WO2014172448A2 - Anticorps diriges contre le recepteur d'activine de type ii (actrii) - Google Patents

Anticorps diriges contre le recepteur d'activine de type ii (actrii) Download PDF

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WO2014172448A2
WO2014172448A2 PCT/US2014/034344 US2014034344W WO2014172448A2 WO 2014172448 A2 WO2014172448 A2 WO 2014172448A2 US 2014034344 W US2014034344 W US 2014034344W WO 2014172448 A2 WO2014172448 A2 WO 2014172448A2
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residue
seq
replaced
chain polypeptide
amino acid
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WO2014172448A3 (fr
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Petra Verdino
Peter Bowers
David King
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Anaptysbio Inc
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Anaptysbio Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 128,165 Byte ASCII (Text) file named "716633_ST25.txt” created on April 15, 2014.
  • Activins are dimeric growth and differentiation factors which belong to the transforming growth factor-beta (TGF-beta) superfamily of structurally related signaling proteins. Activins signal through a heterodimeric complex of receptor serine kinases which include at least two type I receptors (i.e., ALK4 and ALK5) and two type II receptors (i.e., ActRIIA and ActRIIB (also known as "ACVR2A” and “ACVR2B”) receptors).
  • TGF-beta transforming growth factor-beta
  • Activin receptors are transmembrane proteins composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain required for cell signaling through serine/threonine phosphorylation events.
  • Type I receptors are essential for signaling, while type II receptors are required for binding ligands and for expression of type I receptors.
  • Type I and II receptors form a stable complex after ligand binding, which results in the phosphorylation of type I receptors by type II receptors.
  • the activin receptor II B (ActRIIB) is a receptor for myostatin, which is a negative regulator of muscle growth (see, e.g., Lee et al, Proc. Natl. Acad. Sci. USA, 102(50): 18117- 18122 (2005)).
  • the interaction between myostatin and ActRIIB regulates the inhibition of skeletal muscle differentiation via a SMAD-dependent pathway.
  • Inhibition of ActRIIB has been shown to reverse muscle wasting, and leads to increased lean muscle volume and strength in animal models. Inhibition of ActRIIB also has been shown to promote increased muscle volume in human subjects. For example, Bogdanovich et al., Nature, 420: 418-421 (2002), demonstrates that anti-myostatin antibodies can inhibit myostatin, resulting in an increase in muscle mass in a mouse model of Duchenne muscular dystrophy. Recent clinical studies have demonstrated that inhibition of ActRIIB may be an effective strategy for treating muscle wasting disorders.
  • an ActRIIB-Fc fusion protein has been shown in Phase I studies to increase lean body mass and thigh muscle volume in health post-menopausal women (see., e.g., Sako et al, J. Biol. Chem., 285(21): 21037-21048 (2010); Attie et al, Muscle & Nerve, 47(3): 416-423 (2013); and Carlson et al, Muscle & Nerve, 43(5): 694-699 (201 1)).
  • an ActRII-binding agent e.g., an antibody
  • the invention provides such ActllR-binding agents.
  • the invention provides an isolated immunoglobulin heavy chain polypeptide comprising SEQ ID NO: 1 , wherein optionally (a) one or more of residues 5, 12, 18, 23, 31 , 35, 37, 46, 48, 50, 53, 56, 57, 59, 61 , 67, 81 , 83, 97, 98, 99, and 105 of SEQ ID NO: 1 are replaced with a different amino acid residue, and (b) a tyrosine (Y) residue is inserted into SEQ ID NO: 1 after residue 57.
  • the invention provides an isolated immunoglobulin heavy chain polypeptide comprising SEQ ID NO: 34, wherein optionally one or more of residues 24, 28, 31 , 33, 34, 50, 53, 55, 56, 61, 65, 67, 79, 81, and 88 of SEQ ID NO: 34 are replaced with a different amino acid residue.
  • the invention provides an isolated immunoglobulin heavy chain polypeptide comprising SEQ ID NO: 55, wherein optionally one or more of residues of 26, 28, 30, 35, 50, 53, and 59 of SEQ ID NO: 55 are replaced with a different amino acid residue.
  • the invention provides an isolated immunoglobulin heavy chain polypeptide which comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1-68, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 119.
  • the invention provides an isolated immunoglobulin heavy chain polypeptide which comprises at least one complementarity determining region (CDR) of an immunoglobulin heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 1-68, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 119.
  • CDR complementarity determining region
  • the invention also provides an isolated immunoglobulin light chain polypeptide comprising SEQ ID NO: 69, wherein optionally (a) one or more of residues 4, 13, 32, 36, 40, 52, 53, 59, 61, 62, 69, 72, 73, 82, 89, 96, 97, 100, 106, and 110 of SEQ ID NO: 69 are replaced with a different amino acid residue, (b) an amino acid sequence comprising YSS (SEQ ID NO: 98) is inserted into SEQ ID NO: 69 after residue 33, (c) an amino acid sequence comprising NKNYL (SEQ ID NO: 99) is inserted into SEQ ID NO: 69 after residue 39, (d) an amino acid sequence comprising NNNYL (SEQ ID NO: 100) is inserted into SEQ ID NO: 69 after residue 39, or (e) any combination of (a), (b), and (c) or (a), (b), and (d).
  • the invention provides an isolated immunoglobulin light chain polypeptide comprising SEQ ID NO: 101, wherein optionally residue 93 of SEQ ID NO: 101 is replaced with a different amino acid residue.
  • the invention provides an isolated immunoglobulin light chain polypeptide comprising SEQ ID NO: 103, wherein optionally residue 95 of SEQ ID NO: 103 is replaced with a different amino acid residue.
  • the invention provides an isolated immunoglobulin light chain polypeptide which comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of any one of SEQ ID NOs : 69-97, SEQ ID NOs : 101 - 104, SEQ ID NO : 120, SEQ ID NO : 121 , or SEQ ID NO: 122.
  • the invention also provides an isolated immunoglobulin light chain polypeptide which comprises at least one complementarity determining region (CDR) of an immunoglobulin light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 69- 97, SEQ ID NOs: 101-104, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.
  • CDR complementarity determining region
  • the invention provides isolated or purified nucleic acid sequences encoding the foregoing immunoglobulin polypeptides, vectors comprising such nucleic acid sequences, isolated activin receptor type II (ActRII)-binding agents comprising the foregoing immunoglobulin polypeptides, nucleic acid sequences encoding such ActRII-binding agents, vectors comprising such nucleic acid sequences, isolated cells comprising such vectors, compositions comprising such vectors or the ActRII-binding agent and a pharmaceutically acceptable carrier, and a method of treating an ActRII-mediated disorder in a mammal by administering an effective amount of such compositions to the mammal.
  • ActRII activin receptor type II
  • the invention provides an isolated immunoglobulin heavy chain polypeptide and/or an isolated immunoglobulin light chain polypeptide, or a fragment (e.g., antigen-binding fragment) thereof.
  • immunoglobulin or “antibody,” as used herein, refers to a protein that is found in blood or other bodily fluids of vertebrates, which is used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
  • the polypeptide is "isolated” in that it is removed from its natural environment.
  • an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR).
  • the CDRs form the "hypervariable region" of an antibody, which is responsible for antigen binding (discussed further below).
  • a whole immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide.
  • Each of the heavy chains contains one N- terminal variable (V R ) region and three C-terminal constant (C R I , C R 2, and C R 3) regions, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L ) region.
  • the light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda ( ⁇ ), based upon the amino acid sequences of their constant domains.
  • each light chain is linked to a heavy chain by disulphide bonds, and the two heavy chains are linked to each other by disulphide bonds.
  • the light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain.
  • the remaining constant regions of the heavy chains are aligned with each other.
  • variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
  • the V H and V L regions have the same general structure, with each region comprising four framework (FW or FR) regions.
  • framework region refers to the relatively conserved amino acid sequences within the variable region which are located between the hypervariable or complementary determining regions (CDRs).
  • CDRs hypervariable or complementary determining regions
  • the framework regions form the ⁇ sheets that provide the structural framework of the variable region (see, e.g., C.A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)).
  • the framework regions are connected by three complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the three CDRs known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen binding.
  • the CDRs form loops connecting, and in some cases comprising part of, the beta-sheet structure formed by the framework regions.
  • the constant regions of the light and heavy chains are not directly involved in binding of the antibody to an antigen, the constant regions can influence the orientation of the variable regions.
  • the constant regions also exhibit various effector functions, such as participation in antibody-dependent cellular toxicity via interactions with effector molecules and cells.
  • immunoglobulin light chain polypeptide of the invention desirably bind to activin receptor type II.
  • activin type II receptors there are two activin type II receptors: ActRIIA (also referred to as ACVR2A) and ActRIIB (also referred to as ACVR2B).
  • Activin receptors type II are 512 or 513 amino acid proteins that consist of an extracellular domain that specifically binds various ligands of the TGF-beta family, including myostatin, activin A and GDF-11, a single-membrane spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity (see, e.g., Donaldson et al, Biochem.
  • SMAD2 and SMAD3 phosphorylates the SMAD proteins SMAD2 and SMAD3 on serine residues of the C-terminal tail. Soon after their association with the activin receptor and subsequent phosphorylation, SMAD2 and SMAD3 are released into the cytoplasm where they interact with the common partner SMAD4. This SMAD complex translocates into the nucleus where it mediates activin- induced transcription. Signaling through the activin receptor complex is further described in, e.g., Attisano et al, Mol. Cell. Biol, 16: 1066-1073 (1996).
  • inventive isolated immunoglobulin heavy chain polypeptide and the inventive isolated immunoglobulin light chain polypeptide can bind to either ActRIIA or ActRIIB or to both ActRIIA and ActRIIB.
  • inventive isolated immunoglobulin heavy chain polypeptide and the inventive isolated immunoglobulin light chain polypeptide form an agent that binds to both ActRIIA and ActRIIB, the resulting ActRII-binding agent is referred to as a "dual reactive" binding agent (e.g., a dual reactive antibody).
  • the isolated immunoglobulin heavy chain polypeptide comprises, consists of, or consists essentially of SEQ ID NO: 1, wherein optionally (a) one or more of residues 5, 12, 18, 23, 31, 35, 37, 46, 48, 50, 53, 56, 57, 59, 61, 67, 81, 83, 97, 98, 99, and 105 of SEQ ID NO: 1 are replaced with a different amino acid residue, and (b) a tyrosine (Y) residue is inserted into SEQ ID NO: 1 after residue 57.
  • inventive immunoglobulin heavy chain polypeptide consists essentially of SEQ ID NO: 1 and optional amino acid replacements
  • additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation).
  • the inventive immunoglobulin heavy chain polypeptide consists of SEQ ID NO: 1 and optional amino acid replacements
  • the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide).
  • the isolated immunoglobulin heavy chain polypeptide comprises SEQ ID NO: 1, except that residue 50 of SEQ ID NO: 1 is replaced with a different residue, and optionally (a) one or more of residues 5, 12, 18, 23, 31, 35, 37, 46, 48, 53, 56, 57, 59, 61, 67, 81, 83, 97, 98, 99, and 105 of SEQ ID NO: 1 are replaced with a different residue, and/or (b) a tyrosine (Y) residue is inserted into SEQ ID NO: 1 after residue 57.
  • the isolated immunoglobulin heavy chain polypeptide can comprise SEQ ID NO: 1, except that residue 50 of SEQ ID NO: 1 is replaced with a different residue and either (a) one or more of residues 5, 12, 18, 23, 31, 35, 37, 46, 48, 53, 56, 57, 59, 61, 67, 81, 83, 97, 98, 99, and 105 of SEQ ID NO: 1 are replaced with a different residue, or (b) a tyrosine (Y) residue is inserted into SEQ ID NO: 1 after residue 57.
  • the isolated immunoglobulin heavy chain polypeptide can comprise SEQ ID NO: 1, except that residue 50 of SEQ ID NO: 1 is replaced with a different residue and either (a) one or more of residues 5, 12, 18, 23, 31, 35, 37, 46, 48, 53, 56, 57, 59, 61, 67, 81, 83, 97, 98, 99, and 105 of SEQ ID NO: 1 are replaced with a different residue, and (b) a tyrosine (Y) residue is inserted into SEQ ID NO: 1 after residue 57.
  • Residue 50 of SEQ ID NO: 1 can be replaced with any suitable amino acid residue that can be the same or different in each position.
  • each of residues 5, 12, 18, 23, 31, 35, 37, 46, 48, 53, 56, 57, 59, 61, 67, 81, 83, 97, 98, 99, and 105 of SEQ ID NO: 1 can be replaced with any suitable amino acid residue that can be the same or different in each position.
  • the amino acid residue of a first position can be replaced with a first different amino acid residue
  • the amino acid residue of a second position can be replaced with a second different amino acid residue, wherein the first and second different amino acid residues are the same or different.
  • An amino acid "replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence.
  • Amino acids are broadly grouped as “aromatic” or “aliphatic.”
  • An aromatic amino acid includes an aromatic ring.
  • aromatic amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp).
  • Non-aromatic amino acids are broadly grouped as "aliphatic.”
  • aliphatic amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).
  • Aliphatic amino acids may be sub-divided into four sub-groups.
  • the "large aliphatic non-polar sub-group” consists of valine, leucine, and isoleucine.
  • the "aliphatic slightly-polar sub-group” consists of methionine, serine, threonine, and cysteine.
  • the "aliphatic polar/charged sub-group” consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine.
  • the "small-residue sub-group” consists of glycine and alanine.
  • the group of charged/polar amino acids may be sub-divided into three sub-groups: the "positively-charged sub-group” consisting of lysine and arginine, the "negatively-charged sub-group” consisting of glutamic acid and aspartic acid, and the "polar sub-group” consisting of asparagine and glutamine.
  • Aromatic amino acids may be sub-divided into two sub-groups: the "nitrogen ring sub-group” consisting of histidine and tryptophan and the "phenyl sub-group” consisting of phenylalanine and tyrosine.
  • the amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative.
  • the phrase "conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property.
  • a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer- Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra).
  • Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH 2 can be maintained.
  • the isolated immunoglobulin heavy chain polypeptide comprises SEQ ID NO: 1, except that residue 50 of SEQ ID NO: 1 is replaced with a valine (V) residue or a glycine (G) residue.
  • the isolated immunoglobulin heavy chain polypeptide comprises SEQ ID NO: 1, except that residue 50 of SEQ ID NO: 1 is replaced with a valine (V) residue or a glycine (G) residue, and wherein (a) residue 5 of SEQ ID NO: 1 is replaced with a valine (V) residue, (b) residue 12 of SEQ ID NO: 1 is replaced with an isoleucine (I) residue, (c) residue 18 of SEQ ID NO: 1 is replaced with a proline (P) residue, (d) residue 23 of SEQ ID NO: 1 is replaced with a valine (V) residue or a threonine residue (T), (e) residue 31 of SEQ ID NO: 1 is replaced with an asparagine (N) residue, an asparagine (N) residue, an
  • the isolated immunoglobulin heavy chain polypeptide comprises SEQ ID NO: 1 except that a tyrosine (Y) residue is inserted into SEQ ID NO: 1 after residue 56.
  • any one or more of the foregoing replacements can be combined with an insertion of a tyrosine (Y) residue into SEQ ID NO: 1 after residue 57.
  • immunoglobulin heavy chain polypeptides as described above can comprise any one of the following amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117,
  • the isolated immunoglobulin heavy chain polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 34, wherein optionally one or more of residues 24, 28, 31, 33, 34, 50, 53, 55, 56, 61, 65, 67, 79, 81, and 88 of SEQ ID NO: 34 are replaced with a different amino acid residue.
  • inventive immunoglobulin heavy chain polypeptide consists essentially of SEQ ID NO: 34 and optional amino acid replacements
  • additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation).
  • the inventive immunoglobulin heavy chain polypeptide consists of SEQ ID NO: 34 and optional amino acid replacements, the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide).
  • the isolated immunoglobulin heavy chain polypeptide can comprise SEQ ID NO: 34.
  • the isolated immunoglobulin heavy chain polypeptide can comprise SEQ ID NO: 34, except that one or more of residues 24, 28, 31, 33, 34, 50, 53, 55, 56, 61, 65, 67, 79, 81, and 88 of SEQ ID NO: 34 are replaced with a different amino acid residue.
  • residues 24, 28, 31, 33, 34, 50, 53, 55, 56, 61, 65, 67, 79, 81, and 88 of SEQ ID NO: 34 can be replaced with any suitable amino acid residue that can be the same or different in each position.
  • the amino acid residue of a first position can be replaced with a first different amino acid residue
  • the amino acid residue of a second position can be replaced with a second different amino acid residue, wherein the first and second different amino acid residues are the same or different.
  • the isolated immunoglobulin heavy chain polypeptide comprises SEQ ID NO: 34, except that (a) residue 24 of SEQ ID NO: 34 is replaced with a serine (S) residue, (b) residue 28 of SEQ ID NO: 34 is replaced with a serine (S) residue, (c) residue 31 of SEQ ID NO: 34 is replaced with an arginine (R) residue, (d) residue 33 of SEQ ID NO: 34 is replaced with an alanine (A) residue, (e) residue 34 of SEQ ID NO: 34 is replaced with a leucine (L) residue, (f) residue 50 of SEQ ID NO: 34 is replaced with an alanine (A) residue, (g) residue 53 of SEQ ID NO: 34 is replaced with a g
  • Exemplary immunoglobulin heavy chain polypeptides as described above can comprise any one of the following amino acid sequences: SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.
  • the isolated immunoglobulin heavy chain polypeptide comprises SEQ ID NO: 55, wherein optionally one or more of residues of 26, 28, 30, 35, 50, 53, and 59 of SEQ ID NO: 55 are replaced with a different amino acid residue.
  • the isolated immunoglobulin heavy chain polypeptide can comprise SEQ ID NO: 55.
  • the isolated immunoglobulin heavy chain polypeptide can comprise SEQ ID NO: 55, except that one or more of residues of 26, 28, 30, 35, 50, 53, and 59 are replaced with a different residue.
  • Each of residues 26, 28, 30, 35, 50, 53, and 59 of SEQ ID NO: 55 can be replaced with any suitable amino acid residue that can be the same or different in each position.
  • the amino acid residue of a first position can be replaced with a first different amino acid residue
  • the amino acid residue of a second position can be replaced with a second different amino acid residue, wherein the first and second different amino acid residues are the same or different.
  • the isolated immunoglobulin heavy chain polypeptide comprises SEQ ID NO: 55, except that (a) residue 26 of SEQ ID NO: 55 is replaced with an arginine (R) residue, (b) residue 28 of SEQ ID NO: 55 is replaced with a serine (S) residue, (c) residue 30 of SEQ ID NO: 55 is replaced with an arginine (R) residue, (d) residue 35 of SEQ ID NO: 55 is replaced with an asparagine (N) residue or a threonine (T) residue, (e) residue 50 of SEQ ID NO: 55 is replaced with a serine (S) residue or a glycine (G) residue, (f) residue 53 of SEQ ID NO: 55 is replaced with a glycine
  • any suitable amino acids can be inserted into the amino acid sequence of the immunoglobulin heavy chain polypeptide.
  • at least one amino acid e.g., 2 or more, 5 or more, or 10 or more amino acids
  • 20 amino acids e.g., 18 or less, 15 or less, or 12 or less amino acids
  • 1-10 amino acids e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids
  • amino acid(s) can be inserted into SEQ ID NO: l, SEQ ID NO: 34, or SEQ ID NO: 55 in any suitable location.
  • the invention provides an isolated immunoglobulin heavy chain polypeptide which comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%>, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO: 1-68, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 119.
  • Nucleic acid or amino acid sequence is at least 90% identical (e.g., at least 91%>, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO: 1-68, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID
  • identity can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer).
  • a number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs.
  • Such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches).
  • BLAST programs e.g., BLAST 2.1, BL2SEQ, and later versions thereof
  • FASTA programs e.g., FASTA3x, FASTM, and SSEARCH
  • Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al, Proc. Natl. Acad. Sci.
  • the invention also provides an isolated immunoglobulin heavy chain polypeptide which comprises CDRl, CDR2, or CDR3, or any combination of CDRl, CDR2, and/or CDR3, of an immunoglobulin heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NO: 1-68.
  • the isolated immunoglobulin heavy chain polypeptide comprises only one of CDRl, CDR2 or CDR3 of an amino acid sequence comprising any one of SEQ ID NO: 1-68.
  • the isolated immunoglobulin heavy chain polypeptide comprises CDRl and CDR2 , CDRl and CDR3, or CDR2 and CDR3 of an amino acid sequence comprising any one of SEQ ID NO: 1-68.
  • the isolated immunoglobulin heavy chain polypeptide can comprise CDRl, CDR2, and CDR3 of an amino acid sequence of any one of SEQ ID NO: 1-68, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 119.
  • an immunoglobulin light chain polypeptide that binds to ActRII comprises the amino acid sequence of SEQ ID NO: 69.
  • the isolated immunoglobulin light chain polypeptide comprises, consists of, or consists essentially of SEQ ID NO: 69, wherein optionally (a) one or more of residues 4, 13, 32, 36, 40, 52, 53, 59, 61, 62, 69, 72, 73, 82, 89, 96, 97, 100, 106, and 110 of SEQ ID NO: 69 are replaced with a different amino acid residue, (b) an amino acid sequence comprising YSS (SEQ ID NO: 98) is inserted into SEQ ID NO: 69 after residue 33, (c) an amino acid sequence comprising NKNYL (SEQ ID NO: 99) is inserted into SEQ ID NO: 69 after residue 39, (d) an amino acid sequence comprising NNNYL (SEQ ID NO: 100) is inserted into SEQ
  • inventive immunoglobulin light chain polypeptide consists essentially of SEQ ID NO: 69 and optional amino acid insertions and/or replacements
  • additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation).
  • the inventive immunoglobulin light chain polypeptide consists of SEQ ID NO: 69 and optional amino acid insertions and/or replacements
  • the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide).
  • the isolated immunoglobulin light chain polypeptide can comprise SEQ ID NO: 69, except that one or more of residues 4, 13, 32, 36, 40, 52, 53, 59, 61, 62, 69, 72, 73, 82, 89, 96, 97, 100, 106, and 110 of SEQ ID NO: 69 are replaced with a different amino acid residue, an amino acid sequence comprising YSS (SEQ ID NO: 98) is inserted into SEQ ID NO: 69 after residue 33, and an amino acid sequence comprising NKNYL (SEQ ID NO: 99) is inserted into SEQ ID NO: 69 after residue 39.
  • the isolated immunoglobulin light chain polypeptide can comprise or consist of SEQ ID NO: 69, except that one or more of residues 4, 13, 32, 36, 40, 52, 53, 59, 61, 62, 69, 72, 73, 82, 89, 96, 97, 100, 106, and 110 of SEQ ID NO: 69 are replaced with a different amino acid residue, an amino acid sequence comprising YSS (SEQ ID NO: 98) is inserted into SEQ ID NO: 69 after residue 33, and an amino acid sequence comprising NNNYL (SEQ ID NO: 100) is inserted into SEQ ID NO: 69 after residue 39.
  • SEQ ID NO: 98 amino acid sequence comprising YSS
  • NNNYL SEQ ID NO: 100
  • the isolated immunoglobulin light chain polypeptide can comprise SEQ ID NO: 69, except that one or more of residues 4, 13, 32, 36, 40, 52, 53, 59, 61, 62, 69, 72, 73, 82, 89, 96, 97, 100, 106, and 110 of SEQ ID NO: 69 are replaced with a different amino acid residue, and an amino acid sequence comprising NNNYL (SEQ ID NO: 100) is inserted into SEQ ID NO: 69 after residue 39.
  • Each of residues 4, 13, 32, 36, 40, 52, 53, 59, 61, 62, 69, 72, 73, 82, 89, 96, 97, 100, 106, and 110 of SEQ ID NO: 69 can be replaced with any suitable amino acid residue that can be the same or different in each position.
  • the amino acid residue of a first position can be replaced with a first different amino acid residue
  • the amino acid residue of a second position can be replaced with a second different amino acid residue, wherein the first and second different amino acid residues are the same or different.
  • the isolated immunoglobulin light chain polypeptide comprises SEQ ID NO: 69, except that (a) residue 4 of SEQ ID NO: 69 is replaced with a leucine (L) residue, (b) residue 13 of SEQ ID NO: 69 is replaced with a leucine (L) residue, (c) residue 32 of SEQ ID NO: 69 is replaced with a threonine (T) residue or an arginine (R) residue, (d) residue 36 of SEQ ID NO: 69 is replaced with an asparagine (N) residue, (e) residue 40 of SEQ ID NO: 69 is replaced with a glycine (G) residue, (f) residue 52 of SEQ ID NO: 69 is replaced with a valine (V) residue, (g) residue 53 of SEQ ID NO: 69 is replaced with a phenylalanine (F) residue, (h) residue 59 of SEQ ID NO: 69 is replaced with an isoleucine (I) residue, (i) residue
  • Exemplary immunoglobulin light chain polypeptides as described above can comprise any one of the following amino acid sequences: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.
  • one or more amino acids can be inserted into the aforementioned immunoglobulin light chain polypeptide.
  • Any number of any suitable amino acids can be inserted into the amino acid sequence of the immunoglobulin light chain polypeptide.
  • at least one amino acid e.g., 2 or more, 5 or more, or 10 or more amino acids
  • 20 amino acids e.g., 18 or less, 15 or less, or 12 or less amino acids
  • 1-10 amino acids e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids are inserted in to the amino acid sequence of the immunoglobulin light chain polypeptide.
  • the amino acid(s) can be inserted into SEQ ID NO: 69 in any suitable location.
  • the amino acid(s) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of SEQ ID NO: 69.
  • the amino acid(s) are inserted into CDR1 of SEQ ID NO: 69.
  • immunoglobulin light chain polypeptide comprising SEQ ID NO: 69 can include an amino acid insertion alone, or in combination with one or more amino acid replacements and/or deletions described herein.
  • Exemplary immunoglobulin light chain polypeptides as described above can comprise SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 97.
  • the isolated immunoglobulin light chain polypeptide comprises, consists of, or consists essentially of SEQ ID NO: 101, wherein optionally residue 93 of SEQ ID NO: 101 is replaced with a different amino acid residue.
  • inventive immunoglobulin light chain polypeptide consists essentially of SEQ ID NO: 101 and the optional replacement of residue 93 of SEQ ID NO: 101, additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation).
  • the inventive immunoglobulin light chain polypeptide consists of SEQ ID NO: 101 and the optional replacement of residue 93 of SEQ ID NO: 101
  • the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide).
  • the isolated immunoglobulin light chain polypeptide comprises SEQ ID NO: 101.
  • the isolated immunoglobulin light chain polypeptide comprises SEQ ID NO: 101, except that residue 93 of SEQ ID NO: 101 is replaced with a different amino acid residue. Residue 93 of SEQ ID NO: 101 can be replaced with any suitable amino acid residue.
  • residue 93 of SEQ ID NO: 101 is replaced with a histidine (H) residue.
  • An exemplary immunoglobulin light chain polypeptide as described above can comprise SEQ ID NO: 102.
  • the isolated immunoglobulin light chain polypeptide comprises, consists of, or consists essentially of SEQ ID NO: 103, wherein optionally residue 95 of SEQ ID NO: 103 is replaced with a different amino acid residue.
  • inventive immunoglobulin light chain polypeptide consists essentially of SEQ ID NO: 103 and the optional replacement of residue 95 of SEQ ID NO: 103
  • additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation).
  • the inventive immunoglobulin light chain polypeptide consists of SEQ ID NO: 103 and the optional replacement of residue 95 of SEQ ID NO: 103
  • the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide).
  • the isolated immunoglobulin light chain polypeptide comprises SEQ ID NO: 103.
  • the isolated immunoglobulin light chain polypeptide comprises SEQ ID NO: 103, except that residue 95 of SEQ ID NO: 103 is replaced with a different amino acid residue.
  • Residue 95 of SEQ ID NO: 103 can be replaced with any suitable amino acid residue.
  • residue 95 of SEQ ID NO: 103 is replaced with a threonine (T) residue.
  • An exemplary immunoglobulin light chain polypeptide as described above can comprise SEQ ID NO: 104.
  • the invention provides an isolated immunoglobulin light chain polypeptide which comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%>, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO: 69-97, SEQ ID NO: 101-104, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.
  • Nucleic acid or amino acid sequence "identity," as described herein, can be determined using the methods described herein.
  • the invention also provides an isolated immunoglobulin light chain polypeptide which comprises CDR1, CDR2, or CDR3, or any combination of CDR1, CDR2, and/or CDR3, of an immunoglobulin light chain variable region comprising an amino acid sequence of any one of SEQ ID NO: 69-97, SEQ ID NO: 101-104, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.
  • the isolated immunoglobulin light chain polypeptide comprises only one of CDR1, CDR2 or CDR3 of an amino acid sequence comprising any one of SEQ ID NO: 69-97, SEQ ID NO: 101-104, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.
  • the isolated immunoglobulin light chain polypeptide comprises CDRl and CDR2, CDRl and CDR3, or CDR2 and CDR3 of an amino acid sequence comprising any one of SEQ ID NO: 69-97, SEQ ID NO: 101-104, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.
  • the isolated immunoglobulin light chain polypeptide can comprise CDRl, CDR2, and CDR3 of an amino acid sequence of any one of SEQ ID NO: 69-97, SEQ ID NO: 101-104, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.
  • the invention provides an isolated activin receptor type II (ActRII)-binding agent comprising or consisting of the inventive isolated amino acid sequences described herein.
  • activin receptor type II (ActRII)-binding agent is meant a molecule, preferably a proteinaceous molecule, that binds specifically to activin receptor type II (ActRII).
  • ActRII activin receptor type II
  • the ActRII -binding agent can bind to either ActRII A or ActRIIB or to both ActRII A and ActRIIB.
  • the inventive isolated ActRII-binding agent binds to both ActRIIA and ActRIIB, the ActRII-binding agent is referred to as a "dual reactive" binding agent (e.g., a dual reactive antibody).
  • the ActRII-binding agent is an antibody or a fragment (e.g., immunogenic fragment) thereof.
  • the isolated ActRII-binding agent of the invention comprises or consists of the inventive isolated immunoglobulin heavy chain polypeptide and/or the inventive isolated immunoglobulin light chain polypeptide.
  • the isolated ActRII-binding agent comprises or consists of the inventive immunoglobulin heavy chain polypeptide or the inventive immunoglobulin light chain polypeptide.
  • the isolated ActRII-binding agent comprises or consists of the inventive immunoglobulin heavy chain polypeptide and the inventive immunoglobulin light chain polypeptide.
  • the invention is not limited to an isolated ActRII-binding agent comprising or consisting of an immunoglobulin heavy chain polypeptide or light chain polypeptide having replacements, insertions, and/or deletions of the specific amino acid residues disclosed herein.
  • any amino acid residue of the inventive immunoglobulin heavy chain polypeptide and/or the inventive immunoglobulin light chain polypeptide can be replaced, in any combination, with a different amino acid residue, or can be deleted or inserted, so long as the biological activity of the ActRII-binding agent is enhanced or improved as a result of the amino acid replacements, insertions, and/or deletions.
  • the "biological activity" of an ActRII-binding agent refers to, for example, binding affinity for a particular ActRII epitope, neutralization or inhibition of ActRII protein activity in vitro or in vivo (e.g., IC 50 ), pharmacokinetics, and cross-reactivity (e.g., with non-human homo logs or orthologs of the ActRII protein, or with other proteins or tissues).
  • Other biological properties or characteristics of an antigen-binding agent recognized in the art include, for example, avidity, selectivity, solubility, folding, immunotoxicity, expression, formulation, and catalytic activity. The aforementioned properties or characteristics can be observed, measured, and/or assessed using standard techniques including, but not limited to, ELISA, competitive ELISA, BIACORE surface plasmon resonance analysis (SPR), or
  • KINEXATM in vitro or in vivo neutralization assays, receptor binding assays, cytokine or growth factor production and/or secretion assays, and signal transduction and immunohistochemistry assays.
  • the terms "inhibit” or “neutralize,” as used herein with respect to the activity of a ActRII-binding agent, refer to the ability to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, alter, eliminate, stop, or reverse the progression or severity of, for example, the biological activity of an ActRII protein, or a disease or condition associated with an ActRII protein.
  • the isolated ActRII-binding agent of the invention preferably inhibits or neutralizes the activity of an ActRII protein by at least about 20%, about 30%, about 40%, about 50%, about 60%), about 70%), about 80%, about 90%, about 95%, about 100%), or a range defined by any two of the foregoing values.
  • the isolated ActRII-binding agent of the invention can be a whole antibody, as described herein, or an antibody fragment.
  • fragment of an antibody “antibody fragment,” and “functional fragment of an antibody” are used interchangeably herein to mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al, Nat. Biotech., 23(9): 1126-1129 (2005)).
  • the isolated ActRII binding agent can contain any ActRII-binding antibody fragment.
  • the antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof.
  • antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the V L , V H , C L , and CHi domains, (ii) a F(ab') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (iv) a Fab' fragment, which results from breaking the disulfide bridge of an F(ab') 2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.
  • a Fab fragment which is a monovalent fragment consisting of the V L , V H , C L , and CHi domains
  • the isolated ActRII-binding agent comprises a fragment of the immunoglobulin heavy chain or light chain polypeptide
  • the fragment can be of any size so long as the fragment binds to, and preferably inhibits the activity of, an ActRII protein.
  • a fragment of the immunoglobulin heavy chain polypeptide desirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values) amino acids.
  • a fragment of the immunoglobulin light chain polypeptide desirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values) amino acids.
  • the antibody or antibody fragment desirably comprises a constant region (F c ) of any suitable class.
  • the antibody or antibody fragment comprises a constant region that is based upon wild-type IgGl, IgG2, or IgG4 antibodies, or variants thereof.
  • the ActRII-binding agent also can be a single chain antibody fragment.
  • single chain antibody fragments include, but are not limited to, (i) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., V L and V H ) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al, Science, 242: 421-426 (1988); Huston et al, Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al, Nat.
  • scFv single chain Fv
  • a diabody which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a V H connected to a V L by a peptide linker that is too short to allow pairing between the V H and V L on the same polypeptide chain, thereby driving the pairing between the complementary domains on different V H -V L polypeptide chains to generate a dimeric molecule having two functional antigen binding sites.
  • Antibody fragments are known in the art and are described in more detail in, e.g., U.S. Patent Application Publication 2009/0093024 Al .
  • the isolated ActRII-binding agent also can be an intrabody or fragment thereof.
  • An intrabody is an antibody which is expressed and which functions intracellularly. Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Intrabodies include single domain fragments such as isolated V H and V L domains and scFvs. An intrabody can include sub-cellular trafficking signals attached to the N or C terminus of the intrabody to allow expression at high
  • an intrabody Upon interaction with a target gene, an intrabody modulates target protein function and/or achieves phenotypic/functional knockout by mechanisms such as accelerating target protein degradation and sequestering the target protein in a non-physiological sub-cellular compartment.
  • Other mechanisms of intrabody-mediated gene inactivation can depend on the epitope to which the intrabody is directed, such as binding to the catalytic site on a target protein or to epitopes that are involved in protein-protein, protein-DNA, or protein-RNA interactions.
  • the isolated ActRII-binding agent can be, or can be obtained from, a human antibody, a non-human antibody, or a chimeric antibody.
  • chimeric is meant an antibody or fragment thereof comprising both human and non-human regions.
  • the isolated ActRII-binding agent is a humanized antibody.
  • a "humanized” antibody is a monoclonal antibody comprising a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody.
  • Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat).
  • a humanized antibody can comprise, one, two, or three CDRs obtained or derived from a non-human antibody.
  • CDRH3 of the inventive ActRII-binding agent is obtained or derived from a mouse monoclonal antibody, while the remaining variable regions and constant region of the inventive ActRII-binding agent are obtained or derived from a human monoclonal antibody.
  • a human antibody, a non-human antibody, a chimeric antibody, or a humanized antibody can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody recombinantly) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and are described in, for example, Kohler and
  • a human antibody or a chimeric antibody can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes.
  • transgenic mice wherein endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB- MOUSETM, the Kirin TC MOUSETM, and the Kyowa Kirin KM-MOUSETM (see, e.g., Lonberg, Nat.
  • a humanized antibody can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey (2009)), including, e.g., grafting of non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al, Methods, 36(1): 25-34 (2005); and Hou et al, J. Biochem., 144(1): 115-120 (2008)).
  • a humanized antibody can be produced using the methods described in, e.g., U.S. Patent Application Publication
  • the ActRII-binding agent binds an epitope of an ActRII protein which comprises the amino acid sequence of any one of SEQ ID NO: 105-114.
  • the invention also provides an isolated or purified epitope of an ActRII protein, which comprises the amino acid sequence of any one of SEQ ID NO: 105-114.
  • the invention also provides one or more isolated or purified nucleic acid sequences that encode the inventive immunoglobulin heavy chain polypeptide, the inventive
  • nucleic acid sequence is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides.
  • nucleic acid and polynucleotide refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA.
  • the terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped
  • the invention further provides a vector comprising one or more nucleic acid sequences encoding the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, and/or the inventive ActRII-binding agent.
  • the vector can be, for example, a plasmid, episome, cosmid, viral vector (e.g., retroviral or adenoviral), or phage.
  • Suitable vectors and methods of vector preparation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)).
  • the vector preferably comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the coding sequence in a host cell.
  • expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).
  • promoters including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art.
  • Representative sources of promoters include for example, virus, mammal, insect, plant, yeast, and bacteria, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other commercial or individual sources.
  • Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3' or 5' direction).
  • Non- limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter.
  • Inducible promoters include, for example, the Tet system (U.S. Patents 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al, Proc. Natl. Acad.
  • Enhancers refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences. Enhancers can be located upstream, within, or downstream of coding sequences.
  • selectable marker genes are known in the art and described in, e.g., International Patent Application Publications WO 1992/008796 and WO 1994/028143; Wigler et al, Proc. Natl. Acad. Sci. USA, 77: 3567-3570 (1980); O'Hare et al, Proc. Natl. Acad. Sci. USA, 78: 1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072-2076 (1981); Colberre-Garapin et al, J. Mol.
  • the vector is an "episomal expression vector” or "episome,” which is able to replicate in a host cell, and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al, Gene Therapy, 11: 1735-1742 (2004)).
  • Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein Ban- Nuclear Antigen 1 (EBNA1) and the Epstein Barr Virus (EBV) origin of replication (oriP).
  • kits include integrating expression vectors, which may randomly integrate into the host cell's DNA, or may include a recombination site to enable the specific recombination between the expression vector and the host cell's chromosome.
  • integrating expression vectors may utilize the endogenous expression control sequences of the host cell's chromosomes to effect expression of the desired protein.
  • Examples of vectors that integrate in a site specific manner include, for example, components of the flp-in system from Invitrogen (Carlsbad, CA) (e.g., pcDNATM5/FRT), or the cre-lox system, such as can be found in the pExchange-6 Core Vectors from Stratagene (La Jolla, CA).
  • vectors that randomly integrate into host cell chromosomes include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Invitrogen (Carlsbad, CA), and pCI or pFNIOA (ACT) FLEXITM from Promega (Madison, WI).
  • Viral vectors also can be used.
  • Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands), the lentiviral-based pLPl from Invitrogen
  • Nucleic acid sequences encoding the inventive amino acid sequences can be provided to a cell on the same vector (i.e., in cis).
  • a unidirectional promoter can be used to control expression of each nucleic acid sequence.
  • a combination of bidirectional and unidirectional promoters can be used to control expression of multiple nucleic acid sequences.
  • Nucleic acid sequences encoding the inventive amino acid sequences alternatively can be provided to the population of cells on separate vectors (i.e., in trans). Each of the nucleic acid sequences in each of the separate vectors can comprise the same or different expression control sequences. The separate vectors can be provided to cells simultaneously or sequentially.
  • the vector(s) comprising the nucleic acid(s) encoding the inventive amino acid sequences can be introduced into a host cell that is capable of expressing the polypeptides encoded thereby, including any suitable prokaryotic or eukaryotic cell.
  • Preferred host cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently.
  • suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia.
  • Particularly useful prokaryotic cells include the various strains of Escherichia coli (e.g., K12, HB101 (ATCC No. 33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102).
  • the vector is introduced into a eukaryotic cell.
  • Suitable eukaryotic cells include, for example, yeast cells, insect cells, and mammalian cells.
  • suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhino- sporidium, Saccharomyces, and Schizosaccharomyces .
  • Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.
  • Suitable insect cells are described in, for example, Kitts et al, Biotechniques, 14: 810- 817 (1993); Lucklow, Curr. Opin. Biotechnol, 4: 564-572 (1993); and Lucklow et al, J. Virol, 67: 4566-4579 (1993).
  • Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad, CA).
  • mammalian cells are utilized in the invention.
  • suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, VA).
  • suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells (Urlaub et al, Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92).
  • suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70).
  • Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable.
  • Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from the ATCC. Methods for selecting suitable mammalian host cells and methods for transformation, culture, amplification, screening, and purification of cells are known in the art.
  • the mammalian cell is a human cell.
  • the mammalian cell can be a human lymphoid or lymphoid derived cell line, such as a cell line of pre-B lymphocyte origin.
  • human lymphoid cells lines include, without limitation, RAMOS (CRL-1596), Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al, Proc. Natl. Acad. Sci. USA, 85: 1581-1585 (1988)), Raji cells (CCL-86), and derivatives thereof.
  • a nucleic acid sequence encoding the inventive amino acid sequence may be introduced into a cell by "transfection,” “transformation,” or “transduction.”
  • Transfection refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.
  • Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol.
  • Phage or viral vectors can be introduced into host cells, after growth of infectious particles in suitable packaging cells, many of which are commercially available.
  • the invention provides a composition comprising an effective amount of the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive ActRII-binding agent, the inventive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence.
  • the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the inventive amino acid sequences, antigen-binding agent, or vector.
  • a pharmaceutically acceptable composition which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the inventive amino acid sequences, antigen-binding agent, or vector.
  • Any suitable carrier can be used within the context of the invention, and such carriers are well known in the art.
  • compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams &
  • the invention further provides a method of treating an ActRII-mediated disorder in a mammal.
  • the method comprises administering the aforementioned composition to a mammal having an ActRII-mediated disorder, whereupon the ActRII-mediated disorder is treated in the mammal.
  • ActRII-mediated disorder refers to any disease or disorder in which signaling through an ActRII protein causes or contributes to the pathological effects of the disease, or a decrease in ActRII protein levels or activity has a therapeutic benefit in mammals, preferably humans.
  • Examples of ActRII-mediated diseases include, but are not limited to, cancer cachexia, sarcopenia, post-surgery rehabilitation, spontaneous inclusion body myositis (sIBM), and muscular dystrophy.
  • the terms “treatment,” “treating,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease.
  • the inventive method comprises administering a "therapeutically effective amount" of the ActRII-binding agent.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • the therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the ActRII-binding agent to elicit a desired response in the individual.
  • a therapeutically effective amount of an ActRII-binding agent of the invention is an amount which decreases ActRII protein bioactivity in a human.
  • the pharmacologic and/or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents a disease or symptom thereof.
  • the inventive method comprises administering a "prophylactically effective amount" of the ActRII- binding agent.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease onset).
  • a typical dose can be, for example, in the range of 1 pg/kg to 20 mg/kg of animal or human body weight; however, doses below or above this exemplary range are within the scope of the invention.
  • the daily parenteral dose can be about 0.00001 ⁇ g/kg to about 20 mg/kg of total body weight (e.g., about 0.001 ⁇ g /kg, about 0.1 ⁇ g /kg , about 1 ⁇ g /kg, about 5 ⁇ g /kg, about 10 ⁇ g/kg, about 100 ⁇ g /kg, about 500 ⁇ g/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, or a range defined by any two of the foregoing values), preferably from about 0.1 ⁇ g/kg to about 10 mg/kg of total body weight (e.g., about 0.5 ⁇ g/kg, about 1 ⁇ g/kg, about 50 ⁇ g/kg, about 150 ⁇ g/kg, about 300 ⁇ g/kg, about
  • the desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
  • composition comprising an effective amount of the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive ActRII-binding agent, the inventive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the composition preferably is suitable for parenteral administration.
  • parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • the biological activity of the inventive ActRII-binding agent can be measured by any suitable method known in the art.
  • the biological activity can be assessed by determining the stability of a particular ActRII-binding agent.
  • the ActRII-binding agent e.g., an antibody
  • the ActRII-binding agent has an in vivo half life between about 15 minutes and 45 days (e.g., about 15 minutes, about 30 minutes, about 1 hour, about 6 hours, about 10 hours, about 12 hours, about 1 day, about 5 days, about 10 days, about 15 days, about 25 days, about 35 days, about 40 days, about 45 days, or a range defined by any two of the foregoing values).
  • the ActRII- binding agent has an in vivo half life between about 2 hours and 20 days (e.g., about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 2 days, about 3 days, about 7 days, about 12 days, about 14 days, about 17 days, about 19 days, or a range defined by any two of the foregoing values).
  • the ActRII-binding agent has an in vivo half life between about 10 days and about 40 days (e.g., about 10 days, about 13 days, about 16 days, about 18 days, about 20 days, about 23 days, about 26 days, about 29 days, about 30 days, about 33 days, about 37 days, about 38 days, about 39 days, about 40 days, or a range defined by any two of the foregoing values).
  • the biological activity of a particular ActRII -binding agent also can be assessed by determining its binding affinity to an ActRII protein or an epitope thereof.
  • affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (K D ).
  • affinity of a binding agent to a ligand such as affinity of an antibody for an epitope, can be, for example, from about 1 picomolar (pM) to about 1
  • the ActRII-binding agent can bind to an ActRII protein with a K D less than or equal to 1 nanomolar (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values).
  • 1 nanomolar e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values).
  • the ActRII-binding agent can bind to ActRII with a K D less than or equal to 200 pM (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoing values).
  • 200 pM e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1
  • Immunoglobulin affinity for an antigen or epitope of interest can be measured using any art- recognized assay.
  • Such methods include, for example, fluorescence activated cell sorting (FACS), separable beads (e.g., magnetic beads), antigen panning, SPR, KINEXATM, and/or ELISA (see, e.g., Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY, 2001).
  • the ActRII-binding agent of the invention may be administered alone or in combination with other drugs (e.g., as an adjuvant).
  • the ActRII-binding agent can be administered in combination with other agents for the treatment or prevention of the ActRII- mediated diseases disclosed herein.
  • the ActRII-binding agent can be used in combination with at least one other agent that increases muscle mass and/or strength including, for example, IGF-1, IGF-2, or variants of IGF- 1 or IGF-2, an anti-myostatin antibody, a myostatin propeptide, a myostatin decoy protein that binds ActRIIB but does not activate it, a beta 2 agonist, a Ghrelin agonist, a selective androgen receptor modulator (SARM), growth hormone (GH) agonists or mimetics, and/or follistatin.
  • IGF-1 IGF-2
  • IGF-2 an anti-myostatin antibody
  • a myostatin propeptide a myostatin decoy protein that binds ActRIIB but does not activate it
  • a beta 2 agonist a Ghrelin agonist
  • SARM selective androgen receptor modulator
  • GH growth hormone
  • the ActRII-binding agent described herein can be used in diagnostic or research applications.
  • the ActRII-binding agent can be used in a method to diagnose an ActRII -mediated disease or disorder.
  • the ActRII- binding agent can be used in an assay to monitor ActRII protein levels in a subject being tested for an ActRII-mediated disease or disorder.
  • Research applications include, for example, methods that utilize the ActRII-binding agent and a label to detect an ActRII protein in a sample, e.g., in a human body fluid or in a cell or tissue extract.
  • the ActRII-binding agent can be used with or without modification, such as covalent or non-covalent labeling with a detectable moiety.
  • the detectable moiety can be a radioisotope (e.g., 3H, 14C, 32P, 35S, or 1251), a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), or an enzyme (e.g., alkaline phosphatase, beta-galactosidase, or horseradish peroxidase).
  • a radioisotope e.g., 3H, 14C, 32P, 35S, or 1251
  • a fluorescent or chemiluminescent compound e.g., fluorescein isothiocyanate, rhodamine, or luciferin
  • an enzyme e.g., alkaline phosphatase, beta-galactosidase, or
  • any method known in the art for separately conjugating an antigen-binding agent (e.g., an antibody) to a detectable moiety may be employed in the context of the invention (see, e.g., Hunter et al, Nature, 194: 495-496 (1962); David et al, Biochemistry, 13: 1014-1021 (1974); Pain et al, J. Immunol. Meth., 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412 (1982)).
  • ActRII protein levels can be measured using the inventive ActRII-binding agent by any suitable method known in the art. Such methods include, for example, ELISA,
  • RIA radioimmunoassay
  • FACS fluorescence-activated cell sorting assay
  • a sample comprising, or suspected of comprising, an ActRII polypeptide
  • an ActRII-specific antibody under conditions suitable to form an antigen-antibody complex.
  • the antibody is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials (see, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)).
  • the amount of ActRII polypeptide expressed in a sample is then compared with the standard values.
  • the ActRII-binding agent can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a diagnostic assay.
  • the kit desirably includes substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides a detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g., a substrate precursor which provides a detectable chromophore or fluorophore.
  • other additives may be included in the kit, such as stabilizers, buffers (e.g., a blocking buffer or lysis buffer), and the like.
  • the relative amounts of the various reagents can be varied to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders (typically lyophilized), including excipients which on dissolution will provide a reagent solution having the appropriate
  • DNA samples encoding various immunoglobulin heavy chain (HC) and light chain (LC) polypeptides as described herein were prepared by combining the following: maxi-prepped DNA (containing 1 ⁇ g HC plasmid and 1 ⁇ g LC plasmid), 100 ⁇ OPTIMEMTM (Life Technologies Inc.
  • BIACORETM 4000 instrument (GE Healthcare, Waukesha, WI). Positive, negative, and media controls were included in the assay.
  • An anti-human Fc-specific IgG (GE Healthcare, Waukesha, WI) was amine-coupled to a CM5 sensor chip (GE Healthcare, Waukesha, WI) at two different immobilization levels (typically about 10.000 and 3.000 RU) on two spots of a given flow cell, allowing for 2-over-2 analyses. Antibodies of interest were then captured at spots with different immobilization levels resulting in varying antibody capture levels.
  • HBS-EP+ buffer 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% Polysorbate, pH 7.6 (Teknova, Hollister, CA) or buffer alone were then flowed over each antibody at each capture level and monitored for binding interactions.
  • the surface was regenerated with 3 M MgCl 2 for 1-2 minutes.
  • the data set was double reference subtracted and analyzed with a 1 : 1 interaction model with mass transport with the BIACORETM 4000 evaluation software (GE Healthcare,
  • DNA samples encoding various immunoglobulin heavy chain (HC) and light chain (LC) polypeptides as described herein were prepared by combining the following: maxi-prepped DNA (containing 6 ⁇ g HC plasmid and 6 ⁇ g LC plasmid), 1 ml OPTIMEMTM (Life Technologies Inc.
  • the eluate containing the antibodies was concentrated down to an antibody concentration of approximately 0.1-2 mg/ml in Amicon Ultra 10K concentrators (Millipore, Billerica, MA), and buffer was exchanged three times against PBS buffer.
  • Antibody concentration was determined on a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and purity was assessed by SDS- PAGE analysis.
  • HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% Polysorbate, pH 7.6) (Teknova, Hollister, CA) was used to reconstitute antigen at various concentrations (typically starting at 1000 nM and using either two-fold or three-fold serial dilutions thereof). Each antigen concentration was then injected for three minutes over captured antibody at a flow rate of 30 ⁇ / ⁇ , and allowed to dissociate for three minutes. The surface was regenerated with 60 of 3 M MgCl 2 after each cycle.
  • Antibodies also were characterized using a KINEXATM 3000 assay (Sapidyne Instruments, Boise, Idaho). KINEXATM technology measures the amount of unbound/free antibody molecule in solution phase after incubation with varying concentrations of antigen. Measuring binding events in the solution phase with micro beads for maximized surface area avoids mass transport limitations and mobility effects inherent to methods that measure binding to a solid phase. For each experiment, 50 ⁇ g of soluble human ActRIIB or ActRIIA extracellular domain was amine-coupled to 50 mg of UltraLink Biosupport beads (Thermo Fisher Scientific, Waltham, MA).
  • Antibody V regions were assembled as IgG4 (S228P) antibodies to reduce potential Fc receptor-related effector functions.
  • HC and LC polypeptides can form antibodies that bind to human ActRIIB and/or ActRIIA in vitro and inhibit cell signaling by endogenous ActRII ligands, including myostatin.
  • Hygromycin antibiotic (Life Technologies, Carlsbad, CA) . Cells were grown in an incubator at 37 °C and 5% C0 2 and sub-cultured every 3-4 days. Cells were detached using 0.25% Trypsin- EDTA (Life Technologies, Carlsbad, CA) and then split 1 : 10 into a new flask containing fresh medium. Only cells with low passage number (1-3) were used for the assay described below.
  • the (CAGA)12 luciferase reporter construct carries the luciferase gene downstream of a minimal promoter and multiple CAGA boxes which are specific for phosphorylated Smad-2 and Smad-3 in complex with Smad4.
  • the addition of purified myostatin (and also of GDF-11, activin A, or TGF beta) induces Smad phosphorylation and thus binding to CAGA- 12 reporter, thereby leading to luciferase gene expression.
  • HEK293T/17 (CAGA)12-luc cells were detached as described above and diluted in culture medium to a concentration of 2.5xl0 5 cells/ml. Subsequently 50 ⁇ cells per well were seeded into flat-bottom 96 well plates and incubated at 37 °C and 5% C0 2 for 5-6 hours.
  • the anti-ActRII HC and LC pairs set forth in Table 4, an irrelevant isotype control antibody, the anti-myostatin monoclonal antibody LY C1E4 (designated APE2480 LYC1E4 IgG4P), and a recombinant ActRIIB-Fc fusion protein (APE1777-ActRIIB-Fc fusion; positive control) were diluted in DMEM (Life Technologies, Carlsbad, CA) to the desired concentrations by two-fold dilution series. 50 ⁇ of the antibody solutions were added to the seeded cells in triplicate and cultivated for 1 hour to allow for binding of the antibodies to the cell-surface expressed ActRII receptors. 8 ng/ml of myostatin (R&D Systems, Minneapolis, MN) was added to the wells, and the cells were further cultivated over night at 37 °C and 5% C0 2 .
  • HC and LC polypeptides can form antibodies that bind to cell surface expressed human ActRIIB and/or ActRIIA in vitro and inhibit cell signaling by endogenous ActRII ligands such as myostatin.
  • Human skeletal muscle cells (HuSkMC) (Lonza, Basel, Switzerland) were cultured in growth medium (GM), which contains skeletal muscle basal medium (skBM) (Lonza, Basel, Switzerland) supplemented with skGM SINGLEQUOTTM supplement and growth factors (Lonza, Basel, Switzerland). Differentiation was initiated 24 hours after seeding by changing to serum- free differentiation medium consisting of skBM. At the onset of differentiation, IgG4 and IgGl anti- ActRII HC and LC pairs and the anti-myostatin antibody LY C1E4 were added to cells and tested for their ability to reverse inhibition of myoblast differentiation. Cells were differentiated into myotubes for 72 hours. For determination of potency (IC 50 ), each antibody was measured in triplicate in two separate experiments.
  • CK standard curves were freshly prepared using CK from rabbit muscle (Sigma Aldrich, St. Louis, MO). Protein content was determined using a BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL) following the manufacturer's instructions. IC 50 values were calculated from curve fits using a non-linear regression analysis in GraphPad PRISMTM software (GraphPad Software, Inc., La Jolla, CA).
  • HC and LC polypeptides can form antibodies that are more effective in reversing inhibition of myoblast differentiation than an anti-myostatin antibody.

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Abstract

L'invention concerne un polypeptide à chaîne lourde d'immunoglobuline isolé et un polypeptide à chaîne légère d'immunoglobuline isolé qui se lient à une protéine récepteur d'activine de type II (ActRII) (par exemple, l'un ou l'autre et les deux de ActRIIA et/ou ActRIIB). L'invention concerne un agent de liaison à ActRII qui comprend le polypeptide à chaîne lourde d'immunoglobuline et le polypeptide à chaîne légère d'immunoglobuline mentionnés ci-dessus. L'invention concerne également des vecteurs, des compositions et des procédés d'utilisation associés de l'agent de liaison à ActRII pour traiter une maladie à médiation par ActRII.
PCT/US2014/034344 2013-04-17 2014-04-16 Anticorps diriges contre le recepteur d'activine de type ii (actrii) Ceased WO2014172448A2 (fr)

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US10421969B2 (en) 2011-10-04 2019-09-24 Royal Holloway And Bedford New College Oligomers
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US11015200B2 (en) 2015-03-18 2021-05-25 Sarepta Therapeutics, Inc. Antisense-induced exon exclusion in myostatin
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US10947536B2 (en) 2011-10-04 2021-03-16 Royal Holloway And Bedford New College Oligomers
US10421969B2 (en) 2011-10-04 2019-09-24 Royal Holloway And Bedford New College Oligomers
US10662431B2 (en) 2011-10-04 2020-05-26 Royal Holloway And Bedford New College Oligomers
US11015200B2 (en) 2015-03-18 2021-05-25 Sarepta Therapeutics, Inc. Antisense-induced exon exclusion in myostatin
EP3858993A1 (fr) 2015-10-09 2021-08-04 Sarepta Therapeutics, Inc. Compositions et procédés pour le traitement de la dystrophie musculaire de duchenne et de troubles apparentés
WO2017147182A1 (fr) * 2016-02-22 2017-08-31 Acceleron Pharma Inc. Antagonistes d'actrii pour leur utilisation dans l'accroissement de l'activité immunitaire
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JP7058606B2 (ja) 2016-02-22 2022-04-22 アクセルロン ファーマ インコーポレイテッド 増加した免疫活性において使用するためのａｃｔｒｉｉアンタゴニスト
CN109153713B (zh) * 2016-03-10 2022-12-27 艾科赛扬制药股份有限公司 活化素2型受体结合蛋白及其用途
CN109153713A (zh) * 2016-03-10 2019-01-04 艾科赛扬制药股份有限公司 活化素2型受体结合蛋白及其用途
US11000565B2 (en) 2016-03-10 2021-05-11 Acceleron Pharma Inc. Methods of increasing muscle mass by administration of activin type 2 receptor antibodies
EP3426680A4 (fr) * 2016-03-10 2020-03-11 Acceleron Pharma Inc. Protéines de liaison au récepteur de l'activine de type 2 et leurs utilisations
US12042524B2 (en) 2016-03-10 2024-07-23 Acceleron Pharma Inc. Activin type 2 receptor binding proteins methods of making them
CN114127087A (zh) * 2019-05-30 2022-03-01 艾科赛扬制药股份有限公司 Actrii结合蛋白及其用途
JP2022534966A (ja) * 2019-05-30 2022-08-04 アクセレロン ファーマ インコーポレーテッド Ａｃｔｒｉｉ結合性タンパク質及びその使用
EP3976630A4 (fr) * 2019-05-30 2024-03-20 Acceleron Pharma Inc. Protéines de liaison à actrii et leurs utilisations
JP7669291B2 (ja) 2019-05-30 2025-04-28 アクセレロン ファーマ インコーポレーテッド Ａｃｔｒｉｉ結合性タンパク質及びその使用
JP2025118664A (ja) * 2019-05-30 2025-08-13 アクセレロン ファーマ インコーポレーテッド Ａｃｔｒｉｉ結合性タンパク質及びその使用
US12545736B2 (en) 2019-05-30 2026-02-10 Acceleron Pharma Inc ActRII-binding proteins and uses thereof
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