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 18, and such protein binds to ActRIIB and ActRIIA, or has a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0163] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 24, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 25, and (iii) VH-ABR3 having the amino acid sequence of SEQ ID NO: 26. (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 34, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 35, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 36, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0164] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 24, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 25, and (iii) VH-ABR3 having the amino acid sequence of SEQ ID NO: 26. (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 43, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 44, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 45, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0165] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 53, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 54 or 55, and (iii) VH- (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 63, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 64, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 65, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0166] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 24, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 25, and (iii) VH-ABR3 having the amino acid sequence of SEQ ID NO: 26. (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 71, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 72, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 73, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0167] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and comprises a set of ABRs, namely, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, namely, (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 81, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 82, and (iii) VH- (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 89, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 90, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 91, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0168] In a further embodiment, the ActRII binding protein specifically binds to ActRIIB and ActRIIA and comprises a set of ABRs, i.e., VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2, and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, i.e., (i) VH-ABR1 having the amino acid sequence of SEQ ID NO: 6, (ii) VH-ABR2 having the amino acid sequence of SEQ ID NO: 7 or 8, and (iii) ) VH-ABR3 has the amino acid sequence of SEQ ID NO: 9 or 10, (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 16, (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 17, and (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 18, or have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less than 10 amino acid substitutions, deletions, and / or insertions therefrom.

[0169] In some embodiments, the ActRII binding protein specifically binds to ActRII and comprises a VH and VL pair selected from the group consisting of: (a) (i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20, 49, or 77, and (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, 39, 59, 67, or 85, wherein the protein binds to ActRIIB; and (b) (i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, and (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, wherein the protein binds to ActRIIB and ActRIIA.

[0170] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:30.

[0171] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:39.

[0172] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:49 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:59.

[0173] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 67.

[0174] In some embodiments, the ActRII binding protein specifically binds ActRIIB and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:77 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:85.

[0175] In some embodiments, the ActRII binding protein specifically binds to ActRIIB and ActRIIA and comprises a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2 and a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12.

[0176] In further embodiments, the ActRIIB binding protein (a) competes with an ActRII ligand (e.g., activin A, activin B, GDF1, GDF3, GDF8 (myostatin), GDF11, BMP6, BMP7, BMP9, or BMP10) for binding to ActRII; (b) reduces phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRII in the presence of an ActRII ligand (e.g., activin A or GDF8); (c) reduces phosphorylation of ALK4 and / or ALK7 in cells expressing ActRII and ALK4 and / or ALK7 in the presence of an ActRII ligand; and (d) binds ActRII with a K of 1 nM or less and 1 pM or more. D(e.g., as determined by BIACORE® analysis). In some embodiments, the ActRII binding protein has two, three, or four of the above properties. In some embodiments, the ActRII binding protein has at least two or at least three of the above properties.

[0177] In additional embodiments, the ActRII binding protein competes for binding to ActRII with an antibody comprising a VH and VL sequence pair disclosed herein. In some embodiments, the ActRII binding protein binds to the same epitope as an ActRII binding protein disclosed herein.

[0178] In some embodiments, the ActRII binding protein is an antibody that specifically binds to ActRII. In some embodiments, the anti-ActRII specifically binds to ActRIIB and / or ActRIIA. In some embodiments, the anti-ActRII antibody is a murine antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a multispecific antibody, or any combination thereof. In some embodiments, the anti-ActRII antibody is an Fv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a dsFv fragment, a scFv fragment, or a sc(Fv)2 fragment.

[0179] In some embodiments, the ActRII binding protein specifically binds ActRII (e.g., ActRIIA and / or ActRIIB) and blocks the activity of an ActRII ligand (e.g., GDF8 (myostatin) and / or activin). In some embodiments, the ActRII binding protein specifically binds ActRII (for example) and reduces the inhibition of myogenesis or increased adipogenesis associated with the activity of an ActRII ligand (e.g., GDF8 (myostatin and / or activin). In some embodiments, the ActRII binding protein specifically binds ActRII and treats or ameliorates one or more conditions associated with a muscle disorder or a metabolic disorder. In some embodiments, the muscle disorder is muscle wasting due to disease or disuse. In some embodiments, the metabolic disorder is diabetes, obesity, hyperglycemia, or bone loss.

[0180] In certain embodiments, the ActRIIB binding protein (e.g., an anti-ActRIIB antibody or an anti-ActRIIB and ActRIIA antibody) inhibits or reduces GDF8 (myostatin) binding to ActRIIB or GDF8-mediated ActRIIB Smad signaling. In another embodiment, the ActRIIB binding protein reduces myogenesis inhibition or adipogenesis enhancement. In some embodiments, the ActRIIB binding protein binds ActRIIB and inhibits or reduces one or more conditions associated with a muscle disorder or metabolic disorder. In some embodiments, the muscle disorder is muscle wasting due to disease or disuse. In some embodiments, the metabolic disorder is diabetes, obesity, hyperglycemia, or bone loss. Increases muscle mass and strength in a subject.

[0181] In certain embodiments, blocking ActRII (e.g., ActRIIB and / or ActRIIA) activity with the ActRII binding proteins described herein (e.g., anti-ActRIIB and anti-ActRIIA antibodies) inhibits or reduces one or more conditions associated with muscle disorders, such as muscle wasting. In further embodiments, blocking ActRII inhibits or reduces one or more conditions associated with muscle wasting due to disease or disuse. In certain embodiments, the ActRII binding proteins (e.g., anti-ActRIIB antibodies or anti-ActRIIB and ActRIIA antibodies) inhibit or reduce binding of GDF8 to ActRIIB. In another embodiment, the ActRIIB binding proteins inhibit or reduce inhibition of muscle differentiation by the Smad-dependent pathway.

[0182] In some embodiments, the ActRII binding protein specifically binds to ActRIIB and blocks ActRIIB ligand-mediated activity. Some ActRIIB ligands, such as GDF-8, are known to be negative regulators of skeletal muscle tissue, and myostatin signaling is known to lead to muscle mass. ActRIIB ligand-mediated signaling can also regulate the production of muscle-specific enzymes (e.g., creatine kinase), stimulate myoblast proliferation, and regulate preadipocyte differentiation into adipocytes. Increased myostatin activity is associated with muscle wasting disorders, muscle mass loss due to inactivity, and metabolic disorders including diabetes, obesity, hyperglycemia, and bone loss. Increased ActRIIB ligand-mediated activity is also associated with age-related increases in fat-to-muscle ratios and age-related muscle atrophy. In some embodiments, the ActRII binding protein specifically binds to ActRIIB and reduces the myogenesis inhibition or adipogenesis enhancement associated with the activity of some ActRIIB ligands. In some embodiments, the ActRII binding protein specifically binds to ActRIIB and treats or improves one or more conditions associated with muscle disorder or metabolic disorder.In some embodiments, the muscle disorder is muscle wasting due to disease or disuse.In some embodiments, the metabolic disorder is diabetes, obesity, hyperglycemia, or bone loss.ActRIIB ligand-mediated activity can be determined using art-recognized methods, such as those described herein.

[0183] In certain embodiments, blocking ActRII (e.g., ActRIIB and / or ActRIIA) activity with the ActRII binding proteins described herein (e.g., anti-ActRIIB and anti-ActRIIA antibodies) alleviates one or more conditions associated with fibrosis. In certain embodiments, the ActRIIB binding proteins inhibit or reduce biomolecule expression changes (e.g., mRNA or protein expression) associated with ActRIIB-mediated development of fibrotic lesions, weight loss or other clinical symptoms, and / or development of a fibrotic condition. In certain embodiments, the ActRIIA binding proteins inhibit or reduce biomolecule expression changes (e.g., mRNA or protein expression) associated with ActRIIA-mediated development of fibrotic lesions, weight loss or other clinical symptoms, and / or development of a fibrotic condition.

[0184] As described above, anti-ActRII antibodies (e.g., full-length ActRIIB antibodies and ActRII-binding antibody fragments, as well as variants and derivatives thereof) containing a VH and / or VL amino acid sequence that binds ActRII may have at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences described herein. In some embodiments, the VH and / or VL amino acid sequence(s) that bind ActRII include 8, 7, 6, 5, 4, 3, 2, 1 amino acid additions, substitutions (e.g., conservative substitutions) or deletions relative to the sequences described herein. In additional embodiments, the VH and / or VL amino acid sequences that bind ActRII include 1, 2, 3, 4, 5, or more amino acid additions, substitutions (e.g., conservative substitutions) or deletions relative to the sequences described herein. Anti-ActRII antibodies containing VH and VL regions that have a particular percentage of similarity to the VH or VL regions, or VH and VL regions that have one or more substitutions, deletions and / or insertions (e.g., conservative substitutions), can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding the VH and / or VL regions described herein, followed by testing the encoded modified antibodies for binding to ActRII, and optionally testing for retained function using the functional analyses described herein or using assays known in the art that can be routinely modified to test for retained function.

[0185] The affinity or avidity of an ActRII binding protein, such as an anti-ActRIIB antibody (e.g., a full-length ActRIIB antibody and an ActRII-binding antibody fragment, as well as variants and derivatives thereof) for hActRIIB, murActRIIB, can be determined experimentally using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., BIACORE® or KINEXA® analysis). Direct binding assay and competitive binding assay formats can be readily used. (See, e.g., Berzofsky et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Immunology, WH Freeman and Company: New York, NY (1992), and the methods described herein). The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Thus, affinity and other ActRII binding parameters (e.g., K D or Kd, K on , K off ) is measured using standard solutions of ActRII binding protein and ActRII, and the measurements are performed using other standardized conditions and methods described herein or known in the art.

[0186] The present disclosure further provides ActRII binding proteins, such as the anti-ActRIIB and / or anti-ActRIIA antibodies described herein, where the ActRII binding protein is conjugated to a heterologous agent. In certain embodiments, the heterologous agent is an antibacterial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or antibody fragment, a detectable label, or polyethylene glycol (PEG). Heteroconjugate ActRII binding proteins are discussed in more detail elsewhere herein.

[0187] In certain embodiments, the ActRII binding protein is not an anti-ActRII antibody. A variety of methods are known in the art for identifying and generating non-antibody polypeptides that bind to protein targets with high affinity. See, for example, Skerra, Curr. Opin. Biotech. 18:295-304 (2007); Hosse et al., Protein Science 15:14-27 (2006); Gill et al., Curr. Opin. Biotechnol. 17:653-658 (2006); Nygren, FEBS J. 275:2668-2676 (2008); and Skerra, FEBS J. 275:2677-2683 (2008), each of which is incorporated herein by reference in its entirety. In some embodiments, phage display technology can be used to identify / generate ActRII binding proteins. In some embodiments, the ActRII binding protein comprises a protein scaffold based on a species selected from the group consisting of a VASP polypeptide, avian pancreatic polypeptide (aPP), tetranectin (based on CTLD3), affilin (based on gamma B-crystallin / ubiquitin), knottin, an SH3 domain, a PDZ domain, tendamistat, transferrin, a consensus ankyrin repeat domain (e.g., DARPin), a lipocalin protein fold (e.g., anticalins and duocalins), a protein epitope mimetic (PEM), a maxibody / avimer, a domain antibody fibronectin domain (e.g., 10 Fn3, see, e.g., U.S. Patent Publication Nos. 2003 / 0170753 and 20090155275, both of which are incorporated by reference in their entireties), a domain of Protein A (e.g., an affibody), and thioredoxin.

[0188] In some embodiments, the disclosure provides ActRIIA binding proteins (e.g., anti-ActRIIA antibodies, such as full-length anti-ActRIIA antibodies and ActRIIA-binding antibody fragments) that compete for binding to ActRIIA with the anti-ActRIIA antibodies provided herein. In some embodiments, the disclosure provides ActRIIA binding proteins that bind to the same epitope of ActRIIA as the ActRIIA binding proteins provided herein.

[0189] In some embodiments, the present disclosure provides ActRIIB binding proteins (e.g., anti-ActRIIB antibodies, such as full-length anti-ActRIIB antibodies and ActRIIB-binding antibody fragments) that compete for binding to ActRIIB with the anti-ActRIIB antibodies provided herein. In some embodiments, the present disclosure provides ActRIIB binding proteins that bind to the same epitope of ActRIIB as the ActRIIB binding proteins provided herein. The ability of a test ActRII binding protein to inhibit binding to ActRIIB of a reference binding protein, such as an antibody comprising, for example, a VH sequence of SEQ ID NO: 40 and a VL sequence of SEQ ID NO: 9, or a VH sequence of SEQ ID NO: 119 and a VL sequence of SEQ ID NO: 91, demonstrates that the test ActRII binding protein can compete with the reference antibody for binding to ActRIIB. Such an ActRIIB binding protein may, according to non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximal) epitope on ActRIIB as the ActRIIB reference antibody with which it competes. In some embodiments, the ActRIIB binding protein binds to the same epitope on ActRIIB as an antibody comprising the VH sequence of SEQ ID NO:40 and the VL sequence of SEQ ID NO:9.

[0190] ActRII receptors, such as ActRIIB and ActRIIA, are known to phosphorylate ActRI co-receptors (e.g., Alk4 and Alk7) and signal through phosphorylation of Smads (e.g., Smad2 and / or Smad3). In some embodiments, ActRII binding proteins (e.g., anti-ActRIIB and anti-ActRIIA antibodies) can reduce ActRII-mediated phosphorylation of its cognate ActRI receptor. In some embodiments, ActRIIB binding proteins (e.g., anti-ActRIIB antibodies) can reduce ActRIIB-mediated phosphorylation of ALK4 and / or ALK7. In some embodiments, ActRIIA binding proteins (e.g., anti-ActRIIA antibodies) can reduce ActRIIA-mediated phosphorylation of ALK4 and / or ALK7. In some embodiments, the ActRII binding protein can inhibit ActRII-mediated phosphorylation of Smads (e.g., Smad2 and / or Smad3) in ActRII2-expressing cells. In some embodiments, the ActRIIB binding protein (e.g., an anti-ActRIIB antibody) can reduce ActRIIB-mediated phosphorylation of Smads (e.g., Smad2 and / or Smad3) in ActRIIB-expressing cells. In some embodiments, the ActRIIA binding protein (e.g., an anti-ActRIIA antibody) can reduce ActRIIA-mediated phosphorylation of Smads (e.g., Smad2 and / or Smad3) in ActRIIA-expressing cells. In some embodiments, the ActRII receptor-expressing cells are human.

[0191] In some embodiments, the ActRII binding protein (a) competes with activin A for binding to ActRIIA and / or ActRIIB; (b) reduces phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRIIA and / or ActRIIB in the presence of an ActRIIA and / or ActRIIB ligand (e.g., activin A); (c) reduces phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIA and / or ActRIIB and ALK4 and / or ALK7 in the presence of a ligand for ActRIIB and / or ActRIIA; and (d) binds ActRIIA and / or ActRIIB with a K of 1 nM or less and 1 pM or more as determined by BIACORE® or KINEXA®. D and binding at least one of the following:

[0192] In some embodiments, the ActRII binding protein (e.g., an anti-ActRII antibody) inhibits ActRII-mediated phosphorylation of an ActRI receptor (e.g., ALK4 and / or ALK7) or phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRII, as measured using a cell-based assay. In some embodiments, the ActRII binding protein inhibits ActRII-mediated phosphorylation with an IC of less than 500 pM, less than 350 pM, less than 250 pM, less than 150 pM, less than 100 pM, less than 75 pM, less than 60 pM, less than 50 pM, less than 40 pM, less than 30 pM, less than 20 pM, less than 15 pM, less than 10 pM, or less than 5 pM, as measured using a cell-based assay. 50 to suppress it.

[0193] Preparation of ActRII-binding proteins In some embodiments, the ActRII binding protein binds to the extracellular domain of ActRII (e.g., ActRIIB and ActRIIA). In further embodiments, the ActRII binding protein is an anti-ActRIIA antibody and / or an anti-ActRIIB antibody, such as a full-length anti-ActRIIA antibody and a full-length anti-ActRIIB antibody and an ActRII-binding antibody fragment, and variants and derivatives thereof.

[0194] ActRII binding proteins can be easily prepared using known techniques. Monoclonal anti-ActRII (e.g., ActRIIB and ActRIIA) antibodies can be prepared using techniques known in the art, including hybridoma methods, such as those described by Kohler and Milstein, Nature 256:495-497 (1975). Using the hybridoma method, mice, hamsters, or other suitable host animals are immunized as described above to induce lymphocytes to produce antibodies that specifically bind to the immunizing antigen. Lymphocytes can be immunized in vitro. After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line to form hybridoma cells, which can then be selected and separated from unfused lymphocytes and myeloma cells. Hybridomas producing monoclonal antibodies specifically directed against ActRII, such as hActRIIB and hActRIIA, as determined by immunoprecipitation, immunoblotting, or in vitro binding assays (e.g., radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA)), can then be grown in vitro in culture or in vivo as ascites tumors in animals using standard methods (see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986). Monoclonal antibodies can then be purified from the culture medium or ascites fluid as for polyclonal antibodies described above.

[0195] The monoclonal antibodies provided can also be made using recombinant DNA methods such as those described in U.S. Patent No. 4,816,567, where polynucleotides encoding the monoclonal antibody are isolated from mature B cells or hybridoma cells and their sequences determined using known procedures, such as by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody. The isolated polynucleotides encoding the heavy and light chains are then cloned into appropriate expression vectors and introduced into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, Simian COS cells, Chinese hamster ovary (CHO) cells, Per.C6 cells, or myeloma cells (e.g., NS0 cells), which produce the monoclonal antibody. Recombinant anti-ActRII monoclonal antibodies can also be readily isolated from phage display libraries expressing the CDRs of the desired species using known techniques (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991)).

[0196] Anti-ActRII antibodies can optionally be humanized, resurfaced, and engineered to exhibit high affinity for ActRII antigens (e.g., ActRIIB and ActRIIA) and other favorable biological properties. For example, humanized (or human) anti-ActRII antibodies can be readily designed and prepared using commonly available three-dimensional immunoglobulin modeling and known procedures for selecting framework (FW) residues, consensus sequences, and germline sequences to obtain desired antibody properties, such as increased affinity for ActRII.

[0197] Affinity maturation and chain shuffling strategies are known in the art and can be utilized to generate high affinity anti-ActRII (e.g., anti-ActRIIA and / or anti-ActRIIB) antibodies and derivatives and variants of the ActRII binding proteins disclosed herein. See, for example, Marks et al., Bio / Technology 10:779-783 (1992), which is incorporated herein by reference in its entirety. An additional strategy for generating high affinity anti-ActRII (e.g., anti-ActRIIA and / or anti-ActRIIB) antibodies and derivatives and variants of the ActRII binding proteins disclosed herein is to use random mutagenesis of one or more selected VH and / or VL genes to generate mutations within the entire variable domain to generate novel VH or VL regions that retain the CDR-derived sequences of the present disclosure. Such a technique using error-prone PCR is described by Gram et al. (PNAS USA 89:3576-3580 (1992)). In some embodiments, one or two amino acid substitutions are made within a set of VH CDRs and / or VL CDRs. A further strategy used direct mutagenesis of the CDR regions of the VH or VL genes encoding the anti-ActRII antibodies disclosed herein. Examples of such techniques are disclosed by Barbas et al. (PNAS USA 91:3809-3813 (1994)) and Schier et al. (J. Mol. Biol. 263:551-567 (1996)).

[0198] Humanization, resurfacing or engineering of the anti-ActRII antibodies of the disclosure can be carried out using any known method, including those described in Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988); Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987); Carter et al., PNAS USA 89:4285 (1992); Presta et al., J. Immunol. 199: 2296 (1993); al., J. Immunol. 151:2623 (1993), U.S. Patent No. 5,639,641, U.S. Patent No. 5,723,323, U.S. Patent No. 5,976, No. 862, No. 5,824,514, No. 5,817,483, No. 5,814,476, No. 5,763,192, No. 5,723,3 No. 23, No. 5,766,886, No. 5,714,352, No. 6,204,023, No. 6,180,370, No. 5,693,76 No. 2, No. 5,530,101, No. 5,585,089, No. 5,225,539, No. 4,816,567, No. 7,557,189 , 7,538,195, and 7,342,110, International Application Nos. PCT / US98 / 16280, PCT / US96 / 18978, PCT / US91 / 09630, PCT / US91 / 05939, PCT / US94 / 01234, PCT / GB89 / 01334, PCT / GB91 / 01134, PCT / GB92 / 01755, International Application Publication Nos. WO90 / 14443, WO90 / 14424, WO90 / 14430, and European Patent Publication No. EP229246.Similarly, known assays are available to easily select ActRII antibodies that exhibit desirable characteristics (e.g., assays to determine binding affinity to ActRII, cross-blocking assays such as the human ActRII binding protein competitive binding assay using BIACORE® described herein).

[0199] Methods for engineering, humanizing, or resurfacing non-human or human antibodies can also be used and are known in the art. Humanized, resurfaced, or similarly engineered antibodies can have one or more amino acid residues from a source that is non-human, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are typically replaced by residues taken from an "imported" variable, constant, or other domain of a known human sequence, often referred to as "import" residues. Such imported sequences can be used to reduce immunogenicity or to suppress, enhance, or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable property, as known in the art. Preferably, some or all of the non-human or human CDR sequences are maintained, but the non-human sequences in the variable and constant regions can be replaced with human amino acids or other amino acids.

[0200] The nucleic acid(s) encoding the ActRII binding protein, such as a full-length anti-ActRIIA or anti-ActRIIB antibody, can further be modified in several different ways using recombinant DNA technology to generate alternative antibodies. In some embodiments, the nucleic acid(s) encoding the constant domains of the light and heavy chains, for example, of a mouse monoclonal antibody, can (a) replace the coding regions of, for example, a human antibody to generate a chimeric antibody, or (b) replace the nucleic acid(s) encoding a non-immunoglobulin to generate a fusion antibody. In some embodiments, the constant region is truncated or removed to generate the desired antibody fragment of the monoclonal antibody. Site-directed mutagenesis or high-density mutagenesis of the variable region coding sequence can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0201] Anti-ActRII human antibodies can be prepared directly using any of a number of techniques known in the art (see, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147(1): 86-95 (1991); and U.S. Patent No. 5,750,373). Similarly, human anti-ActRII antibodies can be easily obtained from immortalized human B lymphocytes immunized in vitro, or can be easily isolated from immunized individuals producing antibodies directed against ActRII (e.g., ActRIIB and ActRIIA).

[0202] Human anti-ActRII antibodies can also be selected from phage libraries expressing human antibodies, as described, for example, in Vaughan et al., Nat. Biotech. 14:309-314 (1996), Sheets et al., PNAS 95:6157-6162 (1998), Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991), and Marks et al., J. Mol. Biol. 222:581 (1991). Techniques for generating and screening antibody phage libraries are also described in U.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, 6,593,081, 6,300,064, 6,653,068, 6,706,484, and 7,264,963, as well as Rothe et al., J. Mol. Biol. 376(4):1182-1200 (2008), each of which is incorporated by reference in its entirety.

[0203] Human anti-ActRII antibodies can also be made in transgenic mice containing human immunoglobulin loci capable of producing human antibodies upon immunization in the absence of endogenous production of immunoglobulins, as described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016.

[0204] Human anti-ActRII antibodies can also be selected and / or isolated from yeast-based antibody display libraries, e.g., as disclosed in WO012 / 009568, WO09 / 036379, WO10 / 105256, WO03 / 074679, and U.S. Application Publication No. US2002 / 0177170, the contents of each of which are incorporated herein by reference in their entireties. Such libraries are designed in silico to reflect the diversity provided by the human pre-immune repertoire.

[0205] Alternatively, anti-ActRII antibodies can be selected from yeast-displayed antibody libraries, see, e.g., Blaise et al., Gene 342(2):211-218(2004); Boder et al., Nat Biotechnol. 15(6):553-557(1997); Kuroda et al., Biotechnol. Lett. 33(1):1-9(2011). Review; Lauer et al., J. Pharm. Sci. 101(1):102-15(2012); Orcutt KD and Wittrup KD Antibody Engineering, yeast display and selection (2010), 207-233; Rakestraw et al., Protein Engineering, 1999, 2010 ... See Eng. Des. Sel. 24(6):525-30 (2011), and U.S. Pat. Nos. 6,423,538, 6,696,251, and 6,699,658.

[0206] Various techniques are known for the production of antigen-binding antibody fragments. Traditionally, these fragments are derived through proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993), and Brennan et al., Science 229:81 (1985)). In certain embodiments, ActRII-binding antibody fragments are recombinantly produced. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, allowing for the production of large amounts of these fragments. Such ActRII-binding antibody fragments can also be isolated from the antibody phage libraries described above. In some embodiments, the ActRII-binding antibody fragments are linear antibodies, as described in U.S. Pat. No. 5,641,870. Other techniques for the production of antigen-binding antibody fragments are known in the art.

[0207] Known techniques can be readily adapted for the production of single chain antibodies that bind to ActRII (see, e.g., U.S. Patent No. 4,946,778). In addition, known methods can be routinely adapted for the construction of Fab expression libraries (see, e.g., Huse et al., Science 246:1275-1281 (1989)) to allow rapid and efficient identification of monoclonal Fab fragments with the desired specificity for ActRII. ActRII-binding antibody fragments can be produced by techniques known in the art, including, but not limited to, (a) F(ab')2 fragments produced by pepsin digestion of antibodies, (b) Fab fragments produced by reducing disulfide bridges of F(ab')2 fragments, (c) Fab fragments produced by treating anti-ActRII antibodies with papain and a reducing agent, and (d) Fv fragments.

[0208] In certain embodiments, ActRII binding protein (e.g., anti-ActRIIA antibody and / or anti-ActRIIB antibody) can be modified to increase its serum half-life. This can be achieved, for example, by incorporating a salvage receptor binding epitope into ActRII binding protein by mutation of the appropriate region of ActRII binding protein, or by incorporating a salvage receptor epitope into a peptide tag and then fusing it to either end or the middle of ActRIIB binding protein (e.g., by DNA or peptide synthesis). Other methods for extending the serum half-life of ActRII binding protein, such as conjugation to a heterologous molecule such as PEG, are known in the art.

[0209] Heterocomplex ActRII binding proteins (e.g., anti-ActRIIB antibodies, such as full-length anti-ActRIIB antibodies and ActRIIB binding antibody fragments, and variants and derivatives thereof) are also within the scope of the present disclosure. Heterocomplex ActRII binding proteins are composed of two covalently linked proteins. It is contemplated that heterocomplex ActRII binding proteins can be prepared in vitro using known methods of synthetic protein chemistry, including methods involving crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0210] ActRII binding proteins can include any type of variable region that provides for the association of the antibody with ActRII (e.g., ActRIIB and ActRIIA). Such variable regions can be included in or derived from any mammal that can be induced to mount a humoral response to an ActRII antigen and generate immunoglobulins. The variable regions of the anti-ActRII antibody can be derived, for example, from a human, a mouse, a non-human primate (e.g., cynomolgus monkey, macaque monkey, etc.) or a wolf. In some embodiments, both the variable and constant regions of the modified anti-ActRII antibody are human. In other embodiments, the variable regions of a compatible antibody (usually derived from a non-human source) can be engineered or specifically tailored to improve binding characteristics or reduce the immunogenicity of the molecule. In this regard, the variable regions useful according to the present disclosure can be humanized or otherwise altered by including imported amino acid sequences using affinity maturation, mutagenesis procedures, chain shuffling strategies, and / or other methods described herein or known in the art.

[0211] In certain embodiments, both the heavy and light chain variable domains of the anti-ActRII antibody are modified by at least partial replacement of one or more CDRs and / or by replacement and sequence changes of partial framework regions. The CDRs may be derived from antibodies of the same class, or even subclass, as the antibody from which the framework regions are derived, but it is anticipated that the CDRs will be derived from antibodies of a different class, or in certain aspects, from a different species. To transfer the antigen-binding capacity of one variable domain to another, it is not necessary to replace all of the CDRs with the complete CDRs from the donor variable region. Rather, only those residues necessary to maintain the activity of the antigen-binding site need to be transferred. It is well within the capabilities of one of ordinary skill in the art to routinely obtain functional antibodies with low immunogenicity. See, for example, U.S. Patent Nos. 5,585,089, 5,693,761 and 5,693,762.

[0212] Notwithstanding the modifications to the variable regions, one of skill in the art will appreciate that the modified anti-ActRII of the present disclosure includes antibodies in which at least a small portion of one or more of the constant region domains have been deleted or otherwise altered to obtain desired biochemical properties, such as reduced ADCC or increased serum half-life, when compared to antibodies of similar immunogenicity that contain native or unaltered constant regions. In some embodiments, the constant region of the modified anti-ActRII antibody comprises a human constant region. Modifications to the constant region may include the addition, deletion, or substitution of one or more amino acids in one or more domains. The modified anti-ActRII antibodies disclosed herein may include modifications or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or modifications or modifications to the light chain constant domain (CL). In some embodiments, it is contemplated that the modified anti-ActRII antibody comprises a constant region in which one or more domains are partially or completely deleted. In some embodiments, modified anti-ActRII antibodies include domain deleted constructs or variants in which the entire CH2 domain has been removed (ΔCH2 constructs). In some embodiments, the deleted constant region domain can be replaced with a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility typically imparted by the missing constant region.

[0213] It is generally understood that the constant region mediates several effector functions. For example, the binding of the C1 component of complement to an antibody activates the complement system. Complement activation is important for opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Furthermore, antibodies bind to cells via the Fc region, and Fc receptor sites on the antibody Fc region bind to Fc receptors (FcRs) on the cells. There are several Fc receptors specific for different classes of antibodies, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of antibodies to cell surface Fc receptors induces several important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, immune complex removal, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production.

[0214] In certain embodiments, the anti-ActRII antibody has altered effector functions, which affect the biological profile of the administered anti-ActRII antibody. For example, deletion or inactivation (by point mutation or other means) of constant region domains may reduce Fc receptor binding of the circulating modified antibody. In other cases, constant region modifications may reduce complement binding, thereby reducing serum half-life and non-specific association of bound cytotoxins. Still other modifications of the constant region may be used to eliminate disulfide bonds or oligosaccharide moieties, allowing for improved localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region according to the present disclosure may be readily made using biochemical or molecular engineering techniques known to those skilled in the art.

[0215] In some embodiments, the ActRIIB binding protein provided herein is an ActRII antibody that does not have one or more effector functions. For example, in some embodiments, the anti-ActRII antibody does not have antibody-dependent cellular cytotoxicity (ADCC) activity and / or does not have complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the anti-ActRII antibody does not bind to Fc receptors and / or complement factors. In certain embodiments, the anti-ActRII antibody does not have effector functions. Examples of Fc sequence engineering modifications that reduce or eliminate ADCC and / or CDC activity and Fc receptor and / or complement factor binding are described herein or otherwise known in the art, as are assays and procedures for testing them.

[0216] In some embodiments, anti-ActRII antibodies are engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibody. In other constructs, a peptide spacer is inserted between the hinge region and the modified CH2 and / or CH3 domain. For example, compatible constructs can be expressed in which the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is linked to the hinge region by a 5-20 amino acid spacer. Such a spacer can be added, for example, to ensure that the regulatory elements of the constant domain are free and accessible or that the hinge region is flexible. Amino acid spacers have been shown to be immunogenic in some cases and may induce undesired immune responses against the construct. Thus, in certain embodiments, any spacer added to the construct can be relatively non-immunogenic or can be removed entirely to maintain the desired biochemical qualities of the modified anti-ActRII.

[0217] In additional embodiments, the anti-ActRII antibody is modified by partial deletion or substitution of a few or even single amino acids in the constant region. For example, a single amino acid mutation in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding. Similarly, one or more constant region domains that control effector functions (e.g., complement C1Q binding) may be deleted in whole or in part. Such partial deletion of the constant region may improve selected properties of the anti-ActRII antibody (e.g., serum half-life) while leaving other desirable functions associated with the corresponding constant region domain intact. In some embodiments, the constant region of the anti-ActRII antibody is modified through the mutation or substitution of one or more amino acids that enhance the profile of the resulting construct. In this regard, it is possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenicity profile of the modified anti-ActRII antibody. The disclosure also provides anti-ActRII antibodies that contain one or more amino acid additions to the constant region to enhance desirable characteristics, such as increasing or decreasing effector function, or providing a binding site for one or more cytotoxins, labels, or carbohydrate moieties, In such embodiments, it may be desirable to insert or duplicate specific sequences from selected constant region domains.

[0218] The present disclosure also provides ActRII binding proteins (e.g., murine, humanized, and human ActRII binding proteins) that are variants of the ActRIIB and ActRIIA binding proteins provided herein. In certain embodiments, the variant ActRII binding proteins (a) compete with activin A for binding to ActRIIB and / or ActRIIA, (b) reduce phosphorylation of Smads (e.g., Smad2 and / or Smad3) in cells expressing ActRIIB and / or ActRIIA in the presence of a ligand for ActRIIB and / or ActRIIA (e.g., activin A), (c) reduce phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIB and / or ActRIIA and ALK4 and / or ALK7 in the presence of a ligand for ActRIIB and / or ActRIIA, and (d) bind to ActRIIB or ActRIIA with a K of 1 nM or less and 1 pM or more. D (e.g., as determined by BIACORE® analysis). In some embodiments, the ActRII binding protein has two, three, or four of the above properties. In some embodiments, the ActRII binding protein has at least two or at least three of the above properties. In further embodiments, the variant contains conservative amino acid residue substitution mutations compared to the ActRII binding proteins provided herein.

[0219] The provided ActRII binding proteins, such as anti-ActRII antibodies, can be derivatized to contain additional chemical moieties known in the art to improve, for example, the solubility, biological half-life, bioavailability of the ActRII binding protein, and to otherwise improve its stability, formulation and / or therapeutic properties. A non-exhaustive list of such moieties can be found, for example, in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0220] Nucleic acids encoding ActRII binding proteins and their expression Also provided are nucleic acid molecules and combinations of nucleic acid molecules that code for ActRII binding proteins. In some embodiments, the nucleic acid molecules code for anti-ActRII antibodies, such as full-length anti-ActRII antibodies and ActRII-binding antibody fragments. In further embodiments, the present disclosure provides nucleic acid molecules that code for variants or derivatives of the full-length anti-ActRII antibodies or ActRII-binding antibody fragments provided herein.

[0221] The nucleic acid molecules disclosed herein may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, which may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand. In certain embodiments, the nucleic acid molecule is isolated. In additional embodiments, the nucleic acid molecule is substantially pure. In some embodiments, the nucleic acid is cDNA or derived from cDNA. In some embodiments, the nucleic acid is recombinantly produced.

[0222] In some embodiments, the nucleic acid molecule comprises an ActRII binding protein coding sequence that is functionally linked to a control sequence that controls the expression of the coding sequence in a host cell or in vitro.In certain embodiments, the coding sequence is a cDNA.The present disclosure also relates to a vector that contains the nucleic acid molecule that comprises an ActRII binding protein coding sequence that is functionally linked to a control sequence that controls the expression of the coding sequence in a host cell or in vitro.

[0223] In some embodiments, the nucleic acid molecule comprises a coding sequence for a mature ActRII binding protein fused in the same reading frame to a heterologous polynucleotide sequence. In some embodiments, the heterologous polynucleotide sequence encodes a leader peptide sequence that facilitates secretion of the expressed protein from a host cell transformed with a nucleic acid molecule(s) encoding the ActRII binding protein. Proteins containing a leader sequence are called precursor proteins, and the leader sequence can be cleaved by the host cell to form the mature form of the ActRII binding protein. Such leader peptide sequences and their use to facilitate secretion of recombinant proteins in host cells are generally known in the art. In additional embodiments, the heterologous polynucleotide sequence encodes additional 5' amino acid residues that can function, for example, to facilitate purification of the recombinantly expressed ActRII binding protein, to add or improve the stability and / or therapeutic or diagnostic properties of the protein.

[0224] In some embodiments, the disclosure provides isolated nucleic acids, such as cDNA fragments encoding ActRII binding proteins, that are sufficient for use as hybridization probes, PCR primers or sequencing primers.

[0225] In some embodiments, the nucleic acid molecule (a) competes with an ActRII ligand for binding to ActRII, (b) reduces phosphorylation of ALK4 and / or ALK7 in cells expressing ActRII and a cognate ActRI in the presence of an ActRII ligand, (c) reduces phosphorylation of one or more Smads in cells expressing ActRII in the presence of an ActRII ligand, and (d) binds ActRII with a K of 1 nM or less and 1 pM or more. D The ActRII binding protein encodes an ActRII binding protein having at least one property selected from the group consisting of: binding to an ActRII target gene (e.g., as determined by BIACORE® analysis). In some embodiments, the encoded ActRII binding protein has two, three, or four of the above properties. In some embodiments, the encoded ActRII binding protein has at least two or at least three of the above properties. In some embodiments, the encoded ActRII binding protein competes for binding to ActRII with an antibody having an ActRII-binding VH and VL pair disclosed herein. In additional embodiments, the encoded ActRII binding protein binds to the same epitope of ActRII as the antibodies disclosed herein.

[0226] In some embodiments, the nucleic acid molecule encodes an ActRII binding protein that specifically binds ActRIIA and (a) competes with an ActRIIA ligand (e.g., activin A, activin B, GDF1, GDF3, or Nodal); (b) reduces phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIA and ALK4 and / or ALK7 in the presence of an ActRIIA ligand (e.g., activin A); (c) reduces phosphorylation of one or more Smads in cells expressing ActRIIA in the presence of an ActRIIA ligand; and (d) binds ActRIIA with a K of 1 nM or less and 1 pM or more. D(e.g., as determined by BIACORE® analysis). In some embodiments, the encoded ActRIIA binding protein has two, three, or four of the above properties. In some embodiments, the encoded ActRIIA binding protein has at least two or at least three of the above properties. In some embodiments, the encoded ActRIIA binding protein competes for binding to ActRIIA with an antibody having an ActRIIA-binding VH and VL pair disclosed herein. In additional embodiments, the encoded ActRIIA binding protein binds to the same epitope of ActRIIA as an antibody disclosed herein. In further embodiments, a nucleic acid molecule encodes an ActRIIA binding protein that specifically binds to ActRII and comprises a VH and VL.

[0227] In some embodiments, the nucleic acid molecule encodes an ActRII binding protein that specifically binds ActRIIB and (a) competes with activin A and / or GDF8 for binding to ActRIIB, (b) reduces phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIB and ALK4 and / or ALK7 in the presence of an ActRIIB ligand (e.g., activin A and / or GDF8), (c) reduces phosphorylation of one or more Smads in cells expressing ActRIIB in the presence of an ActRIIB ligand, and (d) binds ActRIIB with a K of 1 nM or less and 1 pM or more. D(e.g., as determined by BIACORE® analysis). In some embodiments, the encoded ActRIIB binding protein has two, three, or four of the above properties. In some embodiments, the encoded ActRIIB binding protein has at least two or at least three of the above properties. In some embodiments, the encoded ActRIIB binding protein competes for binding to ActRIIB with an antibody having an ActRIIB-binding VH and VL pair disclosed herein. In additional embodiments, the encoded ActRIIB binding protein binds to the same epitope of ActRIIB as an antibody disclosed herein. In further embodiments, a nucleic acid molecule encodes an ActRIIB binding protein that specifically binds to ActRIIB and comprises a VH and VL.

[0228] In some embodiments, the nucleic acid molecule encodes an ActRII binding protein that specifically binds ActRIIB and ActRIIA and (a) competes with activin A and / or GDF8 for binding to ActRIIB and ActRIIA; (b) reduces phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIA and / or ActRIIB and ALK4 and / or ALK7 in the presence of an ActRIIA and / or ActRIIB ligand (e.g., activin A and / or GDF8); (c) reduces phosphorylation of one or more Smads in cells expressing ActRIIA and / or ActRIIB in the presence of an ActRIIA and / or ActRIIB ligand; and (d) binds ActRIIA or ActRIIB with a K of less than 1 nM and more than 1 pM (e.g., as determined by BIACORE® analysis). DIn some embodiments, the encoded ActRIIB- and ActRIIA-binding protein has at least one property selected from the group consisting of: binding to ActRIIB and ActRIIA at a VH and VL of at least one of the above properties. In some embodiments, the encoded ActRIIB- and ActRIIA-binding protein has two, three, or four of the above properties. In some embodiments, the encoded ActRIIB-binding protein has at least two or at least three of the above properties. In some embodiments, the encoded ActRIIB- and ActRIIA-binding protein competes for binding to ActRIIB and ActRIIA with an antibody having an ActRIIB- and ActRIIA-binding VH and VL pair disclosed herein. In additional embodiments, the encoded ActRIIB-binding protein binds to the same epitope of ActRIIA or ActRIIB as an antibody disclosed herein. In further embodiments, the nucleic acid molecule encodes a protein that specifically binds to ActRIIB and ActRIIA and comprises a VH and VL.

[0229] The present disclosure also provides vectors and sets of vectors containing nucleic acids and sets of nucleic acids encoding the ActRIIB binding proteins provided herein. Host cells transformed with these nucleic acids, sets of nucleic acids, vectors, and sets of vectors are also provided, as are methods of making and using the ActRII binding proteins.

[0230] In some embodiments, the present disclosure provides a host cell comprising a nucleic acid molecule or a combination of nucleic acid molecules or a vector as provided above, where the host cell can optionally express an ActRII binding protein that specifically binds to ActRII (e.g., an anti-ActRII antibody, such as a full-length ActRIIB antibody and an ActRII binding antibody fragment). In a further embodiment, the present disclosure provides a host cell transformed with a nucleic acid molecule or a combination of nucleic acid molecules or a vector as provided above, where the host cell can optionally express an ActRII binding protein that specifically binds to ActRII. Such a host cell can be utilized in a method of making an ActRII binding protein as provided herein, where the method includes (a) culturing the host cell, and (b) isolating the expressed ActRII binding protein from the host cell.

[0231] The present disclosure also provides a method of making an ActRII binding protein, comprising culturing a host cell (e.g., a hybridoma or a transformed mammalian host cell) capable of expressing the ActRII binding protein under suitable conditions, and optionally provides a method for isolating the ActRII binding protein secreted from the host cell. The present disclosure also further provides an ActRII binding protein isolated using the method of the present disclosure.

[0232] In certain embodiments, the polynucleotide comprises a coding sequence(s) for a mature ActRII binding protein(s) (e.g., ActRII antibodies, such as full length antibodies and ActRII binding antibody fragments) fused in the same reading frame to a marker sequence that allows for purification of the encoded polypeptide. For example, the marker sequence can be a hexa-histidine tag (SEQ ID NO: 94) provided by the pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or a hemagglutinin (HA) tag derived from influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used.

[0233] Nucleic acid variants encoding ActRII binding proteins, such as anti-ActRII antibodies and ActRII binding antibody fragments, are also provided. Nucleic acid variants can contain modifications in coding regions, non-coding regions, or both. In some embodiments, nucleic acid variants contain modifications that produce silent substitutions, additions, or deletions, but do not change the properties or activities of the encoded polypeptide. In some embodiments, nucleic acid variants are generated by silent substitutions due to the degeneracy of the genetic code. Nucleic acid variants can be generated for a variety of reasons, such as, for example, to optimize codon expression for a particular host (changing codons in human mRNA to those preferred by bacterial hosts, such as E. coli). Vectors and cells containing the nucleic acids described herein are also provided.

[0234] In some embodiments, nucleic acid sequences encoding ActRII binding proteins (e.g., anti-ActRII antibodies, such as full-length antibodies and ActRII binding antibody fragments) are constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on codon optimization based on the amino acid sequence of the desired polypeptide and host cell preferences. Standard methods can be routinely applied to synthesize isolated polynucleotide sequences encoding ActRII binding proteins.

[0235] Once constructed (by synthesis, site-directed mutagenesis or otherwise), the nucleic acid sequence encoding the ActRII binding protein can be routinely operably linked to appropriate control sequences for expression of the ActRII binding protein in a desired host. In some embodiments, the nucleic acid sequence encoding the ActRII binding protein is inserted into an expression vector and operably linked to appropriate control sequences for expression of the protein in a desired host. To obtain high levels of expression of the transgene in the host, the gene can be operably linked or associated with transcriptional and translational expression control sequences that are functional in the selected expression host.

[0236] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding ActRII binding proteins, such as anti-ActRIIB antibodies, anti-ActRIIA antibodies, ActRIIB-binding antibody fragments, or ActRIIA-binding antibody fragments. A recombinant expression vector is a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of an ActRII binding protein operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally includes a construct of (1) a genetic element(s) that plays a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences, as detailed below. Such regulatory elements may include an operator sequence that controls transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene that facilitates recognition of transformants can be further incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor involved in the secretion of that polypeptide, a promoter is operably linked to a coding sequence if it controls the transcription of that sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned to allow translation. Structural elements intended for use in yeast expression systems include leader sequences that allow extracellular secretion of the translated protein by the host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, the protein may include an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide the final protein.In certain embodiments, the disclosure provides compositions, e.g., pharmaceutical compositions, comprising a nucleic acid or vector as described above or elsewhere herein, and optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0237] Also provided are host cells transformed with the nucleic acid molecules or cDNA molecules and / or vectors disclosed herein. The disclosure also provides host cells transformed with the disclosed nucleic acid molecules or molecules operably linked to a control sequence and optionally inserted into a vector. In some embodiments, the host cell is a mammalian host cell. In further embodiments, the mammalian host cell is an NS0 mouse myeloma cell, a PER.C6® human cell, or a Chinese Hamster Ovary (CHO) cell. In other embodiments, the host cell is a hybridoma.

[0238] In additional embodiments, the present disclosure provides a method for making the ActRII binding proteins provided herein (e.g., anti-ActRII antibodies, such as full-length ActRII antibodies and ActRII-binding antibody fragments, and variants and derivatives thereof), comprising culturing a transformed host cell or hybridoma disclosed herein under suitable conditions to produce the ActRII binding protein. The present disclosure optionally provides isolating the ActRII binding protein secreted from the host cell. The present disclosure also optionally provides ActRII binding proteins produced using this method, as well as pharmaceutical compositions comprising the ActRII binding protein and a pharma- ceutically acceptable carrier.

[0239] The choice of expression control sequences and expression vectors will vary depending on the choice of host. A wide variety of expression host / vector combinations can be utilized. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids such as E. coli derived plasmids including pCR1, pBR322, pMB9 and their derivatives, as well as broad host range plasmids such as M13 and other filamentous single-stranded DNA phages.

[0240] Suitable host cells for expressing ActRII binding proteins include prokaryotic, yeast, insect or higher eukaryotic cells under suitable promoter control. Prokaryotic organisms include gram-negative or gram-positive organisms, such as E. coli or bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be used. Additional information on protein production methods, including antibody production, can be found, for example, in U.S. Patent Publication No. 2008 / 0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO04 / 009823, each of which is incorporated herein by reference in its entirety.

[0241] Various mammalian or insect cell culture systems can also be advantageously used to express recombinant ActRII binding proteins (e.g., anti-ActRII antibodies, such as full-length ActRII antibodies and ActRII binding antibody fragments, and variants and derivatives thereof). Expression of recombinant ActRII binding proteins in mammalian cells is feasible because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, the COS-7 line of monkey kidney cells described in Gluzman (Cell 23:175 (1981)), and other cell lines, such as, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors can include nontranscribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other nontranscribed sequences flanking the 5' or 3', as well as 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, etc. Baculovirus systems for production of heterologous proteins in insect cells are reviewed in Luckow and Summers, BioTechnology 6:47 (1988).

[0242] The ActRII binding protein produced by the transformed host cell or hybridoma can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and size-control column chromatography), centrifugation, differential lysis, or any other standard protein purification technique. Affinity tags such as hexahistidine (SEQ ID NO: 94), maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein so that it can be easily purified by passing through an appropriate affinity column. The ActRII binding protein can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0243] For example, supernatants from systems secreting recombinant ActRII binding proteins into the medium can be first concentrated using commercially available protein concentration filters, e.g., Amicon or Millipore Pellicon ultrafiltration units. Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be used, e.g., a matrix or substrate with pendant diethylaminoethyl (DEAE) groups. The matrix can be acrylamide, agarose, dextran, cellulose, or other types of matrices commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include a variety of insoluble matrices that contain sulfopropyl or carboxymethyl groups. Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps using hydrophobic RP-HPLC media, e.g., silica gel with pendant methyl or other aliphatic groups, can be used to further purify the ActRII binding protein. Some or all of the above purification steps, in various combinations, can also be routinely used to obtain homogeneous recombinant ActRII binding proteins.

[0244] Recombinant ActRII binding proteins (e.g., anti-ActRII antibodies, such as full-length ActRII antibodies and ActRII binding antibody fragments, and variants and derivatives thereof) produced in bacterial culture can be isolated, for example, by initial extraction from a cell pellet, followed by one or more concentration, salting out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be used in a final purification step. Microbial cells used in expression of recombinant proteins can be disrupted in any convenient manner, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0245] Methods known in the art for purifying target binding proteins, such as full-length antibodies and antigen-binding antibody fragments, include, for example, those described in U.S. Application Publication Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is incorporated herein by reference in its entirety.

[0246] In certain embodiments, the ActRII binding protein is not an antibody. A variety of methods are known for identifying and generating non-antibody polypeptides that bind to protein targets with high affinity. See, for example, Skerra, Curr. Opin. Biotechnol. 18:295-304 (2007); Hosse et al., Protein Science 15:14-27 (2006); Gill et al., Curr. Opin. Biotechnol. 17:653-658 (2006); Nygren, FEBS J. 275:2668-2676 (2008); and Skerra, FEBS J. 275:2677-2683 (2008), each of which is incorporated herein by reference in its entirety. In certain embodiments, phage display technology is used to identify / generate ActRII binding proteins. In certain embodiments, the polypeptide comprises a type of protein scaffold selected from the group consisting of protein A, lipocalin, a fibronectin domain (e.g., fibronectin type III (Fn3)), a consensus ankyrin repeat domain, and thioredoxin.

[0247] Methods of Use and Pharmaceutical Compositions The provided ActRII binding proteins (including antibodies, immunoconjugates, and polypeptides) are useful in a variety of applications, including, but not limited to, methods of diagnosing and treating and / or ameliorating various diseases and conditions using ActRII binding proteins (e.g., anti-ActRIIB and ActRIIA antibodies). Methods are provided for the use of ActRII binding proteins (e.g., anti-ActRII antibodies, such as full-length antibodies and ActRII-binding antibody fragments that specifically bind ActRII, and variants and derivatives thereof) to treat subjects with diseases or conditions associated with ActRII (e.g., ActRIIB and / or ActRIIA) signaling and / or increased ActRII expression. In additional embodiments, the present disclosure provides pharmaceutical compositions comprising the ActRII binding proteins provided herein and a pharma- ceutical acceptable carrier. In some embodiments, the present disclosure provides pharmaceutical compositions comprising the ActRII binding proteins provided herein and a pharma-ceutical acceptable carrier for use as a medicament. The present disclosure also provides for the use of the pharmaceutical compositions disclosed herein to treat and / or ameliorate a disease or condition associated with ActRII, increased ActRII expression and / or increased ActRII signaling. In some embodiments, the disease or condition treated using the pharmaceutical compositions provided herein is a muscle disorder, such as muscle wasting due to disease or disuse. In additional embodiments, the disease or condition treated using the pharmaceutical compositions provided herein is a fibrotic condition (e.g., a hepatic, pulmonary, vascular and / or ocular fibrotic condition); an inflammatory, cardiovascular, pulmonary, musculoskeletal, neurological, or metabolic disease or condition; wound healing; or cancer.

[0248] In some embodiments, the pharmaceutical composition contains an ActRII binding protein (e.g., a full-length antibody that specifically binds ActRIIB and a full-length antibody that specifically binds ActRIIA) and a pharma- ceutically acceptable carrier, and further includes a labeling group or an effector group. A "label" refers to one or more elements, isotopes, or chemicals that are attached to allow detection in screening. Labels are generally classified into three classes: (a) isotopic labels, which may be radioactive or heavy isotopes; (b) small molecule labels, which may include molecules such as biotin that allow fluorescent and colorimetric dyes or other labeling methods; and (c) immunolabels, which may be epitopes incorporated as fusion partners recognized by antibodies; a "labeling group" refers to any detectable label. In some embodiments, the labeling group is attached to the ActRII binding protein via a spacer (e.g., a peptide spacer) to reduce potential steric hindrance. The label may be incorporated into the compound at any position, and may be incorporated either in vitro or in vivo during protein expression. Various methods for labeling proteins are known in the art and may be used in practicing the provided methods. In additional embodiments, the labeling group is selected from the group consisting of an isotope label, a magnetic label, an oxidative (reducing fibrosis) active moiety, an optical dye, a biotinylation group, and a polypeptide epitope recognized by a secondary reporter. In some embodiments, the labeling group is a fluorescent protein, such as green fluorescent protein or a derivative thereof (e.g., enhanced GFP, blue fluorescent protein or a derivative thereof (e.g., EBFP (enhanced blue fluorescent protein), EBFP2, Azurite, mKalama1, cyan fluorescent protein or a derivative thereof (e.g., ECFP (enhanced cyan fluorescent protein), Cerulean, CyPet), yellow fluorescent protein or a derivative thereof (e.g., YFP, Citrine, Venus, YPet). In some embodiments, the polypeptide epitope is a member selected from biotin signaling peptide, histidine peptide (his), hemagglutinin (HA), Flag, and gold binding peptide.In additional embodiments, the effector group is selected from the group consisting of a radioisotope, a radionucleotide, a toxin, a therapeutic agent, and a chemotherapeutic agent.

[0249] The ActRII binding protein of the present disclosure has applications in in vitro and in vivo diagnostic and therapeutic utility.For example, the ActRII binding protein can be administered to, for example, cells in culture in vitro or in vivo, or to cells of a subject, to treat, prevent, or diagnose various diseases or conditions.In some embodiments, the ActRII binding protein is a human antibody, a mouse antibody, or a humanized antibody.

[0250] Also provided are methods of blocking ActRII activity. In some embodiments, the methods include contacting ActRII with an ActRII binding protein. In some examples, the methods are performed in vivo. In other cases, the methods are performed in vitro. In some embodiments, the blocked ActRII activity is selected from (a) binding by an ActRII ligand (e.g., activin A, activin B, GDF8 (myostatin), GDF11, BMP6, GDF3, BMP9, or BMP10), (b) phosphorylation of one or more Smads in cells expressing ActRII in the presence of activin A, and (c) phosphorylation of ALK4 and / or ALK7 in cells expressing ActRII and ALK4 and / or ALK7 in the presence of an ActRII ligand.

[0251] In some aspects, a method of blocking ActRIIA activity is provided. In further embodiments, the method includes contacting ActRIIA with an ActRIIA binding protein. In some examples, the method is performed in vivo. In other cases, the method is performed in vitro. In some embodiments, the blocked ActRIIA activity is selected from (a) binding by an ActRIIA ligand (e.g., activin A, activin B, GDF1, GDF3, or Nodal), (b) phosphorylation of one or more Smads in cells expressing ActRIIA in the presence of activin A, and (c) phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIA and ALK4 and / or ALK7 in the presence of an ActRIIA ligand.

[0252] In some embodiments, a method of blocking ActRIIB activity is provided. In further embodiments, the method includes contacting ActRIIB with an ActRIIB binding protein. In some cases, the method is performed in vivo. In other cases, the method is performed in vitro. In some embodiments, the blocked ActRIIB activity is selected from (a) binding by an ActRIIB ligand (e.g., activin A, activin B, GDF8 (myostatin), GDF11, BMP6, GDF3, BMP9, or BMP10); (b) phosphorylation of one or more Smads in cells expressing ActRIIA in the presence of activin A; (c) phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIA and ALK4 and / or ALK7 in the presence of an ActRIIB ligand.

[0253] In one aspect, the disclosure provides treatment, prevention and / or amelioration of a disease or condition comprising administering an ActRII binding protein (e.g., a full-length antibody that specifically binds ActRIIB and a full-length antibody that specifically binds ActRIIA) to a subject having or at risk of developing a disease or condition associated with elevated ActRII expression and / or ActRII signaling. In some embodiments, the treatment comprises administering an ActRII binding protein to isolated tissue or cells from a subject, where the subject has or is at risk of developing a disease or condition associated with ActRII expression or ActRII signaling. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for the treatment of a disease or condition associated with ActRII expression or ActRII signaling.

[0254] The present disclosure provides a pharmaceutical composition comprising an ActRII binding protein and a pharma- ceutically acceptable carrier. Also provided is a method for treating and / or ameliorating a condition associated with ActRII (e.g., ActRIIA or ActRIIB)-mediated activity in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an ActRII binding protein provided herein. In some embodiments, the ActRII binding protein is administered alone. In other embodiments, the ActRII binding protein is administered as a combination therapy. Also provided is a method for reducing ActRII activity in a subject, comprising administering to a subject in need thereof an effective amount of an ActRII binding protein.

[0255] The present disclosure also provides methods of treating and / or ameliorating a disease or condition associated with a muscle disorder. In some embodiments, the muscle disorder is debilitating. In further embodiments, the debilitating is due to disease or disuse. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an ActRII binding protein (e.g., an antibody that specifically binds ActRIIB, an antibody that specifically binds ActRIIA, or an antibody that specifically binds ActRIIB and ActRIIA). In additional embodiments, the ActRII binding protein is administered alone or as a combination therapy.

[0256] According to some embodiments, the present disclosure provides a method of inducing skeletal muscle formation in a subject. In some embodiments, the method comprises administering an ActRIIB binding protein (e.g., an anti-ActRIIB antibody, such as a full-length ActRIIB antibody and an ActRIIB binding antibody fragment) to a subject in need thereof. In some embodiments, the method increases muscle mass and strength in the subject.

[0257] The disclosure also provides methods of treating and / or ameliorating a disease or condition associated with a muscle disorder, such as a degenerative muscle disease, a muscular dystrophy, a muscle atrophy, or a muscle wasting disorder; a fibrotic condition (e.g., a fibrotic condition of the liver, lung, blood vessels, and / or eye, such as myocardial fibrosis, and idiopathic pulmonary fibrosis (IPF)); a metabolic disease (e.g., type II diabetes insulin resistance, hyperglycemia, and obesity); an inflammatory disease or condition, an autoimmune disease, a cardiovascular disease (e.g., congestive heart failure, and hypertension); an eye disease, such as age-related macular degeneration; a pulmonary disease, a musculoskeletal disease, a skeletal disease, such as osteoporosis; a neurological disease; wound healing; weight loss; and a disease or condition associated with cancer (e.g., carcinoma, myeloma, bone loss-induced cancer, pituitary cancer, and gastrointestinal cancer) in a subject. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an ActRII binding protein (e.g., an antibody that specifically binds ActRIIB, an antibody that specifically binds ActRIIA, or an antibody that specifically binds ActRIIB and ActRIIA). In additional embodiments, the ActRII binding protein is administered alone or as a combination therapy. Further provided is an ActRII binding protein for use in treating a subject having or at risk of developing a disease or condition associated with ActRII expression or ActRII signaling.

[0258] The present disclosure also provides a method of reducing the activity of ActRII (e.g., ActRIIA or ActRIIB), such as signal transduction, in a subject. In some embodiments, the method comprises administering to a subject in need thereof (e.g., a subject diagnosed with muscle wasting; a fibrotic condition (e.g., a fibrotic condition of the liver, lung, blood vessel, and / or eye); an inflammatory, cardiovascular, pulmonary, musculoskeletal (i.e., bone and / or muscle), neurological, or metabolic disease or condition; wound healing; or cancer) an effective amount of an ActRII binding protein (e.g., an antibody that specifically binds ActRIIB, an antibody that specifically binds ActRIIA, or an antibody that specifically binds ActRIIB and ActRIIA) or an effective amount of a pharmaceutical composition comprising an ActRII binding protein.

[0259] In one aspect, the disclosure provides a method of treating and / or ameliorating a muscle disorder in a subject. In some cases, the method includes administering an ActRII binding protein (e.g., an antibody that specifically binds ActRIIB, an antibody that specifically binds ActRIIA, or an antibody that specifically binds ActRIIB and ActRIIA) to a subject with a muscle disorder. In other embodiments, the subject is at risk of developing a muscle disorder. In some embodiments, the muscle disorder or condition is muscle atrophy. In further embodiments, the muscle atrophy is a condition associated with glucocorticoid treatment, such as treatment with cortisol, dexamethasone, betamethasone, prednisone, methylprednisolone, or prednisolone. In additional embodiments, the muscle atrophy is a condition associated with nerve trauma or as a result of degenerative, metabolic, or inflammatory neuropathy (e.g., Guillain-Barre syndrome, peripheral neuropathy, or exposure to environmental toxins or drugs). In additional embodiments, the muscle atrophy is a condition associated with adult motor neuron disease, infantile spinal muscular atrophy, amyotrophic lateral sclerosis, juvenile spinal muscular atrophy, autoimmune motor neuropathy with multifocal conductor block, paralysis due to stroke or spinal cord injury, skeletal immobilization due to trauma, prolonged bed rest, voluntary inactivity, forced inactivity, metabolic overload or nutritional deficiency, cancer, AIDS, fasting, thyroid disorders, diabetes, benign congenital hypotonia, central core disease, burns, chronic obstructive pulmonary disease, liver disease (e.g., fibrosis, cirrhosis, etc.), sepsis, congestive heart failure, aging, space travel or time spent in a zero gravity environment.

[0260] In some embodiments, the muscle disorder treated and / or ameliorated is muscle atrophy associated with myopathy. In further embodiments, the myopathy is selected from the group consisting of mitochondrial myopathy; metabolic myopathy caused by glycogen storage disease or lipid storage disease; congenital myopathy, such as nemaline myopathy, multi / minicore myopathy, and myotubular myopathy; myotonia; familial periodic paralysis; and inflammatory myopathy. In additional embodiments, the myopathy is a condition associated with muscular dystrophy syndrome, such as Duchenne, Becker, myotonia, facioscapulohumeral, Fukuyama, limb-girdle, scapulohumeral, Emery-Dreyfus, oculopharyngeal, etc., Charcot-Marie-Tooth disease (CMT), congenital muscular dystrophy, or hereditary distal myopathy. The provided ActRII binding proteins can be used to treat inclusion body myositis, myoglobinuria, rhabdomyolysis, myositis ossificans, polymyositis, or dermatomyositis.Furthermore, the provided ActRII binding proteins can treat or prevent muscle atrophy resulting from glucocorticoid treatment, sarcopenia, prolonged bed rest, skeletal immobilization, sepsis, or congestive heart failure.

[0261] In another aspect, the present disclosure provides a method for treating and / or ameliorating muscular dystrophy. The term "muscular dystrophy" refers to a group of degenerative muscle diseases characterized by gradual weakening and deterioration of skeletal muscles, and sometimes cardiac and respiratory muscles. Exemplary muscular dystrophies that can be treated and / or ameliorated by the ActRII binding proteins and pharmaceutical compositions provided herein include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifes muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSH or FSHD) (also known as Landauzy-Djerin), myotonic muscular dystrophy (MMD) (also known as Steinert disease), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital muscular dystrophy (CMD), and scapulohumeral muscular dystrophy (SMD).

[0262] In another aspect, the disclosure provides a method of treating and / or ameliorating a fibrotic condition (e.g., fibrosis). In some cases, the method includes administering to a subject having a fibrotic condition an ActRII binding protein (e.g., an antibody that specifically binds ActRIIB, an antibody that specifically binds ActRIIA, or an antibody that specifically binds ActRIIB and ActRIIA). In other embodiments, the subject is at risk of developing a fibrotic condition. In further embodiments, the fibrotic condition is DN. In some embodiments, the fibrotic condition being treated is primary fibrosis. In some embodiments, the fibrotic condition being treated is idiopathic. In some embodiments, the fibrotic condition being treated is chronic. In some embodiments, the fibrotic condition being treated is systemic. In other embodiments, the fibrotic disease or condition being treated is a condition associated with (e.g., secondary to) a disease (e.g., an infectious disease, an inflammatory disease, an autoimmune disease, a malignant or cancerous disease, and / or a connective disease); a toxin; an injury (e.g., an environmental hazard (e.g., asbestos, coal dust, polycyclic aromatic hydrocarbons), smoking, a wound); or a medical procedure (e.g., a surgical incision, chemotherapy, or radiation).

[0263] Fibrotic conditions that may be treated and / or ameliorated by the ActRII binding proteins provided herein include, but are not limited to, fibrosis, liver injury (e.g., liver injury caused by alcohol and viral infections such as Hepatitis B and C), pulmonary fibrosis (e.g., cystic fibrosis, IPF or pulmonary fibrosis caused by smoking, environmental hazards and chemotherapy drugs such as bleomycin), radiation-induced fibrosis, injection-associated fibrosis, vascular fibrosis, atherosclerosis, pancreatic fibrosis, musculoskeletal fibrosis, and osteoporosis. These include, but are not limited to, fibrosis of the muscularis (e.g., myofibrosis), myocardial fibrosis, dermal fibrosis, scleroderma, ocular fibrosis (e.g., age-related macular degeneration, diabetic macular edema, diabetic retinopathy, and dry eye), progressive systemic sclerosis (PSS), chronic graft-versus-host disease, Peyronie's disease, urethral stenosis after cystoscopy, retroperitoneal fibrosis, mediastinal fibrosis, progressive massive fibrosis, proliferative fibrosis, neoplastic fibrosis, Dupuytren's disease, stenosis, pleural fibrosis, sarcoidosis, spinal cord injury / fibrosis, and myelofibrosis.

[0264] Also provided is a method of reducing fibrosis in a subject. In some embodiments, the present disclosure provides a method of reducing fibrosis in a subject, comprising administering an ActRII binding protein (e.g., in a pharmaceutical composition as described herein) to a subject with fibrosis. Such reduced fibrosis can be reflected, for example, in fibrosis reduction and a reduction in signs or conditions associated with fibrosis, including, for example, a reduction in the development of fibrotic lesions, a reduction in weight loss or other clinical symptoms, and / or a change in biomolecule expression (e.g., mRNA or protein expression) associated with the development of the fibrotic condition being treated. In some embodiments, the fibrosis is hepatic, muscular, or pulmonary fibrosis. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for the treatment of fibrosis.

[0265] In another aspect, the present disclosure provides a method for reducing fibrosis of a cell or tissue. The method includes contacting a fibrotic cell or tissue with an ActRII binding protein (e.g., alone or in combination with another agent or treatment modality) in an amount sufficient to reduce or inhibit fibrosis. These methods can be performed in vitro or in vivo. In some embodiments, the method is performed in vivo, for example, in a mammalian subject (e.g., an animal model). In some embodiments, the subject is a human. In some embodiments, reducing fibrosis includes (a) reducing or inhibiting the formation or deposition of tissue fibrosis, (b) reducing the size, cellularity (e.g., number of fibroblasts or immune cells), composition; or cellular content of a fibrotic lesion, (c) reducing the collagen content or hydroxyproline content of a fibrotic lesion, (d) reducing the expression or activity of one or more fibrogenic proteins, and / or (e) reducing fibrosis associated with an inflammatory response. In some embodiments, reducing fibrosis includes (a) reducing or inhibiting the formation or deposition of tissue fibrosis; (b) reducing the size, cellularity (e.g., number of fibroblasts or immune cells), composition, or cellular content of fibrotic lesions; (c) reducing the collagen or hydroxyproline content of fibrotic lesions; (d) decreasing the expression or activity of one or more fibrogenic proteins; and / or (e) reducing fibrosis associated with inflammation.

[0266] According to some embodiments, the disclosure provides a method of inhibiting loss of liver or lung function in a subject. In some embodiments, the method comprises administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as a full-length ActRII antibody and an ActRII binding antibody fragment) to a subject in need thereof. In some embodiments, the method inhibits loss of liver function in the subject. In further embodiments, the method inhibits loss of liver function in the subject through reduction of liver fibrosis. In some embodiments, the method inhibits loss of lung function in the subject. In some embodiments, the method inhibits loss of lung function in the subject through reduction of lung fibrosis. In some embodiments, the method inhibits loss of lung function and / or lung fibrosis in a subject having or at risk of developing idiopathic pulmonary fibrosis (IPF).

[0267] Further provided is a method for improving liver or lung function by reducing fibrosis in a subject. In some cases, the method includes administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as full-length ActRII antibody and ActRII binding antibody fragment, and variants and derivatives thereof) or a pharmaceutical composition provided herein to a subject in need thereof. In some embodiments, suppressing or improving loss of liver or lung function includes (a) reducing or inhibiting the formation or deposition of tissue fibrosis in the corresponding organ; (b) reducing the size, cellularity (e.g., the number of fibroblasts or immune cells), composition; or cellular content of fibrotic lesions in the corresponding organ; (c) reducing the collagen content or hydroxyproline content of fibrotic lesions in the corresponding organ; (d) reducing the expression or activity of one or more fibrogenic proteins (e.g., fibrinogen and collagen) in the corresponding organ; (d) reducing the extracellular matrix expression and / or EMT in the corresponding organ; and / or (e) reducing the fibrosis associated with inflammatory response in the corresponding organ.

[0268] The human body responds to trauma and injury by scarring. Fibrosis, a type of disorder characterized by excessive scarring, occurs when the normal wound healing response is disrupted. During fibrosis, the wound healing response continues, causing excessive production and deposition of collagen. In another embodiment, the present disclosure provides a method for treating fibrosis, comprising administering to a subject in need thereof a therapeutically effective amount of an ActRII binding protein (e.g., an antibody that specifically binds ActRIIB or an antibody that specifically binds ActRIIA).

[0269] In some aspects, the present disclosure provides a method for suppressing or improving the loss of liver function or lung function.In some embodiments, the method results in (a) reducing or inhibiting the formation or deposition of tissue fibrosis in corresponding organs; (b) reducing the size, cellularity (e.g., the number of fibroblasts or immune cells), composition; or cellular content of fibrotic lesions in corresponding organs; (c) reducing the collagen content or hydroxyproline content of fibrotic lesions in corresponding organs; (d) reducing the expression or activity of one or more fibrogenic proteins (e.g., fibrinogen and collagen) in corresponding organs; (d) reducing the extracellular matrix expression and / or EMT in corresponding organs; and / or (e) reducing the fibrosis associated with inflammatory response in corresponding organs.

[0270] The present disclosure also provides a method of treating and / or ameliorating a pulmonary fibrotic condition. In some embodiments, the method comprises administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as an antibody and fragment thereof that specifically binds ActRII, and variants and derivatives thereof) to a subject having or at risk of developing a pulmonary fibrotic condition. In some embodiments, the pulmonary fibrosis is idiopathic, pharmacologically induced, radiation induced, chronic obstructive pulmonary disease (COPD), or chronic asthma. The pulmonary fibrotic conditions that may be treated include one or more members of the group consisting of usual interstitial pneumonia (UIP), interstitial lung disease, idiopathic fibrosing alveolitis (CFA), and bronchiectasis. In some embodiments, the pulmonary fibrotic condition to be treated is a condition associated with a pulmonary inflammatory disease, such as asthma, and / or chronic obstructive pulmonary disease (COPD).

[0271] In certain embodiments, the present disclosure provides a method for treating and / or ameliorating pulmonary fibrosis, comprising administering an ActRII binding protein to a subject having or at risk of developing pulmonary fibrosis. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or ameliorating pulmonary fibrosis.

[0272] In some embodiments, the pulmonary fibrotic condition treated by the ActRII binding protein (e.g., and anti-ActRIIA antibodies and anti-ActRIIB antibodies) is a member selected from the group consisting of acute respiratory distress syndrome, chronic asthma, acute pulmonary syndrome, bronchopulmonary dysplasia, pulmonary hypertension (e.g., idiopathic pulmonary hypertension (IPH)), histiocytosis X pneumoconiosis, Kaplan's disease, rheumatoid disease, and systemic sclerosis.

[0273] In some embodiments, the pulmonary fibrotic condition treated by the ActRII binding proteins (e.g., and anti-ActRIIA antibodies and anti-ActRIIB antibodies) provided herein is a condition associated with an autoimmune connective tissue disorder. In some embodiments, the autoimmune connective tissue disorder is selected from the group consisting of sarcoidosis, rheumatoid arthritis, scleroderma, and systemic lupus erythematosus (SLE). In additional embodiments, the pulmonary fibrotic condition is a condition associated with a disease, a toxin, an injury, or a medical treatment. Thus, in some embodiments, the pulmonary fibrotic condition is a condition associated with one or more members of the group consisting of exposure to toxins and irritants, such as workplace hazards (e.g., dust, asbestos, silica, bauxite, iron, cotton, talc, and coal dust), toxins (e.g., amiodarone, carmustine, chloramphenicol, hexamethonium), inhalation of tobacco smoke, and environmental pollutants. In additional embodiments, the pulmonary fibrotic condition treated is a condition associated with an infection. In certain embodiments, the infection is a condition associated with a chronic infection.

[0274] In additional embodiments, the pulmonary fibrotic condition being treated is a condition associated with a medical treatment. In certain embodiments, the medical treatment is selected from surgery, radiation therapy, and drug therapy. In further embodiments, the drug therapy is chemotherapy. In further embodiments, the chemotherapy involves administration of a chemotherapeutic agent selected from bleomycin, methotrexate, amiodarone, busulfan, nitrosoureas, and nitrofurantoin.

[0275] Also provided are methods for treating and / or ameliorating pulmonary hypertension or idiopathic pulmonary fibrosis (IPF). In some cases, the methods include administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as a full-length ActRII antibody and an ActRII binding antibody fragment, and variants and derivatives thereof) to a subject having or at risk of developing pulmonary hypertension or IPF. In some cases, the ActRII binding protein or a pharmaceutical composition comprising the ActRII binding protein is administered to treat, prevent, and / or ameliorate pulmonary hypertension. In some cases, the ActRII binding protein or a pharmaceutical composition comprising the ActRII binding protein is administered to treat, prevent, and / or ameliorate IPF. In some embodiments, the ActRII binding protein or a pharmaceutical composition comprising the ActRII binding protein is administered to a subject having or at risk of developing pulmonary hypertension or IPF.

[0276] The present disclosure also provides a method for treating and / or ameliorating a liver fibrotic condition. In some embodiments, the method comprises administering an ActRII binding protein or an effective amount of a pharmaceutical composition comprising an ActRII binding protein to a subject having or at risk of developing a liver fibrotic condition. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or ameliorating a liver fibrotic condition. The liver fibrotic condition that can be treated using the ActRII binding protein provided herein includes one or more members of the group consisting of steatosis (e.g., nonalcoholic steatohepatitis (NASH)), fatty liver disease, cholestatic liver disease (e.g., primary biliary cirrhosis (PBC)), cirrhosis, alcoholic liver fibrosis, infection-induced liver fibrosis, bile duct injury, bile duct fibrosis, congenital liver fibrosis, autoimmune hepatitis, and cholangiopathy. In further embodiments, the infection-induced liver fibrosis is bacterial-induced or viral-induced.

[0277] In an additional embodiment, the liver fibrotic condition that may be treated by the ActRII binding proteins provided herein is one or more members of the group consisting of liver fibrosis associated with viral infection (e.g., hepatitis (hepatitis C, B and D), autoimmune hepatitis, nonalcoholic fatty liver disease (NAFLD), progressive massive fibrosis, alcoholism, and exposure to toxins or irritants (e.g., alcohol, pharmaceuticals and environmental toxins).

[0278] The present disclosure also provides a method of treating and / or ameliorating myocardial fibrosis. In some embodiments, the method comprises administering an ActRII binding protein or an effective amount of a pharmaceutical composition comprising an ActRII binding protein to a subject having or at risk of developing a cardiovascular fibrotic condition. In some embodiments, the myocardial fibrosis is endomyocardial fibrosis or idiopathic cardiomyopathy. In some embodiments, the skin fibrosis is scleroderma, post-traumatic, post-operative skin scarring, keloids, or skin keloid formation. In some embodiments, the ocular fibrosis is glaucoma, ocular sclerosis, conjunctival scarring, corneal scarring, or pterygium. In some embodiments, the retroperitoneal fibrosis is idiopathic, pharmacologically induced, or radiation induced. In some embodiments, the cystic fibrosis is pancreatic cystic fibrosis or pulmonary cystic fibrosis. In some embodiments, the injection-associated fibrosis occurs as a complication of intramuscular injection. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for the treatment or amelioration of a fibrotic condition of the cardiovascular system.

[0279] Also provided is a method for treating and / or ameliorating an ocular disease or condition, comprising administering an ActRII binding protein to a subject in need thereof. In certain embodiments, the ocular disease or condition is glaucoma. In some embodiments, the ocular disease is retinopathy. In further embodiments, the ocular disease is diabetic retinopathy.

[0280] In additional embodiments, the present disclosure provides a method for treating and / or ameliorating an ocular fibrotic condition (e.g., ocular fibrosis, ocular fibrosis, and fibrosis associated with retinal dysfunction). Thus, in some cases, the method includes administering an ActRII binding protein to a subject having or at risk of developing an ocular fibrotic condition. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or ameliorating a fibrotic condition of a cardiovascular fibrotic condition.

[0281] Ocular fibrotic conditions that may be treated according to the methods provided herein may arise in response to injury, such as mechanical wounds (e.g., fibrosis associated with alkali burns) or various metabolic dysfunctions (including, for example, responses to inflammation, ischemia, and degenerative diseases). In some embodiments, the present disclosure provides methods for treating fibrosis associated with ocular surgery. In further embodiments, the fibrosis is a condition associated with post-operative scarring in ophthalmic conditions. In further embodiments, the post-operative scarring is a condition associated with surgery involving retinal reattachment, cataract extraction, or drainage procedures.

[0282] In some embodiments, the present disclosure provides methods of treating and / or ameliorating ocular fibrotic conditions associated with one or more members of the group consisting of macular edema (e.g., diabetic macular edema), dry eye, lens fibrosis, corneal stromal or endothelial fibrosis, corneal and conjunctival scarring, fibrovascular scarring, retinal fibrosis, and retinal gliosis.

[0283] In some embodiments, the present disclosure provides a method for treating an ocular fibrotic condition associated with macular degeneration. In some embodiments, the fibrotic condition being treated is a condition associated with age-related macular degeneration. In some embodiments, the condition being treated is a condition associated with wet macular degeneration. In other embodiments, the condition being treated is a condition associated with dry macular degeneration.

[0284] In some embodiments, the present disclosure provides a method for treating and / or ameliorating an inflammatory disease or condition, comprising administering an ActRII binding protein to a subject in need thereof. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or ameliorating an inflammatory disease or condition. In some embodiments, the inflammatory disease or condition is an inflammatory cancer, inflammation associated with fibrosis, inflammation associated with atherosclerosis, asthma, or inflammation associated with an autoimmune disorder.

[0285] Further provided is a method for treating and / or improving a cardiovascular disease or condition. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or improving a cardiovascular disease or condition. In some cases, the method comprises treating or improving a cardiovascular disease or condition by administering an ActRII binding protein to a subject in need thereof. In some embodiments, the cardiovascular disease or condition is anemia, congestive heart failure, ventricular dysfunction, vascular calcification, pulmonary hypertension, arterial restenosis, or myocardial fibrosis.

[0286] In some embodiments, the present disclosure provides a method for treating and / or ameliorating a pulmonary disease or condition, comprising administering an ActRII binding protein to a subject in need thereof. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or ameliorating a pulmonary disease or condition.

[0287] In some embodiments, the present disclosure provides a method for treating and / or improving a musculoskeletal disease or condition, comprising administering an effective dose of an ActRII binding protein to a subject in need thereof. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or improving a musculoskeletal disease or condition. Exemplary ActRIIB-associated conditions that can be treated and / or improved by administering an effective dose of an ActRII binding protein (e.g., an anti-ActRIIB antibody) include neuromuscular disorders (e.g., muscular dystrophy and muscle atrophy), congestive obstructive pulmonary disease or emphysema (and associated muscle wasting), muscle wasting syndrome, sarcopenia, cachexia, adipose tissue disorders (e.g., obesity), type 2 diabetes, and bone degenerative diseases (e.g., osteoporosis). Provided herein is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or improving each of these diseases or conditions.

[0288] Other exemplary ActRII-associated conditions that can be treated and / or ameliorated by administering an effective dose of an ActRII binding protein (e.g., an anti-ActRIIB antibody) include muscle degenerative and neuromuscular disorders, and osteoporosis.

[0289] The provided ActRII binding protein provides an effective means for increasing muscle mass in other neuromuscular diseases or conditions that require muscle growth.For example, in amyotrophic lateral sclerosis (ALS).Other neuromuscular diseases that ActRII binding protein can be useful include paralysis caused by spinal cord injury or stroke; denervation caused by trauma or degenerative, metabolic, or inflammatory neuropathy; adult motor neuron disease; autoimmune motor neuropathy with multifocal conductor block; and infantile or juvenile spinal muscular atrophy.

[0290] In other aspects, the present disclosure provides methods for inducing bone and / or cartilage formation, preventing bone loss, increasing bone mineralization, or preventing bone demineralization.For example, the provided ActRII binding proteins are used to treat osteoporosis and heal bone fractures and cartilage defects in subjects (e.g., humans and other animals).In some embodiments, the present disclosure provides methods for healing bone fractures or cartilage in subjects.In some embodiments, the provided methods and compositions are administered to treat conditions that cause bone loss, such as osteoporosis, hyperparathyroidism, Cushing's disease, thyrotoxicosis, chronic diarrheal conditions or malabsorption, or anorexia nervosa.

[0291] In an additional aspect, the present disclosure provides a method for treating neurological disorders or conditions, comprising administering ActRII binding protein to a subject in need thereof.Further provided is the use of ActRII binding protein as provided herein in the manufacture of a medicament for treating or improving neurological disorders or conditions.In some embodiments, the neurological disorders or conditions are associated with neuronal cell death.In some embodiments, the neurological disorders or conditions are Parkinson's disease, ALS, brain atrophy, or dementia.

[0292] In an additional aspect, the present disclosure provides a method for treating a metabolic disorder or condition, comprising administering an ActRII binding protein to a subject in need thereof.Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or improving a metabolic disorder or condition.In some embodiments, the metabolic disorder or condition is a condition associated with diabetes.In some embodiments, the metabolic disorder or condition is obesity.In further embodiments, the metabolic disorder or condition is hypertrophic obesity.In some embodiments, the metabolic disorder or condition is cancer cachexia or muscle wasting.

[0293] In other aspects, the present disclosure provides locations and methods for regulating body fat mass in a subject and for treating or preventing conditions associated therewith, particularly unhealthy conditions associated therewith.

[0294] As provided herein, regulating (managing) weight can refer to losing or gaining weight, decreasing or increasing the rate of weight gain, or increasing or decreasing the rate of weight loss, including actively maintaining or not significantly changing weight (e.g., in response to external or internal influences that may otherwise cause weight to increase or decrease). According to one aspect, the present disclosure provides a method of regulating weight by administering an ActRII binding protein provided herein to a subject (e.g., a human) in need thereof. In one aspect, the present disclosure provides a method for reducing weight and / or reducing weight gain in a subject, more particularly, a method for treating or ameliorating obesity in a patient at risk of or suffering from obesity. In another aspect, the present disclosure provides methods and compounds for treating a subject (e.g., an animal with a wasting syndrome) who is unable to gain or maintain weight. Such methods are effective for increasing weight and / or mass, or for reducing weight and / or mass loss, or for ameliorating conditions associated with or caused by undesirably low (e.g., unhealthy) weight and / or mass. The provided ActRIIB binding proteins can further be used as therapeutic agents for slowing or preventing the onset of type II diabetes and metabolic syndrome.

[0295] In certain aspects, the present disclosure provides a method for treating and / or ameliorating a condition associated with diabetes, comprising administering an ActRII binding protein to a subject having or at risk of developing diabetes and / or a condition associated with diabetes. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or ameliorating diabetes or a condition associated with diabetes. In some embodiments, the condition associated with diabetes is diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, diabetic angiopathy or diabetic microangiopathy.

[0296] In a further aspect, the present disclosure provides a method for promoting wound healing, comprising administering an ActRII binding protein to a subject in need thereof.In some embodiments, the ActRII binding protein is administered to a subject to reduce scar formation associated with wound healing.In some embodiments, the ActRII binding protein is administered to a subject at risk of developing hypertrophic scar or keloid.

[0297] Further provided are methods of antagonizing ActRII activity in pathological conditions associated with ActRII expression and / or ActRII signaling. In some cases, the method includes administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as a full-length anti-ActRII antibody or an ActRII binding antibody fragment) to a subject in need thereof. In some embodiments, the pathological condition is a musculoskeletal disease or disorder, such as muscle atrophy. In some embodiments, the pathological condition is a fibrotic disease, for example, of the lung or liver. In further embodiments, the pathological condition is diabetes. In some embodiments, the pathological condition is obesity (e.g., hypertrophic obesity). In additional embodiments, the pathological condition is pulmonary hypertension or idiopathic pulmonary fibrosis (IPF). In some embodiments, the pathological condition is an ocular disease, such as diabetic retinopathy. In some embodiments, the pathological condition is a cancer, such as carcinoma (e.g., basal cell and squamous cell carcinoma of the skin, head and neck cancer, and renal cell carcinoma), myeloma (e.g., multiple myeloma), colon cancer, or bone loss-inducing cancer.

[0298] Also provided are methods of antagonizing ActRIIB activity in pathological conditions associated with increased ActRIIB expression and / or ActRIIB signaling. In some cases, the methods include administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as a full-length anti-ActRIIB antibody and an ActRIIB binding antibody fragment, and variants and derivatives thereof) to a subject in need thereof. In some embodiments, the pathological condition is a musculoskeletal disease or disorder, such as muscle atrophy. In some embodiments, the pathological condition is a fibrotic disease, for example, of the lung or liver. In further embodiments, the pathological condition is diabetes. In some embodiments, the pathological condition is obesity (e.g., hypertrophic obesity). In additional embodiments, the pathological condition is pulmonary hypertension or idiopathic pulmonary fibrosis (IPF). In some embodiments, the pathological condition is an ocular disease, such as diabetic retinopathy. In some embodiments, the pathological condition is a cancer, such as carcinoma (eg, basal cell and squamous cell carcinoma of the skin, and head and neck cancer), myeloma, renal cell carcinoma, colon cancer, or bone loss-inducing cancer.

[0299] Further provided are methods of antagonizing ActRIIA activity in pathological conditions associated with increased ActRIIA expression and / or ActRIIA signaling. In some cases, the method includes administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as a full-length anti-ActRIIA antibody or an ActRIIA binding antibody fragment) to a subject in need thereof. In some embodiments, the pathological condition is a musculoskeletal disease or disorder, such as muscle atrophy. In some embodiments, the pathological condition is a fibrotic disease. In some embodiments, the pathological condition is a fibrotic disease, e.g., of the lung or liver. In further embodiments, the pathological condition is a fibrotic disease of the lung or liver. In further embodiments, the pathological condition is diabetes. In some embodiments, the pathological condition is obesity (e.g., hypertrophic obesity). In additional embodiments, the pathological condition is pulmonary hypertension or idiopathic pulmonary fibrosis (IPF). In some embodiments, the pathological condition is an ocular disease, such as diabetic retinopathy. In some embodiments, the pathological condition is a cancer, such as carcinoma (e.g., basal cell carcinoma and squamous cell carcinoma of the skin, head and neck cancer), myeloma (e.g., multiple myeloma), colon cancer, or bone loss-inducing cancer.

[0300] Further provided are methods of antagonizing the activity of ActRIIB and ActRIIA in pathological conditions associated with increased ActRIIB and / or ActRIIA expression and / or increased ActRIIB and / or ActRIIA signaling. In some cases, the method comprises administering an ActRII binding protein (e.g., an anti-ActRII antibody, such as a full-length anti-ActRII antibody or an ActRII binding antibody fragment) to a subject in need thereof. In some embodiments, the pathological condition is a musculoskeletal disease or disorder, such as muscle atrophy. In some embodiments, the pathological condition is a fibrotic disease. In some embodiments, the pathological condition is a fibrotic disease, e.g., of the lung or liver. In some embodiments, the pathological condition is a fibrotic disease of the lung or liver. In further embodiments, the pathological condition is diabetes. In some embodiments, the pathological condition is obesity (e.g., hypertrophic obesity). In additional embodiments, the pathological condition is pulmonary hypertension or idiopathic pulmonary fibrosis (IPF). In some embodiments, the pathological condition is an eye disease, such as diabetic retinopathy. In some embodiments, the pathological condition is a cancer, such as carcinoma (e.g., basal cell carcinoma and squamous cell carcinoma of the skin, head and neck cancer), myeloma (e.g., multiple myeloma), colon cancer, or bone loss-inducing cancer.

[0301] In additional embodiments, the disclosure provides a method of treating and / or ameliorating cancer or a condition associated with cancer or its treatment, comprising administering an ActRII binding protein (e.g., an anti-ActRII antibody or an ActRII binding fragment thereof) to a subject in need thereof. In some embodiments, the ActRII binding protein is an anti-ActRIIB antibody or an ActRIIB binding fragment thereof. Further provided is the use of an ActRII binding protein as provided herein in the manufacture of a medicament for treating or ameliorating cancer or a condition associated with cancer. In some embodiments, the ActRII binding protein is an anti-ActRIIA antibody or an ActRIIA binding fragment thereof. In some embodiments, the ActRII binding protein is an antibody that binds ActRIIB and ActRIIA, or an ActRIIB and ActRIIA ActRIIB binding fragment thereof. In some embodiments, the subject has a cancer selected from melanoma, uterine cancer, lung cancer, ovarian cancer, breast cancer, colon cancer, pancreatic cancer, and sarcoma. In certain embodiments, the subject has carcinoma (eg, basal cell carcinoma and squamous cell carcinoma of the skin, and head and neck cancer), myeloma, colon cancer, or bone loss-inducing cancer.

[0302] In some embodiments, the method includes contacting a cancer cell, tumor-associated stromal cell, or endothelial cell expressing ActRII (e.g., ActRIIB and / or ActRIIA) with an ActRII binding protein that specifically binds ActRII. In some cases, the method includes contacting activin A with the ActRII binding protein. In additional embodiments, the tumor cell is from a cancer selected from the group consisting of myelofibrosis, myeloma (e.g., multiple myeloma), and pituitary cancer. In other embodiments, the cancer is breast cancer, gastrointestinal cancer, or carcinoma (e.g., basal cell carcinoma and squamous cell carcinoma). In an additional embodiment, the cancer is a bone loss-inducing cancer. In some embodiments, the tumor cell is from a cancer line.

[0303] The present disclosure provides methods comprising administering a therapeutically effective amount of an ActRII binding protein, alone or in combination with one or more additional therapies (e.g., one or more additional therapeutic agents), to a subject having or at risk of developing a fibrotic condition. The disclosure further provides compositions for use of the ActRII binding proteins, alone or in combination with another agent, for the preparation of one or more medicaments for use in the treatment (e.g., prevention) and / or amelioration of ActRII-mediated diseases and / or conditions (e.g., muscle disorders such as degenerative muscle diseases, muscular dystrophies, muscle atrophy, or muscle wasting disorders; fibrotic conditions (e.g., fibrotic conditions of the liver, lung, blood vessels, and / or eye, such as myocardial fibrosis, and idiopathic pulmonary fibrosis (IPF)); metabolic diseases (e.g., type II diabetes insulin resistance, hyperglycemia, and obesity); inflammatory diseases or conditions, autoimmune diseases, cardiovascular diseases (e.g., congestive heart failure and hypertension); eye diseases such as age-related macular degeneration; pulmonary diseases, musculoskeletal diseases, skeletal diseases, such as osteoporosis; neurological diseases; wound healing; weight loss; and cancer (e.g., carcinoma, myeloma, bone loss-induced cancer, pituitary cancer, and gastrointestinal cancer)).

[0304] Also provided is the use of the ActRII binding proteins provided herein for diagnostically monitoring protein concentrations (e.g., ActRIIB and / or ActRIIA concentrations) in blood or tissue as part of a clinical testing procedure, for example to determine the effectiveness of a given treatment regimen. For example, detection can be facilitated by coupling the ActRII binding protein to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable examples of prosthetic group complexes include streptavidin / biotin and avidin / biotin; suitable examples of fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent substance is luminol; examples of bioluminescent substances include luciferase, luciferin, and aequorin; suitable examples of radioactive substances include 125 I, 131 I, 35 S, or 3 Examples include H.

[0305] Pharmaceutical Compositions and Methods of Administration Methods for preparing and administering ActRII binding proteins to subjects in need thereof are known or easily determined by those skilled in the art. The route of administration of ActRII binding proteins can be, for example, oral, parenteral, by inhalation or topical. The term parenteral includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, intraocular, subcutaneous, rectal, or intravaginal administration. Although all of these administration forms are expressly contemplated to be within the scope of the present disclosure, another example of a form for administration would be a solution for injection, particularly an intravenous or intraarterial injection or infusion. In general, suitable pharmaceutical compositions can include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), optionally stabilizers (e.g., human albumin), and the like. In another method compatible with the teachings of the present specification, the ActRII binding proteins provided herein can be delivered directly to organs and / or sites of fibrosis or tumors, thereby increasing the exposure of affected tissues to therapeutic agents. In some embodiments, administration is directly to the airways, for example by inhalation or intranasal administration.

[0306] As discussed herein, ActRII binding proteins can be administered in a pharma- ceutical effective amount for the in vivo treatment of ActRII-mediated diseases and conditions, including, but not limited to, muscle disorders such as degenerative muscle diseases, muscular dystrophies, muscle atrophy, or muscle wasting disorders; fibrotic conditions (e.g., fibrotic conditions of the liver, lung, blood vessels, and / or eye, such as myocardial fibrosis, and idiopathic pulmonary fibrosis (IPF)); metabolic diseases (e.g., type II diabetes insulin resistance, hyperglycemia, and obesity); inflammatory diseases or conditions, autoimmune diseases, cardiovascular diseases (e.g., congestive heart failure, and hypertension); eye diseases such as age-related macular degeneration; pulmonary diseases, musculoskeletal diseases, skeletal diseases, such as osteoporosis; neurological diseases; wound healing; weight loss; and cancer (e.g., carcinoma, myeloma, bone loss-induced cancer, pituitary cancer, and gastrointestinal cancer). In this regard, it will be appreciated that the disclosed ActRII binding proteins can be formulated to facilitate administration and promote stability of the active agent. Pharmaceutical compositions according to the present disclosure can include pharma- ceutical acceptable, non-toxic, sterile carriers, such as saline, non-toxic buffers, preservatives, and the like. For purposes of this application, a pharma- ceutical effective amount of an ActRII binding protein, whether bound or unbound, means an amount sufficient to achieve effective binding to ActRII and achieve a benefit, such as ameliorating the symptoms of a disease or condition or detecting a substance or cell. Formulations suitable for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

[0307] Certain pharmaceutical compositions provided herein can be orally administered in acceptable dosage forms, including, for example, capsules, tablets, aqueous suspensions or solutions.Certain pharmaceutical compositions can also be administered by nasal aerosol or inhalation.Such compositions can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers that enhance bioavailability, and / or other conventional solubilizing or dispersing agents.

[0308] The amount of ActRII binding protein (e.g., an antibody that specifically binds ActRIIB and / or ActRIIA) that can be combined with the carrier materials to produce a single dosage form will vary depending on the subject being treated and the particular method of administration. The composition can be administered as a single dose, multiple doses, or infusions over a set period of time. Dosage regimens can also be adjusted to obtain the optimum desired response (e.g., a therapeutic or prophylactic response).

[0309] The ActRII binding proteins provided herein can be administered to humans or other subjects in an amount sufficient to produce a therapeutic effect according to the above-mentioned treatment method.The ActRII binding proteins provided herein can be administered to such humans or other animals in a conventional dosage form prepared by combining the ActRII binding protein with a conventional pharma-ceutically acceptable carrier or diluent according to known techniques.The form and characteristics of the pharma-ceutically acceptable carrier or diluent can be determined by the amount of active ingredient to be combined therewith, the route of administration, and other known variables.A cocktail containing one or more different ActRII binding proteins can also be used.

[0310] The therapeutically effective dose of the ActRII binding composition for treating an ActRII-mediated disease or condition, such as a degenerative muscle disease, a muscular dystrophy, a muscle atrophy, or a muscle wasting disorder; a fibrotic condition; an inflammatory, autoimmune, cardiovascular, pulmonary, musculoskeletal, skeletal, ophthalmic, neurological, or metabolic disease or condition; obesity; wound healing; and cancer, will vary depending on many different factors, such as the means of administration, the target site, the physiological state of the subject, whether the subject is a human or an animal, other administered drugs, and whether the treatment is prophylactic or therapeutic. Typically, the subject is a human, but non-human mammals, such as transgenic mammals, can also be treated. The therapeutic dosage can be adjusted using routine methods known to those skilled in the art to optimize safety and efficacy.

[0311] Ameliorating the symptoms of a particular disease or condition by administration of an ActRII binding protein refers to any relief, whether permanent or temporary, persistent or transient, that can be attributed to or associated with administration of the ActRII binding protein.

[0312] The disclosure also provides uses of ActRII binding proteins, such as anti-ActRII antibodies, for example, for the treatment or manufacture of a medicament for a degenerative muscle disease, muscular dystrophy, muscle atrophy, or muscle wasting disorder; a fibrotic condition; an inflammatory, autoimmune, cardiovascular, pulmonary, musculoskeletal, skeletal, ophthalmic, neurological, or metabolic disease or condition; obesity; wound healing; and cancer.

[0313] Combination therapy In some embodiments, the ActRII binding protein (e.g., anti-ActRII antibodies, such as full-length ActRII antibodies and ActRII binding antibody fragments, and variants and derivatives thereof) is administered in combination with one or more other therapies. Such therapies include additional therapeutic agents and other medical interventions. Exemplary therapeutic agents that may be administered in combination with the ActRII binding protein provided herein include, but are not limited to, anti-SDI fibrotic agents, corticosteroids, anti-inflammatory agents, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, diuretics, antidiabetic agents, immunosuppressants, chemotherapeutic agents, metabolic antagonists, and immunomodulatory agents. In various embodiments, the ActRII binding protein is administered to the subject before, during, and / or after a surgical resection / removal procedure.

[0314] Diagnostics The disclosure also provides methods for treating ActRII-mediated diseases and conditions (e.g., muscle disorders such as degenerative muscle diseases, muscular dystrophies, muscle atrophy, or muscle wasting disorders; fibrotic conditions (e.g., fibrotic conditions of the liver, lung, blood vessels, and / or eye, such as myocardial fibrosis, and idiopathic pulmonary fibrosis (IPF)); metabolic diseases (e.g., type II diabetes insulin resistance, hyperglycemia, and obesity); inflammatory diseases or conditions, autoimmune diseases, cardiovascular diseases (e.g., congestive heart failure, and hypertension); eye diseases such as age-related macular degeneration; pulmonary diseases, musculoskeletal diseases, skeletal diseases, such as osteoporosis; neurological diseases; wound healing; weight loss; and cancer (e.g., carcinoma, myeloma, bone loss-induced Also provided is a diagnostic method useful in diagnosing cancers such as ovarian cancer, pituitary cancer, and gastrointestinal cancer, which involves measuring the expression level of ActRII (e.g., ActRIIA or ActRIIB) protein tissue or fluid from an individual and comparing the measured expression level to a standard ActRII (e.g., ActRIIA or ActRIIB) expression level in normal tissue or fluid, whereby an increase in ActRII expression compared to the standard indicates a disorder that can be treated with the ActRII binding proteins provided herein, such as the full-length anti-ActRIIB antibodies and antigen-binding antibody fragments provided herein.

[0315] The ActRII binding proteins provided herein, such as anti-ActRII antibodies (e.g., full-length ActRII antibodies and ActRII-binding antibody fragments, as well as variants and derivatives thereof), can be used to assess ActRII (e.g., ActRIIB and ActRIIA) levels in biological samples using classical immunohistological methods known to those skilled in the art (see, e.g., Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting the expression of ActRII proteins (e.g., ActRIIB and ActRIIA) include immunoassays such as enzyme-linked immunosorbent assays (ELISAs), immunoprecipitation, or Western blotting.

[0316] "Assessing the expression level of ActRII protein" refers to measuring or estimating the ActRII protein concentration in a first biological sample, either directly (e.g., by determining or estimating absolute protein concentration) or relatively (e.g., by comparing with the disease-related polypeptide concentration in a second biological sample), qualitatively or quantitatively. The ActRII protein expression level in a first biological sample can be measured or estimated and compared to a standard ActRII protein concentration, which is measured from a second biological sample obtained from an individual without the disorder or determined by averaging concentrations from a population of individuals without the disorder. As is understood in the art, once the "standard" ActRII protein concentration is known, it can be used repeatedly as a comparison standard.

[0317] By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other cell source that may express ActRII. Methods for obtaining tissue biopsies and body fluids from mammals are known in the art.

[0318] Kits containing ActRII binding proteins The present disclosure further provides kits that can be used to practice the methods described herein, including an ActRII binding protein (e.g., an antibody that specifically binds ActRII, such as a full-length ActRII antibody and an ActRII binding antibody fragment, and variants and derivatives thereof) in suitable packaging, and informational material. The informational material can include any of the following information: instructions for use, discussion of clinical trials, a list of side effects, scientific references, package insert information, clinical trial results, and / or summaries thereof. The informational material can demonstrate or demonstrate the activity and / or benefits of the composition, and / or describe dosing, administration, side effects, drug interactions, or other information useful to health care providers. Such information can be based on the results of various studies, for example, studies using laboratory animals involving in vivo models and / or studies based on human clinical trials. The kit can further include informational material as described above that serves to provide information regarding alternative therapies (e.g., other drugs) and / or other therapies (e.g., other drugs).

[0319] In certain embodiments, the kit comprises at least one purified ActRII binding protein in one or more containers, hi some embodiments, the kit contains all of the components necessary and / or sufficient to perform a detection assay, including all controls, instructions for performing the assay, and any software necessary for analyzing and presenting the results.

[0320] Immunoassays ActRII binding proteins (e.g., antibodies that specifically bind ActRII, and ActRII-binding fragments of antibodies that specifically bind ActRII, as well as variants or derivatives thereof) can be assessed for immunospecific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blot, radioimmunoassay (REA), ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, or protein A immunoassay. Such assays are routine and known in the art (see, for example, Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1, which is incorporated herein by reference in its entirety).

[0321] The ActRII binding proteins provided herein (e.g., antibodies that specifically bind ActRII and ActRII-binding fragments of antibodies that specifically bind ActRII, as well as variants, or derivatives thereof) can be used histologically, as in immunofluorescence, immunoelectron microscopy, or non-immunological assays, for in situ detection of ActRII (e.g., ActRIIB and ActRIIA) or conserved variants or peptide fragments thereof. In situ detection can be achieved according to methods known in the art. Those skilled in the art can determine effective and optimal assay conditions for each determination by using routine experimentation. Methods suitable for determining the binding properties of ActRII binding proteins are described herein or are otherwise known in the art. Devices and software designed for such kinetic analysis are commercially available (e.g., BIACORE®, BIAevaluation® software, GE Healthcare; KINEXA® Software, Sapidyne Instruments).

[0322] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of those in the art. The present invention also provides the following aspects. [1] An isolated activin receptor type II (ActRII) binding protein comprising a set of CDRs, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, and / or a set of ABRs, VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2 and VL-ABR3, wherein the CDRs and / or ABRs are a pair of a heavy chain variable region (VH) and a light chain variable region (VL), i.e. (a)(i) a VH sequence of SEQ ID NO: 20; (ii) a VL sequence of SEQ ID NO: 30, The protein binds to activin receptor type IIB (ActRIIB); (b)(i) a VH sequence of SEQ ID NO: 20; and (ii) the VL sequence of SEQ ID NO: 39, The protein binds to ActRIIB. (c)(i) a VH sequence of SEQ ID NO: 49; and (ii) a VL having the amino acid sequence of SEQ ID NO: 59; The protein binds to ActRIIB. (d)(i) a VH sequence of SEQ ID NO: 20; and (ii) the VL sequence of SEQ ID NO: 67, The protein binds to ActRIIB. (e)(i) a VH sequence of SEQ ID NO: 77; and (ii) the VL sequence of SEQ ID NO: 85, The protein binds to ActRIIB, and (f)(i) a VH sequence of SEQ ID NO:2; (ii) the VL sequence of SEQ ID NO: 12, The protein binds to ActRIIB and activin receptor type IIA (ActRIIA); The isolated activin receptor type II (ActRII) binding protein is present in a pair selected from the group consisting of: [2] An isolated ActRII binding protein comprising a set of CDRs, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, wherein the set of CDRs is a reference set of CDRs, i.e. (a)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 23, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 31, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 32, (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 33, and wherein the protein binds to ActRIIB; (b)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 23, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 40, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 41; (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 42, and wherein the protein binds to ActRIIB; (c)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 50; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 51, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 52, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 60, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 61; (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 62, and wherein the protein binds to ActRIIB; (d)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 23, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 68, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 69, (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 70, and wherein the protein binds to ActRIIB; (e)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 78; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 79, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 80; (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 86, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 87; (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 88, and the protein binds to ActRIIB; or (f)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO:3; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO:4, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO:5, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 13, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 14, (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 15, and the protein binds to ActRIIB and ActRIIA; The isolated ActRII binding protein has a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, less than 10, or zero amino acid substitutions, deletions, and / or insertions from. [3] A set of CDRs, i.e. (a)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 23, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 31, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 32, (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 33, and wherein the protein binds to ActRIIB; (b)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 23, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 40, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 41; (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 42, and wherein the protein binds to ActRIIB; (c)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 50; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 51, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 52, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 60, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 61; (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 62, and wherein the protein binds to ActRIIB; (d)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 21; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 22, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 23, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 68, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 69, (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 70, and wherein the protein binds to ActRIIB; (e)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO: 78; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO: 79, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO: 80; (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 86, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 87; (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 88, and the protein binds to ActRIIB; or (f)(i) VH-CDR1 has the amino acid sequence of SEQ ID NO:3; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO:4, (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO:5, (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 13, (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 14, (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 15, and the protein binds to ActRIIB and ActRIIA; An isolated ActRII binding protein described in [2], comprising: [4] An isolated ActRII binding protein comprising a set of ABRs, i.e., VH-ABR1, VH-ABR2, VH-ABR3, VL-ABR1, VL-ABR2 and VL-ABR3, wherein the set of ABRs is a reference set of ABRs, i.e., (a)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 24; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 25; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 26; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 34; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 35; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 36; and wherein the protein binds to ActRIIB; (b)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 24; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 25; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 26; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 43; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 44; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 45; and wherein the protein binds to ActRIIB; (c)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 53; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 54 or 55; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 56 or 57; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 63; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 64; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 65; and wherein the protein binds to ActRIIB; (d)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 24; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 25; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 26; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 71; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 72; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 73; and wherein the protein binds to ActRIIB; (e)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 81; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 82; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 83; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 89; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 90; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 91; and the protein binds to ActRIIB; or (f)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO:6; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 7 or 8; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 9 or 10; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 16; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 17; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 18; and the protein binds to ActRIIB and ActRIIA; The isolated ActRII binding protein has a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, less than 10, or zero amino acid substitutions, deletions, and / or insertions from. [5] A set of CDRs, i.e. (a)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 24; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 25; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 26; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 34; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 35; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 36; and wherein the protein binds to ActRIIB; (b)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 24; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 25; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 26; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 43; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 44; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 45; and wherein the protein binds to ActRIIB; (c)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 53; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 54 or 55; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 56 or 57; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 63; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 64; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 65; and wherein the protein binds to ActRIIB; (d)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 24; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 25; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 26; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 71; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 72; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 73; and wherein the protein binds to ActRIIB; (e)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO: 81; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 82; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 83; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 89; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 90; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 91; and the protein binds to ActRIIB; or (f)(i) VH-ABR1 has the amino acid sequence of SEQ ID NO:6; (ii) VH-ABR2 has the amino acid sequence of SEQ ID NO: 7 or 8; (iii) VH-ABR3 has the amino acid sequence of SEQ ID NO: 9 or 10; (iv) VL-ABR1 has the amino acid sequence of SEQ ID NO: 16; (v) VL-ABR2 has the amino acid sequence of SEQ ID NO: 17; (vi) VL-ABR3 has the amino acid sequence of SEQ ID NO: 18; and the protein binds to ActRIIB and ActRIIA; An isolated ActRII binding protein described in [4], comprising: [6] An ActRII binding protein, (a)(i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20; (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, wherein the protein binds ActRIIB; (b)(i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20; (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 39, wherein the protein binds ActRIIB; (c)(i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 49; (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 59, wherein the protein binds ActRIIB; (d)(i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20; (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 67, wherein the protein binds ActRIIB; (e)(i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 77; (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 85, wherein the protein binds ActRIIB; or (f) (i) a VH having the amino acid sequence of SEQ ID NO: 2; (ii) a VL having the amino acid sequence of SEQ ID NO: 12; The protein binds to ActRIIB and ActRIIA; The ActRII binding protein comprises a VH and VL pair selected from the group consisting of: [7] The VH and VL pair (a) a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 30, wherein the protein binds to ActRIIB; (b) a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 39, wherein the protein binds to ActRIIB; (c) a VH sequence of SEQ ID NO: 49 and a VL sequence of SEQ ID NO: 59, wherein the protein binds to ActRIIB; (d) a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 67, wherein the protein binds to ActRIIB; (e) a VH sequence of SEQ ID NO: 77 and a VL sequence of SEQ ID NO: 85, wherein the protein binds to ActRIIB; or (f) a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 12, wherein the protein binds to ActRIIB and ActRIIA; The ActRII binding protein according to [6], selected from the group consisting of: [8] An ActRII binding protein, (a)(i) a VH sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VH sequence of SEQ ID NO: 20; (ii) a VL sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VL sequence of SEQ ID NO: 30; The protein binds to ActRIIB. (b)(i) a VH sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VH sequence of SEQ ID NO: 20; and (ii) a VL sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VL sequence of SEQ ID NO: 39; The protein binds to ActRIIB. (c)(i) a VH sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VH sequence of SEQ ID NO: 49; and (ii) a VL sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VL sequence of SEQ ID NO:59; The protein binds to ActRIIB. (d)(i) a VH sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VH sequence of SEQ ID NO: 20; (ii) a VL sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from the reference VL sequence of SEQ ID NO: 67; The protein binds to ActRIIB. (e)(i) a VH sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VH sequence of SEQ ID NO: 77; (ii) a VL sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VL sequence of SEQ ID NO: 85; The protein binds to ActRIIB. (f)(i) a VH sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from a reference VH sequence of SEQ ID NO:2; (ii) a VL sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, less than fifteen, or zero amino acid substitutions, deletions, and / or insertions from the reference VL sequence of SEQ ID NO: 12; The protein binds to ActRIIB and ActRIIA; The ActRII binding protein comprises a VH and VL pair selected from the group consisting of: [9] An ActRII binding protein that binds to the same epitope as the ActRII binding protein described in any one of [1] to [8].

[10] An ActRII-binding protein that competes with the ActRII-binding protein described in any one of [1] to [9] for binding to ActRII.

[11] The ActRII binding protein described in any one of [1] to

[10] , wherein the ActRII binding protein antagonizes ActRII activity.

[12] The binding protein, (a) competes with activin A, activin B, BMP7, BMP9, BMP10, GDF8 (myostatin), GDF11, or Nodal for binding to ActRIIB and / or ActRIIA; (b) decreasing phosphorylation of one or more Smads in cells expressing ActRIIB and / or ActRIIA in the presence of a ligand for ActRIIB or ActRIIA (e.g., activin A); (c) decreasing phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIB and / or ActRIIA and ALK4 and / or ALK7 in the presence of a ligand for ActRIIB and / or ActRIIA; and (d) a K of 1 nM or less and 1 pM or more for ActRIIB and / or ActRIIA (e.g., as determined by BIACORE® analysis); D Join with The ActRII binding protein according to any one of [1] to

[11] , having at least one characteristic selected from the group consisting of the following characteristics:

[13] The ActRII binding protein described in any one of [1] to

[12] , wherein the ActRII binding protein is an antibody that specifically binds to ActRII.

[14] The ActRII binding protein described in

[13] , wherein the antibody is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody, or an ActRII-binding antibody fragment.

[15] The ActRII-binding antibody fragment is a Fab fragment, a Fab' fragment, or a F(ab') 2 The ActRII binding protein according to

[14] , wherein the ActRII binding protein is selected from the group consisting of an antibody fragment, an Fv fragment, a diabody, or a single-chain antibody molecule.

[16] The antibody further comprising: (a) human IgA constant domain; (b) human IgD constant domain, (c) human IgE constant domain, (d) a human IgG1 constant domain; (e) a human IgG2 constant domain, (f) a human IgG3 constant domain, (g) a human IgG4 constant domain, and (h) human IgM constant domain An ActRII binding protein described in any one of

[13] to

[15] , which contains a heavy chain immunoglobulin constant domain selected from the group consisting of:

[17] The antibody further comprising: (a) a human Ig kappa constant domain, and (b) Human Ig lambda constant domain An ActRII binding protein described in any one of

[13] to

[16] , which contains a light chain immunoglobulin constant domain selected from the group consisting of:

[18] An ActRII binding protein described in any one of

[13] to

[17] , wherein the antibody further contains a human IgG1 heavy chain constant domain and a human lambda light chain constant domain.

[19] An isolated nucleic acid molecule or set of nucleic acid molecules encoding an ActRII binding protein described in any one of [1] to

[18] .

[20] An isolated nucleic acid molecule or set of nucleic acid molecules according to

[19] , which is cDNA.

[21] A vector comprising the nucleic acid molecule described in

[19] or

[20] .

[22] A host cell comprising a nucleic acid molecule according to

[19] or

[20] , or a vector according to

[21] .

[23] The host cell described in

[22] , wherein the host cell is a mammalian host cell.

[24] The mammalian host cell according to

[23] , wherein the host cell is an NS0 mouse myeloma cell, a PER.C6 (registered trademark) human cell, or a Chinese hamster ovary (CHO) cell.

[25] A method for producing an ActRII binding protein described in any one of [1] to

[18] , the method comprising culturing a host cell described in

[22] ,

[23] , or

[24] under suitable conditions for producing the ActRII binding protein.

[26] The method according to

[25] , further comprising isolating the ActRII binding protein secreted from the host cell.

[27] An ActRII binding protein produced using the methods described in

[25] or

[26] .

[28] A pharmaceutical composition comprising an ActRII binding protein described in any one of [1] to

[18] or

[27] and a pharma- ceutical acceptable carrier.

[29] The pharmaceutical composition according to

[28] for use as a medicine.

[30] Use of the pharmaceutical composition described in

[29] for treating and / or ameliorating a disease or condition associated with increased ActRII expression or ActRII signaling.

[31] The use of

[30] , wherein the disease or condition is a member selected from degenerative muscle diseases, muscular dystrophy, muscle atrophy, muscle wasting, fibrotic conditions (fibrotic conditions of the liver, lungs, blood vessels or eyes), myocardial fibrosis, idiopathic pulmonary fibrosis, metabolic diseases, type II diabetes, obesity, inflammatory diseases, autoimmune diseases, eye diseases, age-related macular degeneration cardiovascular diseases, congestive heart failure, hypertension, pulmonary diseases, musculoskeletal diseases, skeletal diseases, osteoporosis, neuromuscular diseases, degenerative diseases, wound healing, and cancer.

[32] The pharmaceutical composition according to

[28] , further comprising a labeling group or an effector group.

[33] The pharmaceutical composition of

[32] , wherein the effector group is selected from a radioisotope, a radionuclide, a toxin, a therapeutic agent, and a chemotherapeutic agent.

[34] A method for treating and / or ameliorating a disease or condition associated with increased ActRII expression or ActRII-mediated signaling in a subject, the method comprising administering to a subject in need thereof a composition comprising an ActRII binding protein described in any one of [1] to

[18] or

[27] , or a pharmaceutical composition described in

[28] .

[35] The method of

[34] , wherein the disease or condition is a member selected from degenerative muscle diseases, muscular dystrophy, muscle atrophy, muscle wasting, fibrotic conditions (fibrotic conditions of the liver, lungs, blood vessels or eyes), myocardial fibrosis, idiopathic pulmonary fibrosis, metabolic diseases, type II diabetes, obesity, inflammatory diseases, autoimmune diseases, eye diseases, age-related macular degeneration cardiovascular diseases, congestive heart failure, hypertension, pulmonary diseases, musculoskeletal diseases, skeletal diseases, osteoporosis, neuromuscular diseases, degenerative diseases, wound healing, and cancer.

[36] The method according to

[34] , wherein the ActRII binding protein or pharmaceutical composition is administered alone or as a combination therapy.

[37] A method for reducing ActRII activity in a subject, the method comprising administering an ActRII binding protein described in any one of [1] to

[18] or

[27] , or the pharmaceutical composition described in

[28] .

[0323] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0324] From the foregoing description of specific embodiments, the general nature of the present disclosure is sufficiently clear that others can easily modify and / or adapt such specific embodiments for various applications by applying knowledge within the skill of the art without undue experimentation and without departing from the general concept of the present disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It should be understood that the expressions or terms used herein are for the purpose of description and not limitation, and thus the terms or terms used herein should be interpreted by those skilled in the art in light of the teaching and guidance.

[0325] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0326] All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually indicated to be incorporated by reference for all purposes.

[0327] Example 1. Selection, characterization, and generation of ActRII-binding antibodies A multi-round selection procedure was used to select for human IgG antibodies that bind with high affinity to ActRII and compete with activin A for binding to human ActRII, as detailed below.

[0328] Materials and Methods Antigens (ActRIIA, ActRIIB, ActRIIA-Fc, and ActRIIB-Fc) were biotinylated using Pierce's EZ-Link Sulfo-NHS-Biotinylation Kit. Goat anti-human F(ab')2 kappa-FITC (LC-FITC), Extravidin-PE (EA-PE), and streptavidin-633 (SA-633) were obtained from Southern Biotech, Sigma, and Molecular Probes, respectively. Streptavidin MicroBeads and MACS LC separation columns were purchased from Miltenyi Biotec.

[0329] Naive discovery Each has a diversity of about 10 9 Eight naive human synthetic yeast libraries were grown as previously reported (see, e.g., WO09 / 036379, WO10 / 105256, WO12 / 009568). For the first two rounds of selection, a magnetic bead sorting procedure was performed using the Miltenyi MACs system as previously reported (see, e.g., Siegel et al., J. Immunol. Meth. 286(1-2):141-153 (2004)). Briefly, yeast cells (approximately 10 10Cells / library) and 3 ml of biotinylated monomeric ActRII-Fc antigen (ActRIIB-Fc or ActRIIA-Fc) (10 nM) were incubated in FACS wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) for 15 min at room temperature. After one wash with 50 ml of ice-cold wash buffer, the cell pellet was resuspended in 40 mL of wash buffer and Streptavidin MicroBeads (500 μl) were added to the yeast and incubated at 4 °C for 15 min. The yeast were then pelleted, resuspended in 5 mL of wash buffer and loaded onto a Miltenyi LS column. After 5 mL had been loaded, the column was washed three times with 3 ml of FACS wash buffer. The column was then removed from the magnetic field and the yeast were eluted with 5 mL of growth medium and then grown overnight. The following rounds of sorting were performed using flow cytometry. Approximately 1 × 10 8Yeast were pelleted, washed three times with wash buffer, and incubated at room temperature under equilibration conditions with decreasing concentrations of biotinylated monomeric ActRII-Fc fusion antigen (100 nM to 1 nM). Yeast were then washed twice and stained with LC-FITC (diluted 1:100) and secondary reagents SA-633 (diluted 1:500) or EA-PE (diluted 1:50) for 15 min at 4°C. After washing twice with ice-cold wash buffer, cell pellets were resuspended in 0.4 mL of wash buffer and transferred to strainer-capped sorting tubes. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sorting gates were assigned to select specific binders compared to background controls. Subsequent selection rounds were used to reduce the number of nonspecific reagent binders utilizing soluble membrane proteins from CHO cells (see, e.g., WO14 / 179363 and Xu et al., Protein Eng. Des. Sel. 26(10):663-670 (2013)) and to identify binders with improved affinity for ActRII (ActRIIB or ActRIIA) using ActRII-Fc (ActRIIB-Fc and ActRIIA-Fc antigens, respectively). After the final selection round, yeast were plated and individual colonies were harvested for characterization and clonal assignment for affinity maturation.

[0330] affinity maturation Binding optimization of the naive clones was performed using a three-fold maturation strategy: diversifying the light chain; diversifying CDRH and / CDRH2; and performing sequential mutagenesis of VH and VL.

[0331] Diversification of the light chain: The heavy chain plasmid was a naive product extracted (above) with a diversity of 1 × 10 6 The light chain library was converted to a 100% IgG light chain library. Selections were performed as described above with one round of MACS selection and two rounds of FACS selection using 10 nM or 1 nM biotinylated ActRII-Fc antigen (ActRIIB-Fc or ActRIIA-Fc) in each round.

[0332] Selection of CDRH1 and CDRH2: CDRH3 from clones selected by the light chain d...

Claims

1. an activin receptor type II (ActRII) binding protein, the ActRII binding protein being an antibody which specifically binds to ActRII and comprises a set of CDRs: VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3; (i) VH-CDR1 has the amino acid sequence of SEQ ID NO:3; (ii) VH-CDR2 has the amino acid sequence of SEQ ID NO:4; (iii) VH-CDR3 has the amino acid sequence of SEQ ID NO:5; (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO: 13; (v) VL-CDR2 has the amino acid sequence of SEQ ID NO: 14; (vi) VL-CDR3 has the amino acid sequence of SEQ ID NO: 15; And an ActRII binding protein, which binds to ActRIIB and ActRIIA.

2. (i) a VH having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2; (ii) a VL having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12 2. The ActRII binding protein of claim 1, comprising a VH and VL pair having the following structure:

3. 3. The ActRII binding protein of claim 2, wherein the VH and VL pair has a VH sequence of SEQ ID NO:2 and a VL sequence of SEQ ID NO:12, and the protein binds to ActRIIB and ActRIIA.

4. (i) a VH sequence that has a total of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or zero amino acid substitutions, deletions, and / or insertions from a reference VH sequence of SEQ ID NO:2; (ii) a VL sequence having a total of one, two, three, four, five, six, seven, eight, nine, ten, or zero amino acid substitutions, deletions, and / or insertions from the reference VL sequence of SEQ ID NO:

12.

2. The ActRII binding protein of claim 1, comprising a VH and VL pair having the following structure:

5. An ActRII binding protein that competes for binding to ActRII with the ActRII binding protein of any one of claims 1 to 4.

6. The ActRII binding protein of any one of claims 1 to 5, wherein the ActRII binding protein antagonizes ActRII activity.

7. The binding protein is (a) competes with activin A, activin B, BMP7, BMP9, BMP10, GDF8 (myostatin), GDF11, or Nodal for binding to ActRIIB and / or ActRIIA; (b) decreasing phosphorylation of one or more Smads in cells expressing ActRIIB and / or ActRIIA in the presence of a ligand for ActRIIB or ActRIIA (e.g., activin A); (c) reducing phosphorylation of ALK4 and / or ALK7 in cells expressing ActRIIB and / or ActRIIA, and ALK4 and / or ALK7, in the presence of a ligand for ActRIIB and / or ActRIIA; and (d) a K for ActRIIB and / or ActRIIA of 1 nM or less and 1 pM or more (e.g., as determined by BIACORE® analysis); Ｄ Join with 7. The ActRII binding protein of claim 1, having at least one property selected from the group consisting of:

8. The ActRII binding protein of any one of claims 1 to 7, wherein the antibody is a monoclonal antibody, a recombinant antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody, or an ActRII-binding antibody fragment.

9. The ActRII-binding antibody fragment may be a Fab fragment, a Fab' fragment, an F(ab') ２ 9. The ActRII binding protein of claim 8 selected from the group consisting of an Fv fragment, an Fv fragment, or a diabody.

10. The antibody further comprises (a) a human IgA constant domain; (b) a human IgD constant domain; (c) a human IgE constant domain; (d) a human IgG1 constant domain; (e) a human IgG2 constant domain, (f) a human IgG3 constant domain, (g) a human IgG4 constant domain, and (h) human IgM constant domain 10. The ActRII binding protein of any one of claims 1 to 9, comprising a heavy chain immunoglobulin constant domain selected from the group consisting of:

11. The antibody further comprises (a) a human Ig kappa constant domain, and (b) human Ig lambda constant domain 11. The ActRII binding protein of any one of claims 1 to 10, comprising a light chain immunoglobulin constant domain selected from the group consisting of:

12. The ActRII binding protein of any one of claims 1 to 11, wherein the antibody further comprises a human IgG1 heavy chain constant domain and a human lambda light chain constant domain.

13. 13. An isolated nucleic acid molecule or set of nucleic acid molecules encoding an ActRII binding protein according to any one of claims 1 to 12.

14. 14. The isolated nucleic acid molecule or set of nucleic acid molecules of claim 13 which is a cDNA.

15. A vector comprising the nucleic acid molecule of claim 13 or 14.

16. A host cell comprising a nucleic acid molecule according to claim 13 or 14, or a vector according to claim 15.

17. The host cell of claim 16 , wherein the host cell is a mammalian host cell.

18. 18. The mammalian host cell of claim 17, wherein the host cell is an NSO mouse myeloma cell, a PER.C6® human cell, or a Chinese Hamster Ovary (CHO) cell.

19. A method for making an ActRII binding protein according to any one of claims 1 to 12, comprising culturing a host cell according to claim 16, 17 or 18 under suitable conditions for producing the ActRII binding protein.

20. 20. The method of claim 19, further comprising isolating the ActRII binding protein secreted from the host cell.

21. A pharmaceutical composition comprising an ActRII binding protein according to any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

22. 22. The pharmaceutical composition of claim 21, further comprising a labeling group or an effector group.

23. 23. The pharmaceutical composition of claim 22, wherein the effector group is selected from a radioisotope, a radionuclide, a toxin, a therapeutic agent, and a chemotherapeutic agent.

24. A pharmaceutical composition according to any one of claims 21 to 23 for use as a medicament.

25. 25. A pharmaceutical composition according to claim 21 or 24 for use as a medicament for treating and / or ameliorating a disease or condition associated with elevated ActRII expression or ActRII signalling.

26. 26. The pharmaceutical composition of claim 25, wherein the disease or condition is a member selected from degenerative muscle diseases, muscular dystrophies, muscle atrophy, muscle wasting, fibrotic conditions (fibrotic conditions of the liver, lungs, blood vessels or eyes), myocardial fibrosis, idiopathic pulmonary fibrosis, metabolic diseases, type II diabetes, obesity, inflammatory diseases, autoimmune diseases, eye diseases, age-related macular degeneration, cardiovascular diseases, congestive heart failure, hypertension, pulmonary diseases, musculoskeletal diseases, skeletal diseases, osteoporosis, neuromuscular diseases, degenerative diseases, wound healing, and cancer.

27. 27. The pharmaceutical composition of claim 25 or 26, wherein the pharmaceutical composition is administered alone or as a combination therapy.

28. 25. The pharmaceutical composition of claim 21 or 24 for use as a medicament for reducing ActRII activity in a subject.

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