Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • 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
  • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2121/00—Preparations for use in therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/53—Hinge
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• the transforming growth factor-beta (TGFP) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis,
• TGF family members have diverse, often complementary biological effects. By manipulating the activity of a member of the TGF family, it is often possible to cause significant physiological changes in an organism.
• GDF8 also called myostatin
• inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength [Schuelke et al. (2004) N Engl J Med 350:2682-8]
• Changes in various tissues may be achieved by enhancing or inhibiting intracellular signaling (e.g., SMAD 1, 2, 3, 5, and/or 8) that is mediated by ligands of the TGF family.
• intracellular signaling e.g., SMAD 1, 2, 3, 5, and/or 8
• the TGF superfamily is comprised of over 30 secreted factors including TGF s, activins, nodals, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), and anti-Mullerian hormone (AMH) [Weiss et al. (2013) Developmental Biology, 2(1): 47-63]
• the TGF family can be divided into two phylogenetic branches based on the type I receptors they bind and the Smad proteins they activate.
• the more recently evolved branch which includes, e.g., the TGF s, activins, GDF8, GDF11, GDF9, BMP3 and nodal, which signal through type I receptors that activate Smads 2 and 3 [Hinck (2012) FEBS Letters 586: 1860-1870]
• the other branch comprises the more distantly related proteins of the superfamily and includes, e g., BMP2, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF1, GDF5, GDF6, and GDF7, which signal through Smads 1, 5, and 8.
• the present disclosure provides ActRIIB ⁇ RII heteromultimers that can antagonize a broad range of Smad 2/3 activating ligands.
• the disclosure demonstrates that an ActRIIB:T RII heterodimer inhibits TGF 1.
• TGF 3 activin A, activin B, GDF8, GDF11, and BMP 10- signaling pathways in a cell-based assay.
• ActRIIB and TbIIII homodimers alone inhibit a smaller subset of Smad 2/3 activating ligands.
• the data demonstrate that the ActRIIB:T RII heterodimer is a surprisingly more selective Smad 2/3 ligand antagonists than merely combining the antagonistic profiles of ActRIIB and TbIIII homodimer ligand traps.
• the ActRIIB ⁇ RII heterodimer inhibited activin A, activin B, GDF8, GDF11, and BMP 10- signaling pathways similarly to an ActRIIB homodimer.
• Ao ⁇ IIIIB:TbIIII heterodimer inhibition of BMP9 signaling pathways is significantly reduced compared to the ActRIIB homodimer. ActRIIB :TbIIII
• heteromultimers therefore are more selective antagonists of Smad 2/3 activating ligands compared to ActRIIB homodimers. Accordingly, an ActRIIB ⁇ RII heteromultimer will be more useful than an ActRIIB or TbIIII homodimer, or combination thereof, in certain applications where such broad, yet selective, Smad 2/3 antagonism is advantageous.
• Examples include therapeutic applications where it is desirable to antagonize one or more of T ⁇ RbI, TORb3.
• activin e.g., activin A, activin B, and activin AB
• GDF8 e.g., GDF8
• GDF11 e.g., GDF11 with decreased antagonism of BMP9.
• the disclosure provides for a multispecific binder of T ⁇ Rb- superfamily ligands.
• the multispecific binder protein is capable of binding to a) at least one of T ⁇ RbI and TORb3. and b) at least one of activin A, activin B, activin AB, GDF11, and GDF8.
• the multispecific binder comprises: a) a first portion that is capable of binding to TORb 1 and/or T ⁇ Rb3; and b) a second portion that is capable of binding to at least one of activin A, activin B, activin AB, GDF11, and GDF8.
• the multispecific binder is a heteromultimer comprising an ActRIIB polypeptide and a TbIIII polypeptide.
• the multispecific binder comprises a TbMI polypeptide and a folbstatin or a folbstatin-bke protein domain.
• the multispecific binder comprises a TbIIII polypeptide and an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to one or more of activin A, activin B, activin AB, GDF11, and/or GDF8.
• the multispecific binder comprises a TbIIII polypeptide and an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to GDF8.
• the disclosure provides for a heteromultimer comprising an ActRIIB polypeptide and a TbIIII polypeptide.
• the ActRIIB polypeptide comprises an amino acid sequence that is at least 75% identical to: a) a sequence beginning at any one of positions 20 to 29 of SEQ ID NO: 50, and ending at any one of positions 109 to 134 of SEQ ID NO: 50; b) a sequence beginning at position 20 of SEQ ID NO: 50, and ending at position 134 of SEQ ID NO: 50; c) a sequence beginning at position 29 of SEQ ID NO: 50 and ending at position 109 of SEQ ID NO: 50; d) a sequence beginning at position 25 of SEQ ID NO: 50 and ending at position 131 of SEQ ID NO: 50; e) the sequence of SEQ ID NO: 51; f) the sequence of SEQ ID NO: 52; g) the sequence of SEQ ID NO: 54; h) the sequence of SEQ ID NO: 55; or i)
• the ActRIIB polypeptide comprises an amino acid sequence that is at least 95% identical to: a) a sequence beginning at any one of positions 20 to 29 of SEQ ID NO: 50, and ending at any one of positions 109 to 134 of SEQ ID NO: 50; b) a sequence beginning at position 20 of SEQ ID NO: 50, and ending at position 134 of SEQ ID NO: 50; c) a sequence beginning at position 29 of SEQ ID NO: 50 and ending at position 109 of SEQ ID NO: 50; d) a sequence beginning at position 25 of SEQ ID NO: 50 and ending at position 131 of SEQ ID NO: 50; e) the sequence of SEQ ID NO: 51; f) the sequence of SEQ ID NO: 52; g) the sequence of SEQ ID NO: 54; h) the sequence of SEQ ID NO: 55; or i) the sequence of SEQ ID NO: 109.
• the ActRIIB polypeptide comprises a amino acid sequence is selected from: a) a sequence beginning at any one of positions 20 to 29 of SEQ ID NO: 50, and ending at any one of positions 109 to 134 of SEQ ID NO: 50; b) a sequence beginning at position 20 of SEQ ID NO: 50, and ending at position 134 of SEQ ID NO: 50; c) a sequence beginning at position 29 of SEQ ID NO: 50 and ending at position 109 of SEQ ID NO: 50; d) a sequence beginning at position 25 of SEQ ID NO: 50 and ending at position 131 of SEQ ID NO: 50; e) the sequence of SEQ ID NO: 51; f) the sequence of SEQ ID NO: 52; g) the sequence of SEQ ID NO: 54; h) the sequence of SEQ ID NO: 55; and i) the sequence of SEQ ID NO: 109.
• the ActRIIB polypeptide is a fusion protein comprising: a) a ActRIIB portion comprising an extracellular domain of ActRIIB; and b) a heterologous portion.
• the ActRIIB portion comprises an amino acid sequence that is at least 75% identical to: a) a sequence beginning at any one of positions 20 to 29 of SEQ ID NO: 50, and ending at any one of positions 109 to 134 of SEQ ID NO: 50; b) a sequence beginning at position 20 of SEQ ID NO: 50, and ending at position 134 of SEQ ID NO: 50; c) a sequence beginning at position 29 of SEQ ID NO: 50 and ending at position 109 of SEQ ID NO: 50; d) a sequence beginning at position 25 of SEQ ID NO: 50 and ending at position 131 of SEQ ID NO: 50; e) the sequence of SEQ ID NO: 51; f) the sequence of SEQ ID NO: 52; g) the sequence of SEQ ID NO: 54
• the ActRIIB portion comprises an amino acid sequence that is at least 90% identical to: a) a sequence beginning at any one of positions 20 to 29 of SEQ ID NO: 50, and ending at any one of positions 109 to 134 of SEQ ID NO: 50; b) a sequence beginning at position 20 of SEQ ID NO: 50, and ending at position 134 of SEQ ID NO: 50; c) a sequence beginning at position 29 of SEQ ID NO: 50 and ending at position 109 of SEQ ID NO: 50; d) a sequence beginning at position 25 of SEQ ID NO: 50 and ending at position 131 of SEQ ID NO: 50; e) the sequence of SEQ ID NO: 51; f) the sequence of SEQ ID NO: 52; g) the sequence of SEQ ID NO: 54; h) the sequence of SEQ ID NO: 55; or i) the sequence of SEQ ID NO: 109.
• the ActRIIB portion comprises an amino acid sequence that is at least 95% identical to: a) a sequence beginning at any one of positions 20 to 29 of SEQ ID NO: 50, and ending at any one of positions 109 to 134 of SEQ ID NO: 50; b) a sequence beginning at position 20 of SEQ ID NO: 50, and ending at position 134 of SEQ ID NO: 50; c) a sequence beginning at position 29 of SEQ ID NO: 50 and ending at position 109 of SEQ ID NO: 50; d) a sequence beginning at position 25 of SEQ ID NO: 50 and ending at position 131 of SEQ ID NO: 50; e) the sequence of SEQ ID NO: 51; f) the sequence of SEQ ID NO: 52; g) the sequence of SEQ ID NO: 54; h) the sequence of SEQ ID NO: 55; or i) the sequence of SEQ ID NO: 109.
• the ActRIIB portion comprises an amino acid sequence selected from: a) a sequence beginning at any one of positions 20 to 29 of SEQ ID NO: 50, and ending at any one of positions 109 to 134 of SEQ ID NO: 50; b) a sequence beginning at position 20 of SEQ ID NO: 50, and ending at position 134 of SEQ ID NO: 50; c) a sequence beginning at position 29 of SEQ ID NO: 50 and ending at position 109 of SEQ ID NO: 50; d) a sequence beginning at position 25 of SEQ ID NO: 50 and ending at position 131 of SEQ ID NO: 50; e) the sequence of SEQ ID NO: 51; f) the sequence of SEQ ID NO: 52; g) the sequence of SEQ ID NO: 54; h) the sequence of SEQ ID NO: 55; and i) the sequence of SEQ ID NO: 109.
• the heterologous portion comprises a first or second member of an interaction pair. In some embodiments, the heterologous portion comprises one or more amino acid modifications that promotes heterodimer formation. In some embodiments, the heterologous portion is an immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is a human immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is an immunoglobulin GlFc domain.
• the immunoglobulin Fc domain comprises an amino acid sequence that is at least 75% identical to: a) the amino acid sequence of SEQ ID NO: 68, wherein the sequence comprises a lysine (K) at position 356 and a K at position 399 based on the amino acid positioning of EU numbering scheme of Kabat; b) the amino acid sequence of SEQ ID NO: 69, wherein the sequence comprises a aspartic acid (D) at position 392 and a D at position 409 based on the amino acid positioning of EU numbering scheme of Kabat; c) the amino acid sequence of SEQ ID NO: 72, wherein the sequence comprises a cysteine (C) at position 354 and a tryptophan (W) at position 366 based on the amino acid positioning of EU numbering scheme of Kabat; or d) the amino acid sequence of SEQ ID NO: 73, wherein the sequence comprises a C at position 349, a serine (S) at position 366, an alanine (A)
• the immunoglobulin Fc domain comprises an amino acid sequence that is at least 95% identical to: a) the amino acid sequence of SEQ ID NO: 68, wherein the sequence comprises a lysine (K) at position 356 and a K at position 399 based on the amino acid positioning of EU numbering scheme of Kabat; b) the amino acid sequence of SEQ ID NO: 69, wherein the sequence comprises a aspartic acid (D) at position 392 and a D at position 409 based on the amino acid positioning of EU numbering scheme of Kabat; c) the amino acid sequence of SEQ ID NO: 72, wherein the sequence comprises a cysteine (C) at position 354 and a tryptophan (W) at position 366 based on the amino acid positioning of EU numbering scheme of Kabat; or d) the amino acid sequence of SEQ ID NO: 73, wherein the sequence comprises a C at position 349, a serine (S) at position 366, an alanine (A)
• the immunoglobulin Fc domain comprises an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 68; b) the amino acid sequence of SEQ ID NO: 69; c) the amino acid sequence of SEQ ID NO: 72; and d) the amino acid sequence of SEQ ID NO: 73.
• the linker comprises (GGGGS)n, wherein n1 > 5.
• the ActRIIB fusion protein comprises an amino acid sequence that is at least 75%, 80%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the ActRIIB fusion protein comprises the amino acid sequence of SEQ ID NO: 84.
• the ActRIIB fusion protein comprises an amino acid sequence that is at least 75%, 80%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the ActRIIB fusion protein comprises the amino acid sequence of SEQ ID NO: 90.
• the ActRIIB polypeptide consists of or consists essentially of: a) an ActRIIB polypeptide portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 51 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids; b) a linker portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1 additional amino acids; c) a heterologous portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, or 73 and no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids; and d) optionally a leader sequence (e.g., SEQ ID NO: 23).
• a leader sequence e.g
• the ActRIIB polypeptide consists of or consists essentially of: a) an ActRIIB polypeptide portion comprising the amino acid sequence of SEQ ID NO: 51 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids; b) a linker portion comprising the amino acid sequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1 additional amino acids; c) a heterologous portion comprising an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, or 73 and no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids; and d) optionally a leader sequence (e.g., SEQ ID NO: 23).
• the ActRIIB polypeptide comprises: a) an ActRIIB polypeptide portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to the sequence of SEQ ID NO: 51; b) a heterologous portion, wherein the heterologous portion comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, or 73; and c) a linker portion connecting the ActRIIB polypeptide portion and the heterologous portion; wherein the linker comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 6.
• the ActRIIB polypeptide comprises: a) an ActRIIB polypeptide portion comprising the amino acid sequence of SEQ ID NO: 51; b) a heterologous portion comprising an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, or 73; and c) a linker portion connecting the ActRIIB polypeptide portion and the heterologous portion; wherein the linker comprises the amino acid sequence of SEQ ID NO: 6.
• the ActRIIB polypeptide or ActRIIB fusion protein does not comprise an acidic amino acid at the residue corresponding to position 79 of SEQ ID NO: 50.
• the ActRIIB polypeptide or ActRIIB fusion protein does not comprise a D at the residue corresponding to position 79 of SEQ ID NO: 50.
• the TbBII polypeptide comprises an amino acid sequence that is at least 75% identical to: a) a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1; b) a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2; c) the sequence of SEQ ID NO: 18; d) the sequence of SEQ ID NO: 27; or e) the sequence of any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38; and 39.
• the TbBII polypeptide comprises an amino acid sequence that is at least 90% identical to: a) a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1; b) a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2; c) the sequence of SEQ ID NO: 18; d) the sequence of SEQ ID NO: 27; or e) the sequence of any one of SEQ ID NOs: 28,
• the TbIIII polypeptide comprises an amino acid sequence that is at least 95% identical to: a) a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1; b) a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2; c) the sequence of SEQ ID NO: 18; d) the sequence of SEQ ID NO: 27; or e) the sequence of any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38; and 39.
• the TbKII polypeptide comprises a amino acid sequence is selected from: a) a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1; b) a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2; c) the sequence of SEQ ID NO: 18; d) the sequence of SEQ ID NO: 27; and e) the sequence of any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38; and 39.
• the TbKII polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to the sequence of SEQ ID NO: 18. In some embodiments, the TbMI polypeptide comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the TbMI polypeptide is a fusion protein comprising: a) a TbMI portion comprising an extracellular domain of TbMI; and b) a heterologous portion.
• the TbMI portion comprises an amino acid sequence that is at least 75% identical to: a) a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1; b) a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2; c) the sequence of SEQ ID NO: 18; d) the sequence of SEQ ID NO: 27; or e) the sequence of any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38; and 39.
• the TbKII portion comprises an amino acid sequence that is at least 90% identical to: a) a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1; b) a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2; c) the sequence of SEQ ID NO: 18; d) the sequence of SEQ ID NO: 27; or e) the sequence of any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38; and 39.
• the TbKII portion comprises an amino acid sequence that is at least 95% identical to: a) a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1; b) a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2; c) the sequence of SEQ ID NO: 18; d) the sequence of SEQ ID NO: 27; or e) the sequence of any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38; and 39.
• the TbKII portion comprises an amino acid sequence selected from: a) a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1; b) a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2; c) the sequence of SEQ ID NO: 18; d) the sequence of SEQ ID NO: 27; or e) the sequence of any one of SEQ ID NOs: 28,
• the heterologous portion comprises a first or second member of an interaction pair. In some embodiments, the heterologous portion comprises one or more amino acid modifications that promotes heterodimer formation. In some embodiments, the heterologous portion is an
• the immunoglobulin Fc domain comprises an amino acid sequence that is at least 75% identical to: a) the amino acid sequence of SEQ ID NO: 68, wherein the sequence comprises a lysine (K) at position 356 and a K at position 399 based on the amino acid positioning of the EU numbering scheme of Kabat; b) the amino acid sequence of SEQ ID NO: 69, wherein the sequence comprises a aspartic acid (D) at position 392 and a D at position 409 based on the amino acid positioning of the EU numbering scheme of Kabat; c) the amino acid sequence of SEQ ID NO: 72, wherein the sequence comprises a cysteine (C) at position 354 and a tryptophan (W) at position 366 based on the amino acid
• the immunoglobulin Fc domain comprises an amino acid sequence that is at least 95% identical to: a) the amino acid sequence of SEQ ID NO: 68, wherein the sequence comprises a lysine (K) at position 356 and a K at position 399 based on the amino acid positioning of the EU numbering scheme of Kabat; b) the amino acid sequence of SEQ ID NO: 69, wherein the sequence comprises a aspartic acid (D) at position 392 and a D at position 409 based on the amino acid positioning of the EU numbering scheme of Kabat; c) the amino acid sequence of SEQ ID NO: 72, wherein the sequence comprises a cysteine (C) at position 354 and a tryptophan (W) at position 366 based on the amino acid positioning of the EU numbering scheme of Kabat; or d) the amino acid sequence of SEQ ID NO: 73, wherein the sequence comprises a C at position 349, a serine (S) at position 366, an alanine
• the immunoglobulin Fc domain comprises an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 68; b) the amino acid sequence of SEQ ID NO: 69; c) the amino acid sequence of SEQ ID NO: 72; and d) the amino acid sequence of SEQ ID NO: 73.
• the fusion protein further comprises a linker domain portion positioned between the TbMI portion and the heterologous portion. In some embodiments, the linker is between 10 and 25 amino acids in length.
• the linker comprises (GGGGS)n, wherein n1 > 5.
• the TbMI fusion protein comprises an amino acid sequence that is at least 75%, 80%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the TbMI fusion protein comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the TbINI fusion protein comprises an amino acid sequence that is at least 75%, 80%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the TbMI fusion protein comprises the amino acid sequence of SEQ ID NO: 93.
• the TbMI polypeptide consists of or consists essentially of: a) an TbMI polypeptide portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 18 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids; b) a linker portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1 additional amino acids; c) a heterologous portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, or 73 and no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids; and d) optionally a leader sequence ( e.g ., SEQ ID NO: 23).
• a leader sequence e.g
• the TbMI polypeptide consists of or consists essentially of: a) an TbMI polypeptide portion comprising the amino acid sequence of SEQ ID NO: 18 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids; b) a linker portion comprising the amino acid sequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1 additional amino acids; c) a heterologous portion comprising an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, or 73 and no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids; and d) optionally a leader sequence (e.g., SEQ ID NO: 23).
• a leader sequence e.g., SEQ ID NO: 23
• the TbMI polypeptide comprises: a) an TbMI polypeptide portion comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to the sequence of SEQ ID NO: 18; b) a heterologous portion, wherein the heterologous portion comprises an amino acid sequence that is at least 85%,
• linker portion connecting the TbMI polypeptide portion and the heterologous portion; wherein the linker comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 6.
• the TbMI polypeptide comprises: a) an TbMI polypeptide portion comprising the amino acid sequence of SEQ ID NO: 18; b) a heterologous portion comprising an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, or 73; and c) a linker portion connecting the TbMI polypeptide portion and the heterologous portion;
• the linker comprises the amino acid sequence of SEQ ID NO: 6.
• the heteromultimer comprises one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinylated amino acid, and an amino acid conjugated to a lipid moiety.
• the heteromultimer is glycosylated.
• the heteromultimer has a glycosylation pattern characteristic of expression of the polypeptide in CHO cells.
• the heteromultimer has a glycosylation pattern characteristic of expression of the polypeptide in CHO cells.
• the heteromultimer binds to one or more of: GDF11, GDF8, activin A, activin B, BMP10, T ⁇ RbI, and TORb3. In some embodiments, the heteromultimer inhibits on or more of GDF11, GDF8, activin A, activin B, BMP10, T ⁇ RbI, and T ⁇ Rb3 signaling as determined using a reporter gene assay. In some embodiments, the heteromultimer is a heterodimer. In some embodiments, the heteromultimer is isolated. In some embodiments, the
• the disclosure provides for an isolated polynucleotide comprising a coding sequence for any of the ActRIIB polypeptides or fusion proteins disclosed herein. In some embodiments, the disclosure provides for an isolated polynucleotide comprising a coding sequence for any of the ActRIIB polypeptides or fusion proteins disclosed herein. In some embodiments, the disclosure provides for an isolated polynucleotide comprising a coding sequence for any of the ActRIIB polypeptides or fusion proteins disclosed herein. In some embodiments, the disclosure provides for an isolated
• the disclosure provides for an isolated polynucleotide comprising a coding sequence for any of the ActRIIB polypeptides or fusion proteins disclosed herein and any of the TbIIII polypeptides or fusion proteins disclosed herein.
• the disclosure provides for a recombinant polynucleotide comprising a promotor sequence operably linked to any of the polynucleotides disclosed herein.
• the disclosure provides for a cell comprising the any of the polynucleotides disclosed herein.
• the cell is a CHO cell.
• the disclosure provides for a pharmaceutical preparation comprising any of the polypeptides/heteromultimers disclosed herein and a pharmaceutically acceptable excipient.
• the disclosure provides for a method of making a
• heteromultimer comprising an ActRIIB polypeptide and a TbMI polypeptide comprising culturing a cell under conditions suitable for expression of an ActRIIB polypeptide and a TbIIII polypeptide, wherein the cell comprises any one or more of the polynucleotides disclosed herein.
• the disclosure provides for a method of making a
• heteromultimer comprising an ActRIIB polypeptide and a TbIIII polypeptide comprising culturing a cell under conditions suitable for expression of an ActRIIB polypeptide and a TbIIII polypeptide, wherein the cell comprises any of the polynucleotides disclosed herein.
• the disclosure provides for a method of making a
• heteromultimer comprising an TbIIII polypeptide and an ActRIIB polypeptide comprising: a) culturing a first cell under conditions suitable for expression of an TbIIII polypeptide, wherein the first cell comprises any of the recombinant polynucleotides disclosed herein; b) recovering the TbIIII polypeptide so expressed; c) culturing a second cell under conditions suitable for expression of an ActRIIB polypeptide, wherein the second cell comprises any of the recombinant polynucleotides disclosed herein; d) recovering the ActRIIB polypeptide so expressed; e) combining the recovered TbIIII polypeptide and the recovered ActRIIB polypeptide under conditions suitable for ActRIIB :TbIBI heteromultimer formation.
• the disclosure provides for a method of modulating the response of a cell to a T ⁇ Rb superfamily member, the method comprising exposing the cell to any of the heteromultimers disclosed herein.
• the disclosure provides for a method of treating a disease or condition associated with a T ⁇ Rb superfamily member in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the heteromultimers disclosed herein or any of the pharmaceutical preparations disclosed herein.
• the disclosure provides for a method of treating a muscle-related disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the heteromultimers disclosed herein or any of the pharmaceutical preparations disclosed herein.
• the muscle-related disease or condition is selected from: muscular dystrophy, Duchene muscular dystrophy, Becker muscular dystrophy, Charcot-Marie-Tooth, facioscapulohumeral muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia.
• the disclosure provides for a method of treating a pulmonary-related disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the heteromultimers disclosed herein or any of the pharmaceutical preparations disclosed herein.
• the pulmonary-related disease or condition is selected from pulmonary hypertension, pulmonary arterial hypertension, and idiopathic pulmonary fibrosis.
• the disclosure provides for a method of treating a cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the heteromultimers disclosed herein or any of the pharmaceutical preparations disclosed herein.
• the disclosure provides for a method of treating a kidney-related disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the heteromultimers disclosed herein or any of the pharmaceutical preparations disclosed herein.
• the kidney-related disease or condition is selected from: Alport syndrome, chronic kidney disease, polycystic kidney disease and renal fibrosis.
• the disclosure provides for a method of treating a anemia or an anemia-related disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the heteromultimers disclosed herein or any of the pharmaceutical preparations disclosed herein.
• the anemia-related disease or condition is selected from: thalassemia, myelodysplastic syndrome, myelofibrosis, and sickle cell disease.
• the disclosure provides for a multispecific binder protein comprising a TbMI polypeptide and a follistatin polypeptide.
• the TbMI polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 170, or a biologically active fragment thereof.
• the follistatin polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 111, or a biologically active fragment thereof.
• the binder protein further comprises a heterologous portion.
• the heterologous portion is an Fc domain.
• the Fc domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 163.
• the heterologous portion is between the follistatin polypeptide and the TbINI polypeptide.
• the heterologous portion is conjugated to the follistatin polypeptide directly.
• the heterologous portion is conjugated to the follistatin polypeptide by means of a linker.
• the linker comprises the amino acid sequence of SEQ ID NO: 3.
• the heterologous portion is conjugated to the TbINI polypeptide directly.
• the heterologous portion is conjugated to the TbMI polypeptide by means of a linker.
• the linker conjugating the heterologous portion to the TbMI polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 165.
• the protein comprises, from N- terminus to C-terminus: the follistatin polypeptide, the heterologous domain, and the TbMI polypeptide.
• the protein comprises a leader sequence.
• the leader sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.
• the binder protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 164. In some embodiments, the binder protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 180 or 181.
• the disclosure provides for a multispecific binder protein comprising a TbMI polypeptide and an antibody or antigen-binding fragment capable of binding to GDF8.
• the TbINI polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 170, or a biologically active fragment thereof.
• the antibody or antigen-binding fragment comprises a variable heavy chain and a variable light chain.
• the variable heavy chain comprises CDRs having the amino acid sequence of SEQ ID NOs: 151-153.
• variable light chain comprises CDRs having the amino acid sequence of SEQ ID NOs: 154-156.
• variable heavy chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 167.
• variable light chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 174.
• the antibody or antigen binding fragment comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 168, or a biologically active fragment thereof.
• the antibody or antigen-binding fragment comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 167, or a biologically active fragment thereof.
• the antibody or antigen-binding fragment comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 171, or a biologically active fragment thereof.
• the protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 172.
• the protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 175. In some embodiments, the protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 182.
• the protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 172, and wherein the protein further comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 182.
• the protein comprises a leader sequence.
• the leader sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 176.
• the antibody or antigen binding fragment is also capable of binding to GDF11 and/or activin.
• the disclosure provides for a polynucleotide or collection of polynucleotides capable of expressing any of the multispecific binder proteins disclosed herein. In some embodiments, the disclosure provides for a vector or collection of vectors comprising any of the polynucleotides disclosed herein. In some embodiments, the disclosure provides for a host cell comprising and capable of expressing any of the polynucleotides or vectors disclosed herein. In some embodiments, the disclosure provides for a pharmaceutical composition comprising any of the multispecific binders disclosed herein and a pharmaceutically acceptable carrier.
• the disclosure provides for a method of treating a subject having a muscle disorder with any of the multispecific binders disclosed herein.
• the subject has muscular dystrophy.
• the subject has Duchenne Muscular Dystrophy.
• the subject has Becker Muscular Dystrophy.
• the disorder is associated with muscle fibrosis.
• the disorder is associated with muscle loss or muscle wasting.
• the disclosure provides for a fusion protein comprising an ActRIIB polypeptide and a TbIIII polypeptide.
• the ActRIIB polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 51 or 52.
• the TbMI polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 170.
• the ActRIIB polypeptide portion is N-terminal to the TbIIII polypeptide portion. In some embodiments, the ActRIIB polypeptide portion is C-terminal to the TbIIII polypeptide portion.
• a heterologous portion and/or one or more linker portions separate the ActRIIB and TbIIII polypeptide portions in the fusion protein.
• the heterologous portion is an Fc polypeptide portion comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 163.
• the heterologous portion is an Fc polypeptide portion comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73 (which may optionally lack the C-terminal lysine residue).
• the TbIIII polypeptide portion is fused to the Fc portion by means of a linker.
• the TbIIII polypeptide portion is fused to the Fc portion by means of a glycine-serine-rich linker.
• the linker comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 165.
• the ActRIIB polypeptide portion is fused to the Fc portion by means of a linker.
• the ActRIIB polypeptide portion is fused to the Fc portion by means of a linker comprising a GGG linker.
• the fusion protein comprises a signal sequence.
• the signal sequence comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23.
• the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 183 or 195.
• the fusion protein is a unit of a multimer. In some embodiments, the multimer is a homodimer.
• the multimer is a heteromultimer, wherein the fusion protein is one unit of the heteromultimer, and wherein the heteromultimer comprises a second protein unit.
• the second protein unit comprises an ActRIIB polypeptide portion but lacks a TbMI polypeptide portion.
• the second protein unit comprises a TbMI polypeptide portion but lacks an ActRIIB polypeptide portion.
• each unit of the heteromultimer comprises a member of an interaction pair.
• the members of the interaction pair comprise an Fc domain.
• the Fc domains comprise amino acid modifications that promote heteromultimer formation and/or to inhibit homomul timer formation.
• the Fc domains have been modified to include one or more “knob-in-hole” mutations.
• the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 184 or 196.
• the second unit of the heteromultimer comprises a TbINI polypeptide portion but lacks an ActRIIB polypeptide portion, wherein the second protein unit comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 185 or 197.
• the fusion protein comprises the amino acid sequence of SEQ ID NO:
• the second protein unit comprises the amino acid sequence of SEQ ID NO: 185 or 197.
• the disclosure provides for a fusion protein comprising a TbINI polypeptide portion and a heterologous portion, wherein the TbIIII polypeptide is C-terminal to a heterologous portion.
• a linker connects the TbIIII portion to the heterologous portion.
• the linker comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 165.
• the heterologous portion is an Fc portion.
• the Fc portion comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 73 (which may optionally lack the C- terminal lysine residue), or functional fragments thereof.
• the TbINI portion comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 170, or functional fragments thereof.
• the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 193 or 198.
• the fusion protein is part of a homodimer.
• the homodimer comprises two fusion proteins each comprising the amino acid sequence of SEQ ID NO: 193 or 198.
• the fusion protein is a monomer.
• the fusion protein comprises the amino acid sequence of SEQ ID NO: 193 or 198.
• the fusion protein does not comprise any additional ligand-binding domains.
• the fusion protein does not comprise an ActRIIB portion, an antibody portion, an antigen-binding portion, or a follistatin portion.
• the disclosure provides for an isolated polynucleotide encoding any of the fusion proteins disclosed herein.
• the disclosure provides for a recombinant polynucleotide comprising a promotor sequence operably linked to any of the polynucleotides disclosed herein.
• the disclosure provides for a cell comprising any of the polynucleotides disclosed herein.
• the cell is a CHO cell.
• the disclosure provides for a pharmaceutical preparation comprising any of the fusion proteins disclosed herein and a pharmaceutically acceptable excipient.
• the disclosure provides for a method of modulating the response of a cell to a TGF superfamily member, the method comprising exposing the cell to any of the fusion proteins disclosed herein.
• the disclosure provides for a method of treating a disease or condition associated with a TGF superfamily member in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the fusion proteins disclosed herein.
• the disclosure provides for a method of treating a muscle- related disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the fusion proteins disclosed herein.
• the muscle-related disease or condition is selected from: muscular dystrophy, Duchene muscular dystrophy, Becker muscular dystrophy, Charcot-Marie-Tooth, facioscapulohumeral muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia.
• the disclosure provides for a method of treating a pulmonary- related disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the fusion proteins disclosed herein.
• the pulmonary-related disease or condition is selected from interstitial lung disease, pulmonary hypertension, pulmonary arterial hypertension, and idiopathic pulmonary fibrosis.
• the disclosure provides for a method of treating a cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the fusion protein of any of the fusion proteins disclosed herein.
• the disclosure provides for a method of treating a kidney- related disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the fusion proteins disclosed herein.
• the kidney-related disease or condition is selected from: Alport syndrome, chronic kidney disease, polycystic kidney disease and renal fibrosis.
• the disclosure provides for a method of treating an anemia or an anemia-related disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the fusion proteins disclosed herein.
• the anemia-related disease or condition is selected from: thalassemia, myelodysplastic syndrome, myelofibrosis, and sickle cell disease.
• the disclosure provides for a method of treating a fibrotic or sclerotic disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of any of the fusion proteins disclosed herein.
• the fibrotic or sclerotic disease or condition is any one or more of systemic sclerosis, diffuse systemic sclerosis, systemic sclerosis-interstitial lung disease, myelofibrosis, progressive systemic sclerosis (PSS), or idiopathic pulmonary fibrosis.
• Figure 1 shows the amino acid sequence of native precursor for the B (short) isoform of human TGF receptor type II (IiTbIIP) (NP 003233.4) (SEQ ID NO: 1).
• Solid underline indicates the processed extracellular domain (ECD) (residues 23-159), and double underline indicates valine that is replaced in the A (long) isoform. Dotted underline denotes leader (residues 1-22).
• Figure 2 shows the amino acid sequence of native precursor for the A (long) isoform of human TbMI (NP 001020018.1) (SEQ ID NO: 2).
• Solid underline indicates the processed ECD (residues 23-184), and double underline indicates the splice-generated isoleucine substitution. Dotted underline denotes leader (residues 1-22).
• Figure 3 shows a comparison of the linker sequences of five different TbMI constructs.
• Figures 4A and 4B show in tabular form the binding affinity between T ⁇ EbI and T ⁇ Eb3 and one of several different TbMI-Eo fusion protein constructs.
• Figures 5A and 5C graph the results from reporter gene assays testing the affinity of T ⁇ EbI for one of several different TbIIP-E fusion protein constructs.
• Figures 5B and 5D graph the results from reporter gene assays testing the affinity of the TORb3 for one of several different TbIHI-Ro fusion protein constructs.
• Figures 5E and 5F provide IC50 data from these same experiments in tabular form.
• Figure 6 shows multiple sequence alignment of Fc domains from human IgG isotypes using Clustal 2.1. Hinge regions are indicated by dotted underline. Double underline indicates examples of positions engineered in IgGl Fc to promote asymmetric chain pairing and the corresponding positions with respect to other isotypes IgG2, IgG3 and IgG4.
• Figure 7 shows an alignment of extracellular domains of human ActRIIA and human ActRIIB with the residues that are deduced herein, based on composite analysis of multiple ActRIIB and ActRIIA crystal structures, to directly contact ligand indicated with boxes.
• Figure 8 shows a multiple sequence alignment of various vertebrate ActRIIB precursor proteins (rat (SEQ ID No: 101); pig (SEQ ID NO: 102); mouse (SEQ ID NO: 103); human (SEQ ID NO: 104); cow (SEQ ID NO: 108); and xenopus (SEQ ID NO: 105)) without their intracellular domains human ActRIIA precursor protein (SEQ ID NO: 106) without its intracellular domain, and a consensus ActRII precursor protein (SEQ ID NO: 107).
• rat SEQ ID No: 101
• pig SEQ ID NO: 102
• mouse SEQ ID NO: 103
• human SEQ ID NO: 104
• cow SEQ ID NO: 108
• xenopus SEQ ID NO: 105
• FIGS 9A-9D show schematic examples of heteromeric protein complexes comprising an TbRII polypeptide (e.g. a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain of an TbIIII protein from humans or other species such as those described herein, e.g., SEQ ID Nos: 18, 27, and 28-39) and an ActRIIB polypeptide (e.g.
• TbRII polypeptide e.g. a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain of an TbIIII protein from humans or other species such as those described herein, e.g., SEQ ID Nos: 18, 27, and 28-39
• an ActRIIB polypeptide e.
• polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain of an ActRIIB protein from humans or other species such as those described herein, e.g., SEQ ID Nos: 51, 52, 54, 55, and 109).
• the TbIIII polypeptide (from left to right) is part of a fusion polypeptide that comprises a first member of an interaction pair (“Ci”)
• the ActRIIB polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“C2”).
• Suitable interaction pairs included, for example, heavy chain and/or light chain immunoglobulin interaction pairs, truncations, and variants thereof such as those described herein [e.g., Spiess et al (2015) Molecular Immunology 67(2A): 95-106]
• a linker may be positioned between the TbIIII or ActRIIB polypeptide and the corresponding member of the interaction pair.
• the first and second members of the interaction pair may be unguided, meaning that the members of the pair may associate with each other or self-associate without substantial preference, and they may have the same or different amino acid sequences. See Figure 9A.
• the interaction pair may be a guided (asymmetric) pair, meaning that the members of the pair associate preferentially with each other rather than self-associate. See Figure 9B. Complexes of higher order can be envisioned. See Figure 9C and 9D.
• FIGS 10A-10G show schematic examples of heteromeric protein complexes comprising two TbIIII polypeptides (e.g. polypeptide that are independently at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain of an TbIIII protein from humans or other species such as those described herein, e.g., SEQ ID Nos: 18, 27, and 28-39) and two ActRIIB polypeptides (e.g. two polypeptides that are independently at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,
• TbIIII polypeptides e.g. polypeptide that are independently at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,
• the first TbIIII polypeptide (from left to right) is part of a fusion polypeptide that comprises a first member of an interaction pair (“Ci”) and further comprises an additional first member of an interaction pair (“Ai”); and the second TbIIII polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“C2”) and further comprises an first member of an interaction pair (“A2”).
• the first ActRIIB polypeptide (from left to right) is part of a fusion polypeptide that comprises a second member of an interaction pair (“Bi”); and the second ActRIIB polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“B 2 ”).
• a linker may be positioned between the TbIIII or ActRIIB polypeptide and the corresponding member of the interaction pair as well as between interaction pairs.
• Figure 10A is an example of an association of unguided interaction pairs, meaning that the members of the pair may associate with each other or self-associate without substantial preference and may have the same or different amino acid sequences.
• the first ActRIIB polypeptide (from left to right) is part of a fusion polypeptide that comprises a first member of an interaction pair (“Ci”) and further comprises an additional first member of an interaction pair (“Ai”); and the second ActRIIB polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“B2”).
• the first TbIIII polypeptide (from left to right) is part of a fusion polypeptide that comprises a second member of an interaction pair (“Bi”); and the second TbIIII polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“C2”) and further comprises a first member of an interaction pair (“A2”).
• a linker may be positioned between the TbIIII or ActRIIB polypeptide and the corresponding member of the interaction pair as well as between interaction pairs.
• Figure 1 OB is an example of an association of guided (asymmetric) interaction pairs, meaning that the members of the pair associate preferentially with each other rather than self-associate.
• Suitable interaction pairs included, for example, heavy chain and/or light chain immunoglobulin interaction pairs, truncations, and variants thereof as described herein [e.g., Spiess et al (2015) Molecular Immunology 67(2A): 95-106]
• Complexes of higher order can be envisioned. See Figure 9C-9F.
• interaction pairs may be used to produce ActRIIB ⁇ RII heterodimers that resemble antibody Fab and F(ab’)2 complexes [e.g., Spiess et al (2015) Molecular Immunology 67(2A): 95-106] See Figure 10G.
• Figures 11A and 11B show schematic examples of a heteromeric protein complex comprising an antigen-binding domain of antibody that binds to one or more oGTORb I .
• T ⁇ Rb2, TORb3 and at least one ActRIIB polypeptide domain e.g. a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain of an ActRIIB protein from humans or other species as such as those described herein, e.g., SEQ ID Nos: 51, 52, 54, 55, and 109).
• the first ActRIIB polypeptide is part of a fusion polypeptide that comprises a first member of an interaction pair (“Ci”), and further comprises an additional first member of an interaction pair (“Ai”).
• the second ActRIIB polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“Bi”).
• the variable heavy chain (VH) polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“C2”), and further comprises a first member of an interaction pair (“A2”).
• variable light chain (VL) polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“B2”) ⁇
• a linker may be positioned between the first or second ActRIIB polypeptide and the corresponding member of the interaction pair, between interaction pairs, and between the Vii and VL polypeptides and a member of the interaction pair.
• Ai and A2 may be the same or different; Bi and B2 may be the same or different, and Ci and C2 may be the same or different.
• Figure 11A is an example of a heterodimer comprising a first and second ActRIIB extracellular domain.
• Figure 11B is an example of a heteromultimer comprising a single ActRIIB extracellular domain.
• Figure 12 shows comparative ActRIIB-Fc:T RII-Fc heterodimer compared to an ActRIIB-Fc:ActRIIB-Fc homodimer and T RII-Fc:T RII-Fc homodimer.
• IC50 data was determined by an A-204 Reporter Gene Assay as described herein.
• ActRIIB-Fc:T RII-Fc heterodimer inhibits activin A, activin B, GDF8, GDF11, and BMP 10- signaling pathways similarly to the ActRIIB-Fc:ActRIIB-Fc homodimer.
• ActRIIB-Fc:T RII-Fc heterodimer inhibition of BMP9 signaling pathways is significantly reduced compared to the ActRIIB-Fc:ActRIIB-Fc homodimer.
• ActRIIB-Fc:T RII-Fc heterodimers are more selective antagonists of activin A, activin B, GDF8, GDF11 and BMP10 compared to corresponding ActRIIB-Fc:ActRIIB-Fc homodimers.
• the ActRIIB-Fc:T RII-Fc heterodimer inhibits TGFP 1 and TGFp3 signaling pathways similarly to the T RII-Fc:T RII-Fc homodimer.
• Figure 13 shows the amino acid sequence for a truncated, variant ActRIIB (25-131, L79D) domain (SEQ ID NO: 109).
• Figures 14A-14D show schematic examples of heteromeric protein complexes comprising an TbKII polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain of an TbMI protein from humans or other species such as those described herein, e.g., SEQ ID Nos: 18, 27, and 28-39) and an ActRIIB polypeptide (e.g.
• TbKII polypeptide e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain of an TbMI protein from humans
• polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain of an ActRIIB protein from humans or other species such as those described herein, e.g., SEQ ID NOs: 51, 52, 53, 54, and 109).
• the TbIIII ActRIIB single-chain polypeptide (is part of a fusion polypeptide that comprises a first member of an interaction pair (“Ci”), and the ActRIIB ⁇ RII polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“C2”).
• Suitable interaction pairs included, for example, heavy chain and/or light chain immunoglobulin interaction pairs, truncations, and variants thereof such as those described herein [e.g., Spiess et al (2015) Molecular Immunology 67(2A): 95-106]
• a linker may be positioned between the TbIIII and/or ActRIIB polypeptide and the corresponding member of the interaction pair.
• the first and second members of the interaction pair may be unguided, meaning that the members of the pair may associate with each other or self-associate without substantial preference, and they may have the same or different amino acid sequences. See Figure 14A.
• the interaction pair may be a guided (asymmetric) pair, meaning that the members of the pair associate preferentially with each other rather than self-associate.
• Figure 14B Additional protein complexes can be envisioned. See Figure 14C and 14D.
• Figure 15A shows a schematic example of a representative multispecific binder comprising a TbIIII (referred to here as a TGFBRII) polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 170) and a follistatin polypeptide (e.g. a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 111).
• a TbIIII referred to here as a TGFBRII
• Figure 15B shows a schematic example of multispecific binder comprising a TbIIII polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 170) and a GDF8 antigen binding fragment (e.g. a polypeptide comprising the heavy chain and light chain CDRs of SEQ ID NOs: 151-156).
• a TbIIII polypeptide e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 170
• GDF8 antigen binding fragment e.g. a polypeptide comprising the heavy chain and light chain CDRs of SEQ ID NOs: 151-156.
• FIG 16A shows a simplified schematic of a representative“four arm” homodimer comprising two fusion proteins, with each fusion protein comprising an ActRIIB extracellular domain (IIB ECD), a GGG linker, an Fc portion comprising CH2-CH3 Fc domains, a (G4S)4G linker, and a TGF RII extracellular domain (TGF RII ECD).
• IIB ECD ActRIIB extracellular domain
• GGG linker an Fc portion comprising CH2-CH3 Fc domains
• G4S G4S4G linker
• TGF RII ECD TGF RII extracellular domain
• Figure 16B shows a simplified schematic of a representative“three-arm” heteromultimer comprising two fusion proteins, where the first fusion protein comprises an ActRIIB extracellular domain (IIB ECD), a GGG linker, an Fc portion comprising CH2-CH3 Fc domains with“knob substitutions”, a (G4S)4G linker, and a TGF RII extracellular domain (TGF RII ECD); and where the second fusion protein comprises an Fc portion comprising CH2-CH3 Fc domains with“hole substitutions”, a (G4S)4G linker, and a TGF RII extracellular domain (TGF RII ECD).
• the first fusion protein comprises an ActRIIB extracellular domain (IB ECD), a GGG linker, an Fc portion comprising CH2-CH3 Fc domains with“knob substitutions”, a (G4S)4G linker, and a TGF RII extracellular domain (TGF RII ECD);
• FIG 17A shows a simplified schematic of a representative“two-arm” homodimer comprising two fusion proteins, with each fusion protein comprising (from N-terminus to C- terminus) an Fc portion comprising CH2-CH3 Fc domains with“hole substitutions,” and a TGF RII extracellular domain (TGF RII ECD).
• Figure 17B shows a simplified schematic of a representative“single-arm” comprising only a single fusion protein, with the fusion protein comprising (from N-terminus to C-terminus) an Fc portion comprising CH2-CH3 Fc domains with“hole substitutions,” and a TGF RII extracellular domain (TGF RII ECD).
• Figure 18 is a table providing IC50 data (in pM) for different constructs against GDF11, Activin A, TGF l or TGF 3.
• the disclosure provides for novel binders of TGF -superfamily ligands.
• the disclosure provides for a multispecific binder of TGF - superfamily ligands.
• the multispecific binder protein is capable of binding to a) at least one of TGF l and TGFP3. and b) at least one of activin A, activin B, activin AB, GDF11, and GDF8.
• the multispecific binder comprises: a) a first portion that is capable of binding to TGF l and/or TGFP3; and b) a second portion that is capable of binding to at least one of activin A, activin B, activin AB, GDF11, and GDF8.
• the multispecific binder is a heteromultimer comprising an ActRIIB polypeptide and a TbIIII polypeptide.
• the multispecific binder comprises a TbMI polypeptide and a follistatin or a follistatin-like protein domain.
• the multispecific binder comprises a TbIIII polypeptide and an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to one or more of activin A, activin B, activin AB, GDF11, and/or GDF8.
• the multispecific binder comprises a TbIIII polypeptide and an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to GDF8.
• the disclosure provides heteromultimers that comprise an ActRIIB polypeptide and a TbIIII polypeptide.
• such ActRIIB polypeptides comprise a ligand-binding domain of an ActRIIB receptor and such TbIIII polypeptides comprise a ligand-binding domain of a TbIIII receptor.
• ActRIIB :TbIIP heteromultimers of the disclosure are soluble.
• Ao ⁇ IBIB:TbIIII heteromultimers of the disclosure have an altered TORb superfamily ligand specificity compared to a corresponding sample of a homomultimer (e.g, an ActRIIB :TbIIII heterodimer compared to an ActRIIB: ActRIIB homodimer or an
• the T ⁇ Rb superfamily is comprised of over 30 secreted factors including TORb£. activins, nodals, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), and anti-Mullerian hormone (AMH) [Weiss et al. (2013) Developmental Biology, 2(1): 47-63]
• BMPs bone morphogenetic proteins
• GDFs growth and differentiation factors
• AH anti-Mullerian hormone
• T ⁇ Rb superfamily proteins are key mediators of stem cell self-renewal, gastrulation, differentiation, organ morphogenesis, and adult tissue homeostasis. Consistent with this ubiquitous activity, aberrant T ⁇ Rb superfamily signaling is associated with a wide range of human pathologies including, for example, autoimmune disease, cardiovascular disease, fibrotic disease, and cancer.
• Ligands of the T ⁇ Rb superfamily share the same dimeric structure in which the central 3-1/2 turn helix of one monomer packs against the concave surface formed by the beta-strands of the other monomer.
• the majority oGTORb family members are further stabilized by an intermolecular disulfide bond. This disulfide bonds traverses through a ring formed by two other disulfide bonds generating what has been termed a‘cysteine knot’ motif [Lin et al. (2006) Reproduction 132: 179-190; and Hinck et al. (2012) FEBS Letters 586: 1860-1870]
• TGF superfamily signaling is mediated by heteromeric complexes of type I and type II serine/threonine kinase receptors, which phosphorylate and activate downstream SMAD proteins (e.g ., SMAD proteins 1, 2, 3, 5, and 8) upon ligand stimulation [Massague (2000) Nat. Rev. Mol. Cell Biol. 1 : 169-178]
• SMAD proteins e.g ., SMAD proteins 1, 2, 3, 5, and 8
• type I receptors mediate intracellular signaling while the type II receptors are required for binding TGF superfamily ligands.
• Type I and II receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors.
• the TGF family can be divided into two phylogenetic branches based on the type I receptors they bind and the Smad proteins they activate.
• One is the more recently evolved branch, which includes, e.g., the TGF s, activins, GDF8, GDF9, GDF11, BMP3 and nodal, which signal through type I receptors that activate Smads 2 and 3 [Hinck (2012) FEBS Letters 586: 1860-1870]
• the other branch comprises the more distantly related proteins of the superfamily and includes, e.g., BMP2, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF1, GDF5, GDF6, and GDF7, which signal through Smads 1, 5, and 8.
• TGF isoforms are the founding members of the TGF superfamily, of which there are 3 known isoforms in mammals designated as TGF l, TGF 2 and TGF 3. Mature bioactive TGF ligands function as homodimers and predominantly signal through the type I receptor ALK5, but have also been found to additionally signal through ALK1 in endothelial cells [Goumans et al. (2003) Mol Cell 12(4): 817-828] TGF l is the most abundant and ubiquitously expressed isoform. TGF l is known to have an important role in wound healing, and mice expressing a constitutively active TGF l transgene develop fibrosis
• TGF l is also involved in T cell activation and maintenance of T regulatory cells [Li et al. (2006) Immunity 25(3): 455-471] TGF 2 expression was first described in human glioblastoma cells, and is occurs in neurons and astroglial cells of the embryonic nervous system. TGF 2 is known to suppress interleukin-2-dependent growth of T lymphocytes. TGF 3 was initially isolated from a human rhabdomyosarcoma cell line and since has been found in lung adenocarcinoma and kidney carcinoma cell lines. TGF 3 is known to be important for palate and lung
• Activins are members of the TGF superfamily and were initially discovered as regulators of secretion of follicle-stimulating hormone, but subsequently various reproductive and non-reproductive roles have been characterized.
• the human genome also encodes an activin C and an activin E, which are primarily expressed in the liver, and heterodimeric forms containing bo or b b are also known.
• activins are unique and multifunctional factors that can stimulate hormone production in ovarian and placental cells, support neuronal cell survival, influence cell-cycle progress positively or negatively depending on cell type, and induce mesodermal differentiation at least in amphibian embryos [DePaolo et al. (1991) Proc Soc Ep Biol Med. 198:500-512; Dyson et al. (1997) Curr Biol. 7:81-84; and Woodruff (1998) Biochem Pharmacol. 55:953-963] In several tissues, activin signaling is antagonized by its related heterodimer, inhibin.
• activin promotes FSH synthesis and secretion, while inhibin reduces FSH synthesis and secretion.
• Other proteins that may regulate activin bioactivity and/or bind to activin include follistatin (FS), follistatin-related protein (FSRP, also known as FLRG or FSTL3), and a2-macroglobulin.
• agents that bind to“activin A” are agents that specifically bind to the bA subunit, whether in the context of an isolated bA subunit or as a dimeric complex (e.g., a bAbA homodimer or a bAb b heterodimer).
• agents that bind to“activin A” are specific for epitopes present within the bA subunit, but do not bind to epitopes present within the hoh-bA subunit of the complex (e.g., the b b subunit of the complex).
• agents disclosed herein that antagonize (inhibit) “activin A” are agents that inhibit one or more activities as mediated by a bA subunit, whether in the context of an isolated bA subunit or as a dimeric complex (e.g., a bAbA homodimer or a bAb b heterodimer).
• agents that inhibit“activin A” are agents that specifically inhibit one or more activities of the bA subunit, but do not inhibit the activity of the hoh-bA subunit of the complex (e.g., the b b subunit of the complex).
• Agents disclosed herein that antagonize“activin AB” are agents that inhibit one or more activities as mediated by the bA subunit and one or more activities as mediated by the b b subunit.
• the BMPs and GDFs together form a family of cysteine-knot cytokines sharing the characteristic fold of the TGF superfamily [Rider et al. (2010) Biochem J., 429(1): 1-12]
• This family includes, for example, BMP2, BMP4, BMP6, BMP7, BMP2a, BMP3, BMP3b (also known as GDF10), BMP4, BMP5, BMP6, BMP7, BMP 8, BMP 8 a, BMP8b, BMP9 (also known as GDF2), BMP 10, BMP11 (also known as GDF11), BMP 12 (also known as GDF7), BMP 13 (also known as GDF6), BMP 14 (also known as GDF5), BMP15, GDF1, GDF3 (also known as VGR2), GDF8 (also known as myostatin), GDF9, GDF15, and decapentaplegic.
• BMP/GDFs display morphogenetic activities in the development of a wide range of tissues.
• BMP/GDF homo- and hetero-dimers interact with combinations of type I and type II receptor dimers to produce multiple possible signaling complexes, leading to the activation of one of two competing sets of SMAD transcription factors.
• BMP/GDFs have highly specific and localized functions. These are regulated in a number of ways, including the
• BMP/GDF developmental restriction of BMP/GDF expression and through the secretion of several specific BMP antagonist proteins that bind with high affinity to the cytokines. Curiously, a number of these antagonists resemble TGF superfamily ligands.
• GDF8 Growth and differentiation factor-8
• GDF8 is a negative regulator of skeletal muscle mass and is highly expressed in developing and adult skeletal muscle.
• the GDF8 null mutation in transgenic mice is characterized by a marked hypertrophy and hyperplasia of skeletal muscle [McPherron et al. Nature (1997) 387:83-90] Similar increases in skeletal muscle mass are evident in naturally occurring mutations of GDF8 in cattle and, strikingly, in humans [Ashmore et al. (1974) Growth, 38:501-507;
• GDF8 can modulate the production of muscle-specific enzymes (e.g., creatine kinase) and modulate myoblast cell proliferation [International Patent Application Publication No.
• the GDF8 propeptide can noncovalently bind to the mature GDF8 domain dimer, inactivating its biological activity [Miyazono et al. (1988) J. Biol. Chem, 263: 6407-6415; Wakefield et al. (1988) J. Biol. Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3: 35-43]
• Other proteins which bind to GDF8 or structurally related proteins and inhibit their biological activity include follistatin, and potentially, follistatin-related proteins [Gamer et al. (1999) Dev. Biol., 208: 222-232]
• GDF11 also known as BMP11, is a secreted protein that is expressed in the tail bud, limb bud, maxillary and mandibular arches, and dorsal root ganglia during mouse development [McPherron et al. (1999) Nat. Genet., 22: 260-264; and Nakashima el al. (1999) Mech. Dev., 80: 185-189] GDF11 plays a unique role in patterning both mesodermal and neural tissues [Gamer et al. (1999) Dev Biol., 208:222-32] GDF11 was shown to be a negative regulator of chondrogenesis and myogenesis in developing chick limb [Gamer et al.
• GDF11 in muscle also suggests its role in regulating muscle growth in a similar way to GDF8.
• the expression of GDF11 in brain suggests that GDF11 may also possess activities that relate to the function of the nervous system.
• GDF11 was found to inhibit neurogenesis in the olfactory epithelium [Wu et al. (2003) Neuron., 37: 197-207]
• inhibitors GDF11 may have in vitro and in vivo applications in the treatment of diseases such as muscle diseases and neurodegenerative diseases (e.g., amyotrophic lateral sclerosis).
• BMP7 also called osteogenic protein-l (OP-l)
• OP-l osteogenic protein-l
• BMP7 regulates a wide array of physiological processes.
• BMP7 may be the osteoinductive factor responsible for the phenomenon of epithelial osteogenesis. It is also found that BMP7 plays a role in calcium regulation and bone homeostasis.
• BMP7 binds to type II receptors, ActRIIA and ActRIIB.
• BMP7 and activin recruit distinct type I receptors into heteromeric receptor complexes.
• the major BMP7 type I receptor observed was ALK2, while activin bound exclusively to ALK4 (ActRIIB).
• BMP7 and activin elicited distinct biological responses and activated different SMAD pathways [Macias-Silva et al. (1998) J Biol Chem. 273:25628-36]
• an ActRIIB :Tb ⁇ HI heterodimer can antagonize a broad range of Smad 2/3 activating ligands.
• the disclosure demonstrates that an ActRIIB:T RII heterodimer inhibits TGF 1.
• TGF 3 activin A, activin B, GDF8, GDF11, and BMP 10-signaling pathways in a cell-based assay.
• ActRIIB and TbIIII homodimers alone inhibit a smaller subset of Smad 2/3 activating ligands.
• the data demonstrate that the ActRIIB ⁇ RII heterodimer is a surprisingly more selective Smad 2/3 ligand antagonists that merely combining the antagonistic profiles of ActRIIB and Tb ⁇ HI homodimer ligand traps.
• the ActRIIB:T RII heterodimer inhibited activin A, activin B, GDF8, GDF11, and BMP10- signaling pathways similarly to an ActRIIB homodimer.
• ActRIIB:T RII heterodimer inhibition of BMP9 signaling pathways is significantly reduced compared to the ActRIIB homodimer.
• ActRIIB :TbIIII heteromultimers therefore are more selective antagonists of Smad 2/3 activating ligands compared to ActRIIB homodimers. Accordingly, an ActRIIB:I ⁇ RII heterodimer will be more useful than an ActRIIB or TbIIII homodimer, or combination thereof, in certain applications where such broad, yet selective, Smad 2/3 antagonism is advantageous.
• sequence similarity in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
• sequence similarity when modified with an adverb such as“highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
• Percent (%) sequence identity or“percent (%) identical” with respect to a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to the amino acid residues (or nucleic acids) in the reference polypeptide (nucleotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
• ALIGN-2 sequence comparison computer program ALIGN-2.
• the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office,
• the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
• “Agonize”, in all its grammatical forms, refers to the process of activating a protein and/or gene (e.g., by activating or amplifying that protein’s gene expression or by inducing an inactive protein to enter an active state) or increasing a protein’s and/or gene’s activity.
• “Antagonize”, in all its grammatical forms, refers to the process of inhibiting a protein and/or gene (e.g., by inhibiting or decreasing that protein’s gene expression or by inducing an active protein to enter an inactive state) or decreasing a protein’s and/or gene’s activity.
• the term “and/or” as used in a phrase 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 (alone); B (alone); and C (alone).
• the word“comprise” or variations such as“comprises” or“comprising” will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.
• the term“comprises” also encompasses the use of the narrower terms“consisting” and “consisting essentially of.”
• the term“appreciable affinity” as used herein means binding with a dissociation constant (KD) of less than 50 nM.
• polypeptide oligopeptide
• peptide and “protein” are used interchangeably herein to refer to chains of amino acids of any length.
• the chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non amino acids.
• the terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
• polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
• the polypeptides can occur as single chains or associated chains.
• heteromer or“heteromultimer” is a complex comprising at least a first polypeptide chain and a second polypeptide chain, wherein the second polypeptide chain differs in amino acid sequence from the first polypeptide chain by at least one amino acid residue.
• the heteromer can comprise a“heterodimer” formed by the first and second polypeptide chains or can form higher order structures where one or more polypeptide chains in addition to the first and second polypeptide chains are present.
• Exemplary structures for the heteromultimer include heterodimers, heterotrimers, heterotetramers and further oligomeric structures.
• Heterodimers are designated herein as X:Y or equivalently as X-Y, where X represents a first polypeptide chain and Y represents a second polypeptide chain. Higher-order heteromers and oligomeric structures are designated herein in a corresponding manner.
• a heteromultimer is recombinant (e.g., one or more polypeptide components may be a recombinant protein), isolated and/or purified.
• the term“capable of’ e.g., capable of binding to means that something has the ability to perform a particular action, but does not necessarily need to be performing that action at any particular point in time.
• a protein is“capable of binding to a ligand”
• the term“binds to” means that something is“capable of binding to.”
• the disclosure provides for novel binders of TGFP-superfamily ligands.
• the binder is capable of binding to at least one of TGFpl and TGFp3.
• the binder comprises a TbINI polypeptide and a heterologous domain (e.g., an Fc domain).
• the disclosure provides for a multispecific binder of TGF - superfamily ligands.
• the multispecific binder is capable of binding to a) at least one of TGF l and TGF 3. and b) at least one of activin A, activin B, activin AB, GDF11, and GDF8.
• the multispecific binder comprises: a) a first portion that is capable of binding to TGF l and/or TGF 3; and b) a second portion that is capable of binding to at least one of activin A, activin B, activin AB, GDF11, and GDF8.
• the multispecific binder is a heteromultimer comprising an ActRIIB polypeptide and a TbMI polypeptide.
• the multispecific binder comprises a TbMI polypeptide and a follistatin or a follistatin-like protein domain.
• the multispecific binder comprises a TbINI polypeptide and an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to one or more of activin A, activin B, activin AB, GDF11, and/or GDF8.
• the multispecific binder comprises a TbINI polypeptide and an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to GDF8.
• the present disclosure relates to heteromultimers comprising one or more ActRIIB receptor polypeptides (e.g., SEQ ID NOs: 51, 52, 54, 55, 82, 84, 88, 90, and 109) and one or more TbBII receptor polypeptides (e.g., SEQ ID NOs: 9, 11, 13, 15, 17, 18, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 44, 45, 85, 87, 91, 93, 94, 95, 96, 97, 98, 99. and 100) which are generally referred to herein as ActRIIB:T RII heteromultimer complexes” or“ActRIIB ⁇ RII heteromul timers”.
• heteromultimers of the disclosure are soluble, for example, a heteromultimer may comprises a soluble portion (domain) of a TbKII receptor and a soluble portion (domain) of an ActRIIB receptor.
• a heteromultimer may comprises a soluble portion (domain) of a TbKII receptor and a soluble portion (domain) of an ActRIIB receptor.
• the extracellular domains of TbIIII and ActRIIB correspond to a soluble portion of these receptors. Therefore, in some embodiments, heteromultimers of the disclosure comprise an extracellular domain of a TbIIII receptor and an extracellular domain of an ActRIIB receptor.
• Example extracellular domains TbIIII and ActRIIB receptors are disclosed herein and such sequences, as well as fragments, functional variants, and modified forms thereof, may be used in accordance with the inventions of the disclosure (e.g., Ao ⁇ IIIIB:TbIIII heteromultimer compositions and uses thereof).
• Ao ⁇ IIIIB:TbIIII Ao ⁇ IIIIB:TbIIII
• heteromultimers of the disclosure include, e.g., heterodimers, heterotrimers, heterotetramers and higher order oligomeric structures. See, e.g., Figures 9 and 10.
• heteromultimers of the disclosure are ActRIIB ⁇ RII heterodimers.
• ActRIIB ⁇ RII heteromultimers of the disclosure bind to one or more TORb superfamily ligands.
• ActRIIB ⁇ RII heteromultimers may bind to one or more of activin (e.g., activin A, activin B, activin C, activin E, activin AC, activin AB, activin BC, activin AE, and activin BE), GDF8, GDF11, BMP 10, T ⁇ RbI, and TORb3.
• activin e.g., activin A, activin B, activin C, activin E, activin AC, activin AB, activin BC, activin AE, and activin BE
• GDF8 GDF11 e.g., GDF11, BMP 10, T ⁇ RbI, and TORb3.
• ActRIIB :TbIBI heteromultimers do not bind to, or no not substantially bind to BMP9 (e.g., have indeterminate Ka or Kd due to the transient nature of the interaction between BMP9 and an Ao ⁇ IIIIB:TbIIII heteromultimer).
• ActRIIB :TbIBI heteromultimers do not bind to, or no not substantially bind to BMP9 (e.g., have indeterminate Ka or Kd due to the transient nature of the interaction between BMP9 and an Ao ⁇ IIIIB:TbIIII heteromultimer).
• ActRIIB :TbIBI heteromultimers may be used to inhibit (antagonize) signaling (e.g., Smad 2/3) mediated by one or more T ⁇ Rb superfamily ligands.
• ActRIIB ⁇ RII heteromultimers of the disclosure may be used to inhibit intracellular signaling by one or more T ⁇ Rb superfamily ligands in, for example, a cell-based assay such as those described herein.
• ActRIIB :TbIIII heteromultimers may inhibit signaling mediated by one or more of activin (e.g., activin A, activin B, activin C, activin E, activin AC, activin AB, activin BC, activin AE, and activin BE), GDF8, GDF11, BMP10, T ⁇ RbI, and TORb3 in a cell-based assay.
• activin e.g., activin A, activin B, activin C, activin E, activin AC, activin AB, activin BC, activin AE, and activin BE
• GDF8 GDF11
• BMP10 T ⁇ RbI
• T ⁇ RbI TORb3
• Tb ⁇ BI refers to a family of transforming growth factor beta receptor II (TbEII) proteins from any species and variants derived from such TbIIII proteins by mutagenesis or other modification.
• TbIIII herein is understood to be a reference to any one of the currently identified forms.
• TbMI family are generally transmembrane proteins, composed of a ligand-binding extracellular domain comprising a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
• TbMI polypeptide includes polypeptides comprising any naturally occurring polypeptide of an TbMI family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
• human TbMI occurs naturally in at least two isoforms - A (long) and B (short) - generated by alternative splicing in the extracellular domain (ECD) ( Figures 1 and 2 and SEQ ID NOS: 1 and 2).
• SEQ ID NO: 18, which corresponds to residues 23-184 of SEQ ID NO: 2 depicts the native full-length extracellular domain of the long isoform of TbKII.
• amino acid position numbering with regard to variants based on the TbMI short and long isoforms refers to the corresponding position in the native precursors, SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
• TbINI polypeptides may bind to and inhibit the function of a T ⁇ Eb superfamily member, such as but not limited to, T ⁇ EbI or TOEb3.
• TbINI polypeptides may include a polypeptide consisting of, or comprising, an amino acid sequence at least 70% identical, and optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a truncated ECD domain of a naturally occurring TbMI polypeptide, whose C-terminus occurs at any of amino acids 153-159 of SEQ ID NO: 1.
• TbMI polypeptides may include a polypeptide consisting of, or comprising, an amino acid sequence at least 70% identical, and optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a truncated ECD domain of a naturally occurring TbINI polypeptide, whose C-terminus occurs at any of amino acids 178-184 of SEQ ID NO: 2.
• the TbINI polypeptides comprise an amino acid sequence at least 70% identical, and optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18.
• a TbINI polypeptide does not include more than 5 consecutive amino acids, or more than 10, 20, 30, 40, 50, 52, 60, 70, 80, 90, 100, 150 or 200 or more consecutive amino acids from a sequence consisting of amino acids 160-567 of SEQ ID NO: 1 or from a sequence consisting of amino acids 185-592 of SEQ ID NO: 2.
• the TbMI polypeptide does not include amino acids 160-567 of SEQ ID NO:
• the TbMI polypeptide does not include amino acids 1-22 of SEQ ID NO: 1. In some embodiments, the TbMI polypeptide does not include amino acids 1-22 and 160-567 of SEQ ID NO: 1. In some embodiments, the TbMI polypeptide does not include amino acids 185-592 of SEQ ID NO: 2. In some embodiments, the TbMI polypeptide does not include amino acids 1-22 of SEQ ID NO: 2. In some embodiments, the TbMI polypeptide does not include amino acids 1-22 and 185-592 of SEQ ID NO: 2.
• the unprocessed TbMI polypeptide may either include or exclude any signal sequence, as well as any sequence N-terminal to the signal sequence.
• the N-terminus of the processed TbMI polypeptide may occur at any of amino acids 23-35 of SEQ ID NO: 1 or 23- 60 of SEQ ID NO: 2.
• processed TbMI polypeptides include, but are not limited to, amino acids 23-159 of SEQ ID NO: 1 (set forth in SEQ ID NO: 27), amino acids 29-159 of SEQ ID NO: 1 (set forth in SEQ ID NO: 28), amino acids 35-159 of SEQ ID NO: 1 (set forth in SEQ ID NO: 29), amino acids 23-153 of SEQ ID NO: 1 (set forth in SEQ ID NO: 30), amino acids 29-153 of SEQ ID NO: 1 (set forth in SEQ ID NO: 31), amino acids 35-153 of SEQ ID NO: 1 (set forth in SEQ ID NO: 32), amino acids 23-184 of SEQ ID NO: 2 (set forth in SEQ ID NO: 18), amino acids 29-184 of SEQ ID NO: 2 (set forth in SEQ ID NO: 33), amino acids 60-184
• TbIPI long isoform of TbIPI will include nucleotide sequences encoding the 25-amino acid insertion along with a conservative Val-Ile substitution at the flanking position C-terminal to the insertion.
• the TbMI polypeptides accordingly may include isolated extracellular portions of TbMI polypeptides, including both the short and the long isoforms, variants thereof (including variants that comprise, for example, no more than 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35 amino acid substitutions in the sequence corresponding to amino acids 23-159 of SEQ ID NO: 1 or amino acids 23-184 of SEQ ID NO: 2), fragments thereof, and fusion proteins comprising any of the foregoing, but in each case preferably any of the foregoing TbMI polypeptides will retain substantial affinity for at least one of, or both of, TORb 1 or TOEb3.
• a TbMI polypeptide will be designed to be soluble in aqueous solutions at biologically relevant temperatures, pH levels, and osmolarity.
• the variant TbMI polypeptides of the disclosure comprise one or more mutations in the extracellular domain that confer an altered ligand binding profile.
• a TbMI polypeptide may include one, two, five or more alterations in the amino acid sequence relative to the corresponding portion of a naturally occurring TbMI polypeptide.
• the mutation results in a substitution, insertion, or deletion at the position corresponding to position 70 of SEQ ID NO: 1.
• the mutation results in a substitution, insertion, or deletion at the position corresponding to position 110 of SEQ ID NO: 1.
• TbMI polypeptides include, but are not limited to, the sequences set forth in SEQ ID NOs: 36-39.
• a TbMI polypeptide may comprise a polypeptide or portion thereof that is encoded by any one of SEQ ID NOs: 8, 10, 12, 14, 16, 46 or 47, or silent variants thereof or nucleic acids that hybridize to the complement thereof under stringent hybridization conditions.
• a TbINI polypeptide may comprise a polypeptide or portion thereof that is encoded by any one of SEQ ID NO: 12, or silent variants thereof or nucleic acids that hybridize to the complement thereof under stringent hybridization conditions.
• the variant TbMI polypeptides of the disclosure further comprise an insertion of 36 amino acids (SEQ ID NO: 41) between the pair of glutamate residues (positions 151 and 152 of SEQ ID NO: 1, or positions 176 and 177 of SEQ ID NO:
• TbKP polypeptides can be modified to selectively antagonize TbINI ligands.
• the N70 residue represents a potential glycosylation site.
• the TbKP polypeptides are aglycosylated.
• the TbKP polypeptides are aglycosylated or have reduced glycosylation at position Asnl57.
• the TbKP polypeptides are aglycosylated or have reduced glycosylation at position Asn73.
• a TbKP polypeptide binds to TOEbI, and the TbKP polypeptide does not show substantial binding to TOEb3. In certain embodiments, a TbKP polypeptide binds to T ⁇ Eb3, and the TbKP polypeptide does not show substantial binding to TOEbI. Binding may be assessed using purified proteins in solution or in a surface plasmon resonance system, such as a BiacoreTM system. In certain embodiments, a T RII polypeptide inhibits TORb 1 cellular signaling, and the TbMI polypeptide has an intermediate or limited inhibitory effect on TORb3 signaling.
• a TbMI polypeptide inhibits TORb3 cellular signaling, and the TbMI polypeptide has an intermediate or limited inhibitory effect on TORb 1 signaling. Inhibitory effect on cell signaling can be assayed by methods known in the art.
• an active portion of a TbIIII polypeptide may comprise amino acid sequences 23-153, 23-154, 23-155, 23-156, 23-157, or 23-158 of SEQ ID NO: 1, as well as variants of these sequences starting at any of amino acids 24-35 of SEQ ID NO: 1.
• an active portion of a TbMI polypeptide may comprise amino acid sequences 23-178, 23- 179, 23-180, 23-181, 23-182, or 23-183 of SEQ ID NO: 2, as well as variants of these sequences starting at any of amino acids 24-60 of SEQ ID NO: 2.
• TbMI polypeptides comprise amino acid sequences 29-159, 35-159, 23-153, 29-153 and 35-153 of SEQ ID NO: 1 or amino acid sequences 29-184, 60-184, 23-178, 29-178 and 60-178 of SEQ ID NO: 2. Variants within these ranges are also contemplated, particularly those having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding portion of SEQ ID NO: 1 or SEQ ID NO: 2.
• a TbINI polypeptide may be selected that does not include the sequence consisting of amino acids 160-567 of SEQ ID NO: 1 or amino acids 185-592 of SEQ ID NO: 2.
• the TbMI polypeptides comprise an amino acid sequence at least 70% identical, and optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18.
• ActRIIB refers to a family of activin receptor type IIB (ActRIIB) proteins from any species and variants derived from such ActRIIB proteins by mutagenesis or other modification.
• ActRIIB activin receptor type IIB
• Reference to ActRIIB herein is understood to be a reference to any one of the currently identified forms.
• Members of the ActRIIB family are generally transmembrane proteins, composed of a ligand-binding extracellular domain comprising a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
• ActRIIB polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ActRIIB family member as well as any variants thereof
• the human ActRIIB precursor protein sequence is as follows:
• the signal peptide is indicated with a single underline: the extracellular domain is indicated in bold font; and the potential, endogenous N-linked glycosylation sites are indicated with a double underline.
• the processed extracellular ActRIIB polypeptide sequence is as follows:
• the protein may be produced with an“SGR...” sequence at the N-terminus.
• the C-terminal“tail” of the extracellular domain is indicated by a single underline.
• the sequence with the“tail” deleted is as follows:
• a form of ActRIIB with an alanine at position 64 of SEQ ID NO: 1 (A64) is also reported in the literature. See, e.g., Hilden et al. (1994) Blood, 83(8): 2163-2170. Applicants have ascertained that an ActRIIB-Fc fusion protein comprising an extracellular domain of ActRIIB with the A64 substitution has a relatively low affinity for activin and GDF11. By contrast, the same ActRIIB-Fc fusion protein with an arginine at position 64 (R64) has an affinity for activin and GDF11 in the low nanomolar to high picomolar range. Therefore, sequences with an R64 are used as the“wild-type” reference sequence for human ActRIIB in this disclosure.
• the signal peptide is indicated by single underline and the extracellular domain is indicated by bold font.
• the processed extracellular ActRIIB polypeptide sequence of the alternative A64 form is as follows:
• the protein may be produced with an“SGR...” sequence at the N-terminus.
• the C-terminal“tail” of the extracellular domain is indicated by single underline.
• the sequence with the“tail” deleted is as follows:
• a nucleic acid sequence encoding the human ActRIIB precursor protein is shown below (SEQ ID NO: 56), representing nucleotides 25-1560 of Genbank Reference Sequence NM_00l 106.3, which encode amino acids 1-513 of the ActRIIB precursor.
• the sequence as shown provides an arginine at position 64 and may be modified to provide an alanine instead.
• the signal sequence is underlined.
• polypeptide is as follows (SEQ ID NO: 57). The sequence as shown provides an arginine at position 64, and may be modified to provide an alanine instead.
• FIG. 7 An alignment of the amino acid sequences of human ActRIIB extracellular domain and human ActRIIA extracellular domain are illustrated in Figure 7. This alignment indicates amino acid residues within both receptors that are believed to directly contact ActRII ligands.
• the composite ActRII structures indicated that the ActRIIB-ligand binding pocket is defined, in part, by residues Y31, N33, N35, L38 through T41, E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87, A92, and E94 through F101. At these positions, it is expected that conservative mutations will be tolerated.
• Figure 8 depicts a multi-sequence alignment of a human ActRIIB extracellular domain compared to various ActRIIB orthologs. Many of the ligands that bind to ActRIIB are also highly conserved. Accordingly, from these alignments, it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ActRIIB-ligand binding activities as well as to predict amino acid positions that are likely to be tolerant of substitution without significantly altering normal ActRIIB-ligand binding activities.
• an active, human ActRIIB variant polypeptide useful in accordance with the presently disclosed methods may include one or more amino acids at corresponding positions from the sequence of another vertebrate ActRIIB, or may include a residue that is similar to that in the human or other vertebrate sequences.
• L46 in the human extracellular domain is a valine in Xenopus ActRIIB (SEQ ID NO: 105), and so this position may be altered, and optionally may be altered to another hydrophobic residue, such as V, I or F, or a non- polar residue such as A.
• E52 in the human extracellular domain is a K in Xenopus, indicating that this site may be tolerant of a wide variety of changes, including polar residues, such as E, D, K, R, H, S, T, P, G, Y and probably A.
• T93 in the human extracellular domain is a K in Xenopus, indicating that a wide structural variation is tolerated at this position, with polar residues favored, such as S, K, R, E, D, H, G, P, G and Y.
• F108 in the human extracellular domain is a Y in Xenopus, and therefore Y or other hydrophobic group, such as I, V or L should be tolerated.
• El 11 in the human extracellular domain is K in Xenopus, indicating that charged residues will be tolerated at this position, including D, R, K and H, as well as Q and N.
• Rl 12 in the human extracellular domain is K in Xenopus, indicating that basic residues are tolerated at this position, including R and H.
• a at position 119 in the human extracellular domain is relatively poorly conserved, and appears as P in rodents and V in Xenopus, thus essentially any amino acid should be tolerated at this position.
• ActRII proteins have been characterized in the art in terms of structural and functional characteristics, particularly with respect to ligand binding [Attisano et al. (1992) Cell 68(l):97-l08; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20: 7643-7648; Thompson et al. (2003) The EMBO Journal 22(7): 1555-1566; as well as U.S.
• a defining structural motif known as a three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine residues located at varying positions within the extracellular domain of each monomeric receptor [Greenwald et al. (1999) Nat Struct Biol 6: 18-22; and Hinck (2012) FEBS Lett 586: 1860-1870] Accordingly, the core ligand-binding domains of human ActRIIB, as demarcated by the outermost of these conserved cysteines, corresponds to positions 29-109 of SEQ ID NO: 50 (ActRIIB precursor).
• the structurally less-ordered amino acids flanking these cysteine-demarcated core sequences can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 residues at the N-terminus and/or by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues at the C-terminus without necessarily altering ligand binding.
• Exemplary ActRIIB extracellular domains for N-terminal and/or C-terminal truncation include SEQ ID NOs: 51, 52, 54, 55, and 109. Attisano et al.
• an ActRIIB(20-l29)-Fc protein retains similar, but somewhat reduced activity, relative to the wild-type, even though the proline knot region is disrupted.
• ActRIIB extracellular domains that stop at amino acid 134, 133, 132, 131, 130 and 129 are all expected to be active, but constructs stopping at 134 or 133 may be most active.
• mutations at any of residues 129-134 are not expected to alter ligand-binding affinity by large margins.
• mutations of P129 and P130 do not substantially decrease ligand binding.
• an ActRIIB polypeptide of the present disclosure may end as early as amino acid 109 (the final cysteine), however, forms ending at or between 109 and 119 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119) are expected to have reduced ligand binding.
• Amino acid 119 (with respect to present SEQ ID NO: 50) is poorly conserved and so is readily altered or truncated.
• ActRIIB polypeptides ending at 128 (with respect to SEQ ID NO: 50) or later should retain ligand-binding activity.
• ActRIIB polypeptides ending at or between 119 and 127 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, or 127), with respect to SEQ ID NO: 50, will have an intermediate binding ability. Any of these forms may be desirable to use, depending on the clinical or experimental setting.
• ActRIIB polypeptides beginning at position 20, 21, 22, 23, and 24 should retain general ligand-biding activity
• ActRIIB polypeptides beginning at positions 25, 26, 27, 28, and 29 are also expected to retain ligand-biding activity. It has been demonstrated, e.g, U.S. Patent No. 7,842,663, that, surprisingly, an ActRIIB construct beginning at 22, 23, 24, or 25 will have the most activity.
• ActRIIB polypeptides may, for example, comprise, consist essentially of, or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIB beginning at a residue corresponding to any one of amino acids 20-29 (e.g., beginning at any one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 50 and ending at a position corresponding to any one amino acids 109-134 (e.g., ending at any one of amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,
• SEQ ID NO: 50 examples include polypeptides that begin at a position from 20-29 (e.g., any one of positions 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) or 21-29 (e.g., any one of positions 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 50 and end at a position from 119-134 (e.g., any one of positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), 119-133 (e.g, any one of positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133), 129-134 (e.g, any one of positions 129, 130, 131, 132, 133, or 134), or 129-134 (e.g, any one of positions 129, 130, 131, 132
• constructs that begin at a position from 20-24 (e.g., any one of positions 20, 21, 22, 23, or 24), 21-24 (e.g., any one of positions 21, 22, 23, or 24), or 22-25 (e.g., any one of positions 22, 22, 23, or 25) of SEQ ID NO: 50 and end at a position from 109-134 (e.g., any one of positions 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
• 109-134 e.g., any one of positions 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
• 119-134 e.g., any one of positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134
• 129-134 e.g, any one of positions 129, 130, 131, 132, 133, or 134 of SEQ ID NO: 50.
• Variants within these ranges are also contemplated, particularly those having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding portion of SEQ ID NO: 50.
• ActRIIB variants comprise no more than 1, 2, 5, 6, 7, 8, 9, 10 or 15 conservative amino acid changes in the ligand-binding pocket, and zero, one, or more non conservative alterations at positions 40, 53, 55, 74, 79 and/or 82 in the ligand-binding pocket.
• Sites outside the binding pocket, at which variability may be particularly well tolerated, include the amino and carboxy termini of the extracellular domain (as noted above), and positions 42-46 and 65-73 (with respect to SEQ ID NO: 50).
• N65A Asparagine-to-alanine alteration at position 65
• R64K is well-tolerated, and thus another basic residue, such as H may be tolerated at position 64
• results of the mutagenesis program described in the art indicate that there are amino acid positions in ActRIIB that are often beneficial to conserve.
• SEQ ID NO: 50 these include position 80 (acidic or hydrophobic amino acid), position 78 (hydrophobic, and particularly tryptophan), position 37 (acidic, and particularly aspartic or glutamic acid), position 56 (basic amino acid), position 60 (hydrophobic amino acid, particularly phenylalanine or tyrosine).
• position 80 acidic or hydrophobic amino acid
• position 78 hydrophobic, and particularly tryptophan
• position 37 acidic, and particularly aspartic or glutamic acid
• position 56 basic amino acid
• position 60 hydrophobic amino acid, particularly phenylalanine or tyrosine.
• positions that may be desirable to conserve are as follows: position 52 (acidic amino acid), position 55 (basic amino acid), position 81 (acidic), 98 (polar or charged, particularly E, D, R or K), all with respect to SEQ ID NO: 50.
• ActRIIB polypeptides of the disclosure comprise the naturally occurring leucine at the position 79 with respect to SEQ ID NO: 50. In some embodiments, ActRIIB polypeptides of the disclosure comprise an acidic amino acid (e.g., a naturally occurring D or E amino acid residue or an artificial acidic amino acid) at the position 79 with respect to SEQ ID NO: 50. In alternative embodiments, ActRIIB polypeptides of the disclosure do not comprise an acidic amino acid (e.g., a naturally occurring D or E amino acid residue or an artificial acidic amino acid) at the position 79 with respect to SEQ ID NO: 50.
• an acidic amino acid e.g., a naturally occurring D or E amino acid residue or an artificial acidic amino acid
• the disclosure provides T RII or ActRIIB polypeptides sharing a specified degree of sequence identity or similarity to a naturally occurring TbIIII or ActRIIB polypeptide.
• the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non- homologous sequences can be disregarded for comparison purposes).
• the amino acid residues at corresponding amino acid positions are then compared.
• amino acid “identity” is equivalent to amino acid “homology”
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com).
• the following parameters are used in the GAP program: either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
• the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al, Nucleic Acids Res.
• Exemplary parameters include using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Unless otherwise specified, percent identity between two amino acid sequences is to be determined using the GAP program using a Blosum 62 matrix, a GAP weight of 10 and a length weight of 3, and if such algorithm cannot compute the desired percent identity, a suitable alternative disclosed herein should be selected.
• the percent identity between two amino acid sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
• Another embodiment for determining the best overall alignment between two amino acid sequences can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. ⁇ Comp. App. Biosci., 6:237-245 (1990)).
• the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is presented in terms of percent identity.
• amino acid sequence identity is performed using the FASTDB computer program based on the algorithm of Brutlag et al. ⁇ Comp. App. Biosci., 6:237-245 (1990)).
• Polypeptides of the disclosure may additionally include any of various leader sequences at the N-terminus. Such a sequence would allow the peptides to be expressed and targeted to the secretion pathway in a eukaryotic system. See, e.g., Ernst et al, U.S. Pat. No. 5,082,783 (1992).
• a native signal sequence e.g., native TbIIII or ActRIIB signal sequence
• Possible leader sequences include native leaders, tissue plasminogen activator (TP A) and honeybee mellitin (SEQ ID NOs. 22-24, respectively).
• TbIIII-E ⁇ and ActRIIB- Fc fusion proteins incorporating a TPA leader sequence include SEQ ID NOs: 11, 13, 15, 17, 82, 85, 88, and 91. Processing of signal peptides may vary depending on the leader sequence chosen, the cell type used and culture conditions, among other variables, and therefore actual N-terminal start sites for processed polypeptides may shift by 1, 2, 3, 4 or 5 amino acids in either the N-terminal or C-terminal direction. It will be understood by one of skill in the art that corresponding variants based on the long isoform of TbIIII will include the 25-amino acid insertion along with a conservative Val-Ile substitution at the flanking position C- terminal to the insertion.
• the present disclosure contemplates specific mutations of the polypeptides (e.g., TbIIII or ActRIIB polypeptides) so as to alter the glycosylation of the polypeptide.
• Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites.
• Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X- threonine (or asparagine-X-serine) (where“X” is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes.
• the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type polypeptide (for O-linked glycosylation sites).
• a variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence.
• Another means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide.
• the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine.
• arginine and histidine free carboxyl groups
• free sulfhydryl groups such as those of cysteine
• free hydroxyl groups such as those of serine, threonine, or hydroxyproline
• aromatic residues such as those of phenylalanine, tyrosine, or tryptophan
• the amide group of glutamine are described in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259
• deglycosylation may involve, for example, exposure of the polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N- acetylgalactosamine), while leaving the amino acid sequence intact.
• deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal. Biochem. 118: 131.
• Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350.
• the sequence of a polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide.
• polypeptides for use in humans will be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes, and insect cells are expected to be useful as well.
• a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes, and insect cells are expected to be useful as well.
• This disclosure further contemplates a method of generating mutants, particularly sets of combinatorial mutants of a polypeptide (e.g., TbIIII or ActRIIB polypeptides as well as heteromultimers thereof), as well as truncation mutants; pools of combinatorial mutants are especially useful for identifying functional variant sequences.
• the purpose of screening such combinatorial libraries may be to generate, for example, polypeptide variants which can act as either agonists or antagonist, or alternatively, which possess novel activities all together.
• a variety of screening assays are provided below, and such assays may be used to evaluate variants.
• a ActRIIB :TbIIII heteromultimer comprising an ActRIIB and/or TbMI polypeptide variant may be screened for ability to bind to an AcRIIB or TbIIII ligand, to prevent binding of an ActRIIB or TbIIII ligand to an ActRIIB or TbIIII polypeptide or to interfere with signaling caused by an ActRIIB or TbIIII ligand.
• Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to a polypeptide (e.g., TbIIII or ActRIIB polypeptides) comprising an extracellular domain of a naturally occurring polypeptide.
• a polypeptide e.g., TbIIII or ActRIIB polypeptides
• mutagenesis can give rise to variants which have serum half-lives dramatically different than the corresponding wild-type polypeptide.
• the altered protein can be rendered either more stable or less stable to proteolytic degradation or other processes which result in destruction of, or otherwise elimination or inactivation of, a native TbIIII polypeptide.
• Such variants, and the genes which encode them can be utilized to alter TbIIII polypeptide levels by modulating the half-life of the TbIIII polypeptides.
• a short half-life can give rise to more transient biological effects and can allow tighter control of recombinant polypeptide levels within the patient.
• mutations may be made in the linker (if any) and/or the Fc portion to alter the half-life of the protein.
• a combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential polypeptide (e.g., TbIIII or ActRIIB polypeptides) sequences.
• potential polypeptide e.g., TbIIII or ActRIIB polypeptides
• a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential polypeptide nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
• the library of potential polypeptide (e.g., TbIIII or ActRIIB polypeptide) variants can be generated from a degenerate oligonucleotide sequence.
• Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate vector for expression.
• the synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al, (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed.
• polypeptide (e.g., TbMI or ActRIIB polypeptide) variants can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al, (1994) Biochemistry 33: 1565-1572; Wang et al, (1994)
• combinatorial mutagenesis of polypeptides typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
• Preferred assays include ligand binding assays and ligand- mediated cell signaling assays.
• polypeptides e.g., TbIIII or ActRIIB polypeptides
• the polypeptides of the disclosure may further comprise post-translational modifications in addition to any that are naturally present in the native polypeptides.
• post-translational modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, pegylation
• the modified polypeptides may contain non amino acid elements, such as polyethylene glycols, lipids, mono- or poly-saccharides, and phosphates. Effects of such non-amino acid elements on the functionality of a polypeptide may be tested as described herein for other polypeptide variants.
• post-translational processing may also be important for correct folding and/or function of the protein.
• Different cells such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK-293 have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the polypeptides.
• the disclosure provides for fusion proteins (e.g., TbMI or ActRIIB fusion proteins), and in some embodiments, a first portion (e.g., a TbIIII or ActRIIB polypeptide portion) is connected to a heterologous portion (e.g., Fc portion) by means of a linker.
• the linkers are glycine and serine rich linkers. Other near neutral amino acids, such as, but not limited to, Thr, Asn, Pro and Ala, may also be used in the linker sequence.
• the linker comprises various permutations of amino acid sequences containing Gly and Ser. In some embodiments, the linker is greater than 10 amino acids in length.
• the application teaches the surprising finding that proteins comprising a TbIBI portion and a heterologous portion fused together by means of a
• GGGGS GGGGS4 linker
• n is not greater than 4 in a (GGGGS)n linker.
• n 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, or 5-6.
• n 3, 4, 5, 6, or 7.
• n 4.
• a linker comprising a (GGGGS)n sequence also comprises an N-terminal threonine.
• the linker is any one of the following:
• T GGGGS GGGGS GGGGS (SEQ ID NO: 5)
• T GGGGS GGGGS GGGGS (SEQ ID NO: 6)
• the linker comprises the amino acid sequence of TGGGPKSCDK (SEQ ID NO: 7). In some embodiments, the linker is any one of SEQ ID NOs: 21, 4-7, 25-26 or 40 lacking the N-terminal threonine.
• a linker may be rich in glycine (e.g., 2-10, 2-5, 2-4, 2-3 glycine residues) and may, for example, contain a single sequence of threonine/serine and glycines or repeating sequences of threonine/serine and/or glycines, e.g., GGG (SEQ ID NO: 63), GGGG (SEQ ID NO: 64), TGGGG(SEQ ID NO: 65), SGGGG(SEQ ID NO: 66), or SGGG(SEQ ID NO: 67) singlets, or repeats.
• the linker does not comprise the amino acid sequence of SEQ ID NO: 26 or 40.
• the TbMI polypeptides comprise an amino acid sequence that is at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 94-100, or biologically active fragments thereof. In some embodiments, the TbMI polypeptides comprise an amino acid sequence that is at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 94, or biologically active fragments thereof.
• the TbMI polypeptides comprise an amino acid sequence that is at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 98, or biologically active fragments thereof.
• the disclosure provides for fusion proteins comprising any of the TbMI polypeptides disclosed herein (e.g., a TbMI comprising the amino acid sequence of SEQ ID NO: 170) and any of the heterologous portions disclosed herein (e.g., any of the Fc portions disclosed herein).
• the TbMI portion is N-terminal to the heterologous portion (e.g., Fc portion). In some embodiments, the TbMI portion is C- terminal to the heterologous portion (e.g., Fc portion).
• the TbINI portion is C-terminal to the heterologous portion (e.g., Fc portion), and a linker is used to fuse the TbMI portion to the heterologous portion (e.g., Fc portion).
• the linker is any of the linkers disclosed herein.
• the linker comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 165.
• the heterologous portion is an Fc portion.
• the Fc portion comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 73 (which may optionally lack the C-terminal lysine residue), or functional fragments thereof.
• the TbMI portion comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 170, or functional fragments thereof.
• the fusion protein comprises nine amino acids from CH1 (e.g., SNTKVDKRV— SEQ ID NO: 189), followed by a linker (e.g., TGGG), followed by an Fc portion (e.g., SEQ ID NO: 73), followed by a linker (e.g., SEQ ID NO: 165), followed by a TGFBRII polypeptide portion (e.g., SEQ ID NO: 170).
• the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 193.
• the fusion protein is part of a homodimer, wherein each subunit of the homodimer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 193.
• the fusion protein is a monomer comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 193.
• the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 198.
• the fusion protein is part of a homodimer, wherein each subunit of the homodimer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 198.
• the fusion protein is a monomer comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 198.
• the Fc-T RII fusion protein does not comprise any additional binding domains (e.g., does not comprise an ActRIIB portion, an antibody portion, an antigen-binding portion, or a follistatin portion).
• the disclosure provides for a nucleic acid encoding any of the Fc-T RII fusion proteins disclosed herein.
• the nucleic acid comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 194, or fragments thereof.
• functional variants or modified forms of the TbMI or ActRIIB polypeptides include fusion proteins having at least a portion of the TbMI or ActRIIB polypeptides and one or more heterologous portions.
• heterologous portions include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin.
• a heterologous portion may be selected so as to confer a desired property. For example, some heterologous portions are particularly useful for isolation of the fusion proteins by affinity chromatography.
• relevant matrices for affinity chromatography such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used.
• Many of such matrices are available in“kit” form, such as the Pharmacia GST purification system and the QIAexpressTM system (Qiagen) useful with (HI Sr,) fusion partners.
• a heterologous portion may be selected so as to facilitate detection of the TbMI or ActRIIB polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as“epitope tags,” which are usually short peptide sequences for which a specific antibody is available.
• heterologous portions have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the heterologous portion by subsequent chromatographic separation.
• a TbMI or ActRIIB polypeptide is fused with a domain that stabilizes the TbIIII or ActRIIB polypeptide in vivo (a“stabilizer” domain).
• stabilizing is meant anything that increases serum half life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an
• immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties.
• heterologous portions include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains.
• fusion proteins may be arranged in any manner that is consistent with the desired functionality.
• a TbIIII or ActRIIB polypeptide may be placed C-terminal to a heterologous domain, or, alternatively, a heterologous domain may be placed C-terminal to a TbIIII or ActRIIB polypeptide.
• the TbIIII or ActRIIB polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N- terminal to either domain or between the domains.
• an immunoglobulin Fc domain or simply“Fc” is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof.
• an immunoglobulin Fc region may comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region.
• the immunoglobulin Fc region comprises at least an immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably lacks the CH1 domain. In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region.
• the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igy) (g subclasses 1, 2, 3, or 4).
• polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
• GlFc 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 58.
• Naturally occurring variants in GlFc would include E134D and M136L according to the numbering system used in SEQ ID NO: 58 (see Uniprot P01857).
• the IgGl Fc domain has one or more mutations at residues such as Asp- 265, lysine 322, and Asn-434.
• the mutant IgGl Fc domain having one or more of these mutations e.g., Asp-265 mutation
• the mutant Fc domain having one or more of these mutations has increased ability of binding to the MHC class I-related Fc-receptor (FcRN) relative to a wild-type IgGl Fc domain.
• polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 59.
• VECPPCPAPP VAGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSHEDPEVQ
• G3Fc Two examples of amino acid sequences that may be used for the Fc portion of human IgG3 (G3Fc) are shown below.
• the hinge region in G3Fc can be up to four times as long as in other Fc chains and contains three identical l5-residue segments preceded by a similar l7-residue segment.
• the first G3Fc sequence shown below (SEQ ID NO: 60) contains a short hinge region consisting of a single l5-residue segment
• SEQ ID NO: 61 contains a full-length hinge region.
• dotted underline indicates the hinge region
• solid underline indicates positions with naturally occurring variants according to UniProt P01859.
• polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 60 or 61.
• variant WIS is lacking most of the V region and all of the CH1 region. It has an extra interchain disulfide bond at position 7 in addition to the 11 normally present in the hinge region.
• variant ZUC lacks most of the V region, all of the CH1 region, and part of the hinge.
• variant OMM may represent an allelic form or another gamma chain subclass.
• the present disclosure provides additional fusion proteins comprising G3Fc domains containing one or more of these variants.
• polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 62.
• a given amino acid position in an immunoglobulin sequence consisting of hinge, CH2, and CH3 regions (e.g., SEQ ID NOs: 58, 59, 60, 61, and 62) will be identified by a different number than the same position when numbering encompasses the entire IgGl heavy-chain constant domain (consisting of the OHI , hinge, CH2, and CH3 regions) as in the Uniprot database.
• immunoglobulin IgA (Iga), IgD (Ig6). IgE (Igs) and IgM (Igp), may be used.
• IgA immunoglobulin
• IgD Ig6
• IgE immunoglobulin heavy chain constant region
• IgM Igp
• the portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of a hinge domain, and preferably at least a portion of a CEE domain of Fc gamma or the homologous domains in any of IgA, IgD, IgE, or IgM.
• substitution or deletion of amino acids within the immunoglobulin heavy chain constant regions may be useful in the practice of the methods and compositions disclosed herein.
• One example would be to introduce amino acid substitutions in the upper CH2 region to create an Fc variant with reduced affinity for Fc receptors (Col e et al. (1997) J. Immunol. 159:3613).
• the application further provides Fc fusion proteins with engineered or variant Fc regions.
• Such antibodies and Fc fusion proteins may be useful, for example, in modulating effector functions, such as, antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Additionally, the modifications may improve the stability of the antibodies and Fc fusion proteins.
• Amino acid sequence variants of the antibodies and Fc fusion proteins are prepared by introducing appropriate nucleotide changes into the DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibodies and Fc fusion proteins disclosed herein.
• amino acid changes also may alter post- translational processes of the antibodies and Fc fusion proteins, such as changing the number or position of glycosylation sites.
• Antibodies and Fc fusion proteins with reduced effector function may be produced by introducing changes in the amino acid sequence, including, but are not limited to, the Ala- Ala mutation described by Bluestone et al. (see WO 94/28027 and WO 98/47531; also see Xu et al. 2000 Cell Immunol 200; 16-26).
• Fc fusion proteins of the disclosure with mutations within the constant region including the Ala- Ala mutation may be used to reduce or abolish effector function.
• antibodies and Fc fusion proteins may comprise a mutation to an alanine at position 234 or a mutation to an alanine at position 235, or a combination thereof.
• the antibody or Fc fusion protein comprises an IgG4 framework, wherein the Ala-Ala mutation would describe a mutation(s) from phenylalanine to alanine at position 234 and/or a mutation from leucine to alanine at position 235.
• the antibody or Fc fusion protein comprises an IgGl framework, wherein the Ala- Ala mutation would describe a mutation(s) from leucine to alanine at position 234 and/or a mutation from leucine to alanine at position 235.
• the antibody or Fc fusion protein may alternatively or additionally carry other mutations, including the point mutation K322A in the CH2 domain (Hezareh et al. 2001 J Virol. 75: 12161-8).
• the antibody or Fc fusion protein may be modified to either enhance or inhibit complement dependent cytotoxicity (CDC).
• Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions in an Fc region (see, e.g., U.S. Pat. No. 6,194,551).
• cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
• the homodimeric antibody thus generated may have improved or reduced internalization capability and/or increased or decreased complement-mediated cell killing. See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992), W099/51642, Duncan & Winter Nature 322: 738-40 (1988); U.S.
• heteromultimers described herein comprise at least one TbINI polypeptide associated, covalently or non-covalently, with at least one ActRIIB polypeptide.
• polypeptides disclosed herein form heterodimeric complexes, although higher order heteromultimeric complexes are also included such as, but not limited to, heterotrimers, heterotetramers, and further oligomeric structures (see, e.g., Figures 9 and 10).
• TbMI and/or ActRIIB polypeptides comprise at least one multimerization domain.
• multimerization domain refers to an amino acid or sequence of amino acids that promote covalent or non-covalent interaction between at least a first polypeptide and at least a second polypeptide.
• Polypeptides disclosed herein may be joined covalently or non-covalently to a multimerization domain.
• a multimerization domain promotes interaction between a first polypeptide (e.g., a TbKII polypeptide) and a second polypeptide (e.g., an ActRIIB polypeptide) to promote
• heteromultimer formation e.g., heterodimer formation
• optionally hinders or otherwise disfavors homomultimer formation e.g., homodimer formation
• homomultimer formation e.g., homodimer formation
• non-naturally occurring disulfide bonds may be constructed by replacing on a first polypeptide (e.g. , a TbIIII polypeptide) a naturally occurring amino acid with a free thiol-containing residue, such as cysteine, such that the free thiol interacts with another free thiol-containing residue on a second polypeptide (e.g., an ActRIIB polypeptide) such that a disulfide bond is formed between the first and second polypeptides.
• a first polypeptide e.g. , a TbIIII polypeptide
• a naturally occurring amino acid with a free thiol-containing residue, such as cysteine, such that the free thiol interacts with another free thiol-containing residue on a second polypeptide (e.g., an ActRIIB polypeptide) such that a disulfide bond is formed between the first and second polypeptides.
• interactions to promote heteromultimer formation include, but are not limited
• a multimerization domain may comprise one component of an interaction pair.
• the polypeptides disclosed herein may form protein complexes comprising a first polypeptide covalently or non-covalently associated with a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of a TbMI polypeptide and the amino acid sequence of a first member of an interaction pair; and the second polypeptide comprises the amino acid sequence of an ActRIIB polypeptide and the amino acid sequence of a second member of an interaction pair.
• the interaction pair may be any two polypeptide sequences that interact to form a complex, particularly a
• One member of the interaction pair may be fused to a TbMI or ActRIIB polypeptide as described herein, including for example, a polypeptide sequence comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID NOs: 18, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 51, 52, 54, 55, and 109.
• An interaction pair may be selected to confer an improved property/activity such as increased serum half-life, or to act as an adaptor on to which another moiety is attached to provide an improved
• a polyethylene glycol moiety may be attached to one or both components of an interaction pair to provide an improved property /activity such as improved serum half-life.
• the first and second members of the interaction pair may be an asymmetric pair, meaning that the members of the pair preferentially associate with each other rather than self associate. Accordingly, first and second members of an asymmetric interaction pair may associate to form a heterodimeric complex (see, e.g., Figures 9 and 10).
• the interaction pair may be unguided, meaning that the members of the pair may associate with each other or self-associate without substantial preference and thus may have the same or different amino acid sequences. Accordingly, first and second members of an unguided interaction pair may associate to form a homodimer complex or a heterodimeric complex.
• the first member of the interaction pair (e.g., an asymmetric pair or an unguided interaction pair) associates covalently with the second member of the interaction pair.
• the first member of the interaction pair (e.g., an asymmetric pair or an unguided interaction pair) associates non-covalently with the second member of the interaction pair.
• a problem that arises in large-scale production of asymmetric immunoglobulin-based proteins from a single cell line is known as the“chain association issue”.
• the chain association issue concerns the challenge of efficiently producing a desired multichain protein from among the multiple combinations that inherently result when different heavy chains and/or light chains are produced in a single cell line [Klein et al (2012) mAbs 4:653-663]
• This problem is most acute when two different heavy chains and two different light chains are produced in the same cell, in which case there are a total of 16 possible chain combinations (although some of these are identical) when only one is typically desired. Nevertheless, the same principle accounts for diminished yield of a desired multichain fusion protein that incorporates only two different (asymmetric) heavy chains.
• protuberance-into-cavity knock-into-holes
• protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., a first interaction pair) with larger side chains (e.g., tyrosine or tryptophan).
• Complementary“cavities” of identical or similar size to the protuberances are optionally created on the interface of the second polypeptide (e.g., a second interaction pair) by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
• the IgGl CH3 domain interface comprises four unique charge residue pairs involved in domain-domain interactions: Asp356-Lys439’, Glu357-Lys370’, Lys392- Asp399’, and Asp399-Lys409’ [residue numbering in the second chain is indicated by C)
• C Cyclone numbering in the second chain
• the numbering scheme used here to designate residues in the IgGl CH3 domain conforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetry present in the CH3-CH3 domain interactions, each unique interaction will represented twice in the structure (e.g., Asp-399-Lys409’ and Lys409-Asp399’).
• K409-D399’ favors both heterodimer and homodimer formation.
• a single mutation switching the charge polarity (e.g., K409E; positive to negative charge) in the first chain leads to unfavorable interactions for the formation of the first chain homodimer. The unfavorable interactions arise due to the repulsive interactions occurring between the same charges (negative-negative; K409E-D399’ and D399-K409E’).
• a similar mutation switching the charge polarity (D399K’; negative to positive) in the second chain leads to unfavorable interactions (K409’-D399K’ and D399K-K409’) for the second chain homodimer formation. But, at the same time, these two mutations (K409E and D399K’) lead to favorable interactions (K409E-D399K’ and D399-K409’) for the heterodimer formation.
• the electrostatic steering effect on heterodimer formation and homodimer discouragement can be further enhanced by mutation of additional charge residues which may or may not be paired with an oppositely charged residue in the second chain including, for example, Arg355 and Lys360.
• additional charge residues which may or may not be paired with an oppositely charged residue in the second chain including, for example, Arg355 and Lys360.
• the table below lists possible charge change mutations that can be used, alone or in combination, to enhance ActRIIB:T RII heteromultimer formation.
• one or more residues that make up the CH3-CH3 interface in a fusion protein of the instant application are replaced with a charged amino acid such that the interaction becomes electrostatically unfavorable.
• a positive-charged amino acid in the interface e.g., a lysine, arginine, or histidine
• a negatively charged amino acid e.g., aspartic acid or glutamic acid
• a negative-charged amino acid in the interface is replaced with a positive-charged amino acid.
• the amino acid is replaced with a non-naturally occurring amino acid having the desired charge characteristic.
• One of a pair of Fc sequences with electrostatic complementarity can be arbitrarily fused to the TbMI or ActRIIB polypeptide of the construct, with or without an optional linker, to generate an ActRIIB :TbKII heteromultimer.
• This single chain can be coexpressed in a cell of choice along with the Fc sequence complementary to the first Fc to favor generation of the desired multi chain construct (e.g., ActRIIB ⁇ RII heteromultimer).
• SEQ ID NO: 68 human GlFc(E356K/D399K)
• SEQ ID NO: 69 human GlFc(K392D/K409D)
• T ⁇ Rb superfamily type I or type II receptor polypeptide of the construct can be fused to either SEQ ID NO: 68 or SEQ ID NO: 69, but not both.
• the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains using Fc sequences engineered for steric complementarity.
• the disclosure provides knobs-into-holes pairing as an example of steric complementarity.
• One of a pair of Fc sequences with steric complementarity can be arbitrarily fused to the TbIIII or ActRIIB polypeptide of the construct, with or without an optional linker, to generate an Ao ⁇ IIIIB:TbIIII heteromultimer.
• This single chain can be co-expressed in a cell of choice along with the Fc sequence complementary to the first Fc to favor generation of the desired multi-chain construct.
• SEQ ID NO: 70 SEQ ID NO: 70
• Fc complementarity based on knobs-into-holes pairing combined with an engineered disulfide bond is disclosed in SEQ ID NO: 72 [hGlFc(Sl32C/Tl44W)] and SEQ ID NO: 73 [hGlFc(Yl27C/Tl44S/Ll46A/Yl85V)].
• the engineered amino acid substitutions in these sequences are double underlined, and the TGF superfamily type I or type II polypeptide of the construct can be fused to either SEQ ID NO: 72 or SEQ ID NO: 73, but not both.
• the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains using Fc sequences engineered to generate interdigitating b-strand segments of human IgG and IgA CH3 domains.
• Fc sequences engineered to generate interdigitating b-strand segments of human IgG and IgA CH3 domains include the use of strand-exchange engineered domain (SEED) CH3 heterodimers allowing the formation of SEEDbody fusion proteins [Davis et al. (2010) Protein Eng Design Sel 23: 195-202]
• SEED strand-exchange engineered domain
• One of a pair of Fc sequences with SEEDbody complementarity can be arbitrarily fused to the TbMI or ActllB of the construct, with or without an optional linker, to generate a TbMI or ActRIIB fusion polypeptide.
• This single chain can be co-expressed in a cell of choice along with the Fc sequence complementary to the first Fc to favor generation of the desired multi-chain construct.
• SEQ ID NO: 74 [hGlFc(SbAG)]
• SEQ ID NO: 75 [hGlFc(SbGA)] are examples of complementary IgG Fc sequences in which the engineered amino acid substitutions from IgA Fc are double underlined, and the TbMI or ActRIIB polypeptide of the construct can be fused to either SEQ ID NO: 74 or SEQ ID NO: 75, but not both.
• the disclosure provides desired pairing of asymmetric Fc-containing
• one of a pair of Fc sequences attached to a leucine zipper-forming strand can be arbitrarily fused to the TbMI or ActRIIB polypeptide of the construct, with or without an optional linker, to generate a TbMI or ActRIIB fusion polypeptide.
• This single chain can be co-expressed in a cell of choice along with the Fc sequence attached to a complementary leucine zipper-forming strand to favor generation of the desired multi-chain construct.
• SEQ ID NO: 76 [hGlFc-Apl (acidic)] and SEQ ID NO: 77 [hGlFc-Bpl (basic)] are examples of complementary IgG Fc sequences in which the engineered complimentary leucine zipper sequences are underlined, and the TbKP or ActRIIB polypeptide of the construct can be fused to either SEQ ID NO: 76 or SEQ ID NO: 77, but not both.
• the disclosure relates to TbIIII polypeptides (e.g., TbIBI-Ro fusion proteins) comprising one or more amino acid modifications that alter the isoelectric point (pi) of the TbIIII polypeptide and/or ActRIIB polypeptides (e.g., ActRIIB-Fc fusion proteins) comprising one or more amino acid modifications that alter the isoelectric point of the ActRIIB polypeptide.
• one or more candidate domains that have a pi value higher than about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 are selected for construction of the full multidomain protein.
• one or more candidate domains that have a pi value less than about 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, or 5.0 are selected for construction of the full multidomain protein. It will be understood by one skilled in the art that a single protein will have multiple charge forms. Without wishing to be bound by any particular theory, the charge of a protein can be modified by a number of different mechanisms including but not limited to, amino acid substitution, cationization, deamination, carboxyl-terminal amino acid heterogeneity, phosphorylation and glycosylation.
• the pi of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see for example Bjellqvist et al, 1993, Electrophoresis 14: 1023). In one embodiment, pi is determined using a
• Pharmacia Biotech Multiphor 2 electrophoresis system with a multi temp refrigerated bath recirculation unit and an EPS 3501 XL power supply.
• Pre-cast ampholine gels e.g., Amersham Biosciences, pi range 2.5-10
• Broad range pi marker standards e.g., Amersham, pi range 3-10, 8 .mu.L
• Electrophoresis may be performed, for example, at 1500 V, 50 mA for 105 minutes.
• the gel is fixed using, for example, a Sigma fixing solution (5x) diluted with purified water to lx Staining is performed, for example, overnight at room temperature using Simply Blue stain (Invitrogen).
• Destaining is carried out, for example, with a solution that consisted of 25% ethanol, 8% acetic acid and 67% purified water. Isoelectric points are determined using, for example, a Bio-Rad Densitometer relative to calibration curves of the standards.
• the one or more metrics may further include metrics characterizing stability of the domain under one or more different conditions selected from the group consisting of different pH values, different temperatures, different shear stresses, and different freeze/thaw cycles.
• the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains by methods described above in combination with additional mutations in the Fc domain which facilitate purification of the desired heteromeric species.
• An example is complementarity of Fc domains based on knobs-into-holes pairing combined with an engineered disulfide bond, as disclosed in SEQ ID NOs: 72-73, plus additional substitution of two negatively charged amino acids (aspartic acid or glutamic acid) in one Fc-containing polypeptide chain and two positively charged amino acids (e.g., arginine) in the complementary Fc-containing polypeptide chain (SEQ ID NOs: 78-79).
• Another example involves complementarity of Fc domains based on knobs-into-holes pairing combined with an engineered disulfide bond, as disclosed in SEQ ID NOs: 72-73, plus a histidine-to-arginine substitution at position 213 in one Fc-containing polypeptide chain (SEQ ID NO: 80).
• This substitution (denoted H435R in the numbering system of Kabat et al.) facilitates separation of desired heteromer from undesirable homodimer based on differences in affinity for protein A.
• the engineered amino acid substitution is indicated by double underline, and the TbKII or ActRIIB polypeptide of the construct can be fused to either SEQ ID NO: 80 or SEQ ID NO: 73, but not both.
• ActRIIB :TbIIII heteromultimers may be generated using a combination of heavy and light chain fusion proteins comprising either an TbMI or ActRIIB polypeptide.
• a TbMI polypeptide may be fused, with or without a linker domain, to an immunoglobulin heavy chain (IgGl, IgG2, IgG3, IgG4, IgM, IgAl, or IgA2) that comprises at least a portion of the CM domain.
• an ActRIIB polypeptide may be fused, with or without a linker domain, to an immunoglobulin light chain (kappa or lambda) that comprises at least a portion of the light chain constant domain (CL).
• an ActRIIB polypeptide may be fused, with or without a linker domain, to an immunoglobulin heavy chain (IgGl, IgG2, IgG3, IgG4, IgM, IgAl, or IgA2) that comprises at least a portion of the Cn 1 domain
• an TbMI polypeptide may be fused, with or without a linker domain, to an immunoglobulin light chain (kappa or lambda) that comprises at least a portion of the light chain constant domain (CL).
• heterodimerization of a heavy and light chain occurs between the CM with the CL, which is generally stabilized by covalent linking of the two domains via a disulfide bridge.
• Constructs employing the full-length heavy chain, or at least a portion of the heavy chain comprising the hinge region, could give rise to antibody -like molecules comprising two“light chains” and two“heavy chains”. See Figure 10.
• a potential advantage of this design is that it may more closely mimic the naturally occurring TbIIII-1 ⁇ 3 ⁇ - ActRIIB complex and may display higher affinity for the ligand than comparable single heterodimers.
• this design may be modified by incorporating various heavy chain truncations including, for example, truncations that comprise the CM domain and some or all of the hinge domain (giving rise to F(ab’)2-like molecules) as well as truncations that only comprise the CM domain or a fragment thereof (giving rise to Fab-like molecules).
• various heavy chain truncations including, for example, truncations that comprise the CM domain and some or all of the hinge domain (giving rise to F(ab’)2-like molecules) as well as truncations that only comprise the CM domain or a fragment thereof (giving rise to Fab-like molecules). See Figure 10G.
• Various methods for designing such heteromultimer constructs are described in US 2009/0010879, Klein et al [(2012) mAbs 4:653-663], and Spiess et al [(2015) Molecular Immunology 67(2A): 95-106] the contents of which are incorporated in their entirety herein.
• it is desirable to generate antibody-like ActRIIB:T RII heterodimers comprising at least one branch of the complex comprising an TbMI- CL:ActRIIB-CHl heterodimer pair and at least a second branch comprising an ActRIIB- CL:T RII-CI I 1 heterodimer pair.
• Such heterodimer complexes can be generated, for example, using combinations of heavy chain and light chain asymmetrical pairing technologies [Spiess et al (2015) Molecular Immunology 67(2A): 95-106] For example, in CrossMab technology, [Schaefer et al (2011). Proc. Natl. Acad. Sci. U.S.A.
• heterodimerization strategy to facilitate efficient IgG production in a single host cell.
• Electrostatic steering may also be used to generate orthogonal Fab interfaces to facilitate the construction of such heterodimers.
• Peptide linkers may be used to ensure cognate pairing of light and heavy chains in a format known as“LUZ-Y” [Wranik et al (2012) J. Biol. Chem. 287: 43331-43339], wherein heavy chain heterodimerization is accomplished using leucine zippers which may be subsequently removed by proteolysis in vitro.
• the disclosure provides for TbIIII polypeptides fusion proteins, as well as ActRIIB ⁇ RII heteromultimers comprising the same, comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 11, 13, 15, 17, 18, 27, 85, 87, 91, and 93 or biologically active fragments thereof.
• the TbIIII polypeptides fusion proteins, as well as ActRIIB ⁇ RII heteromultimers comprising the same comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 85, 87, 91, and 93, or biologically active fragments thereof.
• heteromultimers comprising the same, comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NO: 87, or a biologically active fragment thereof.
• the TbMI polypeptides fusion protein, as well as ActRIIB ⁇ RII heteromultimers comprising the same comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 93, or a biologically active fragment thereof.
• the disclosure provides for ActRIIB polypeptides fusion proteins, as well as ActRIIB :TbIIII heteromultimers comprising the same, comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 51, 52, 54, 55, 82, 84, 88, 90, and 109 or biologically active fragments thereof.
• the ActRIIB polypeptides fusion proteins as well as Ao ⁇ IBIB:TbIIII heteromultimers comprising the same, comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 82, 84, 88, and 90, or biologically active fragments thereof.
• the ActRIIB polypeptides fusion proteins as well as Ao ⁇ IBIB:TbIIII heteromultimers comprising the same, comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NO: 84, or a biologically active fragment thereof.
• the ActRIIB polypeptides fusion protein as well as ActRIIB :TbIBI heteromultimers comprising the same, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 90, or a biologically active fragment thereof.
• the TbIIII fusion proteins described herein have improved binding affinity for T ⁇ EbI and TOEb3.
• a TbIIII fusion protein comprising a linker at least 10 amino acids in length e.g., a fusion protein having the amino acid sequence of any one of SEQ ID NOs: 11, 13 and 15
• has improved binding affinity for T ⁇ EbI and TORb3 as compared to a reference TbIIII fusion protein e.g., a TbIIII fusion protein having the amino acid sequence of SEQ ID NO: 9).
• the TbIIII fusion protein binds to TGF l with a KD of less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM, less than 50 pM or less than 25 pM. In some embodiments, the fusion protein binds to TGF 3 with a KD of less than 75 pM, less than 70 pM, less than 60 pM, less than 50 pM, less than 40 pM, less than 35 pM, less than 25 pM, less than 15, less than 10, or less than 5 pM.
• any of the TbMI polypeptides, as well as ActRIIB:T RII heteromultimers comprising the same, disclosed herein inhibits one or more of activin (e.g., activin A, activin B, activin C, activin E, activin AC, activin AB, activin BC, activin AE, and activin BE), GDF8, GDF11, BMP10, TGF l, and TGF 3 in a measurable assay.
• the reporter gene assay is a CAGA reporter assay.
• the CAGA assay is based on a human lung carcinoma cell line transfected with a
• CAGA pGL3(CAGA)l2 reporter plasmid
• pRLCMV Renilla reporter plasmid
• the CAGA motif is present in the promoters of TGF -responsive genes (for example, PAI-l), so this vector is of general use for factors signaling through SMAD2 and SMAD3. See, e.g., Example 2.
• any of the fusion polypeptides disclosed herein comprises the following components: a) any of the TbIIII or ActRIIB polypeptides disclosed herein (“A”), b) any of the linkers disclosed herein (“B”), c) any of the heterologous portions disclosed herein (“C”), and optionally a linker (“X”).
• the fusion polypeptide may be arranged in a manner as follows (N -terminus to C-terminus): A-B-C or C-B-A.
• the fusion polypeptide may be arranged in a manner as follows (N- terminus to C-terminus): X- A-B-C or X-C-B-A.
• the fusion polypeptide comprises each of A, B and C (and optionally a leader sequence such as the amino acid sequence of SEQ ID NO: 23), and comprises no more than 100, 90, 80, 70, 60,
• the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows (N-terminus to C-terminus): X-A-B-C, and the fusion polypeptide comprises 1, 2, 3, 4, or 5 amino acids between X and A.
• the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows (N-terminus to C-terminus): X-C-B-A, and the fusion polypeptide comprises 1, 2, 3, 4, or 5 amino acids between X and C.
• the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows (N-terminus to C-terminus): X-A-B-C, and the fusion polypeptide comprises an alanine between X and A.
• the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows (N- terminus to C-terminus): X-C-B-A, and the fusion polypeptide comprises an alanine between X and C.
• the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows (N-terminus to C-terminus): X-A-B-C, and the fusion polypeptide comprises a glycine and an alanine between X and A.
• the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows (N-terminus to C-terminus): X-C-B-A, and the fusion polypeptide comprises a glycine and an alanine between X and C.
• the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows (N-terminus to C-terminus): X-A-B-C, and the fusion polypeptide comprises a threonine between X and A.
• the fusion polypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows (N- terminus to C-terminus): X-C-B-A, and the fusion polypeptide comprises a threonine between X and C.
• the TbMI fusion polypeptide as well as ActRIIB:T RII heteromultimers comprising the same, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the TbMI polypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 18 or 27), wherein the TbMI polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post- translational modifications, such as PEGylation and/or glycosylation).
• SEQ ID NO: 18 or 27 any of the TbMI polypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 18 or 27)
• the TbMI polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further
• the TbMI fusion polypeptide as well as ActRIIB ⁇ RII heteromultimers comprising the same, comprises an amino acid sequence that is at least 70%, 75%, 80%,
• linker portion of the fusion polypeptide comprises no more than 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or
• the TbMI fusion polypeptide as well as
• ActRIIB ⁇ RII heteromultimers comprising the same, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the heterologous portion sequences disclosed herein (e.g., SEQ ID NOs:
• heterologous portion of the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation).
• the TbMI fusion polypeptide as well as ActRIIB ⁇ RII heteromultimers comprising the same, comprises any of the TbMI polypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 18 or 27), wherein the TbINI polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation).
• the TbMI fusion polypeptide as well as ActRIIB ⁇ RII heteromultimers comprising the same, comprises any of the linker sequences disclosed herein (e.g., SEQ ID NO: 6), wherein the linker portion of the fusion polypeptide comprises no more than 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation).
• the TbMI fusion polypeptide as well as ActRIIB ⁇ RII heteromultimers comprising the same, comprises any of the heterologous portion sequences disclosed herein (e.g., SEQ ID NO: 68, 69, 72, or 73), wherein the heterologous portion of the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation).
• the ActRIIB fusion polypeptide, as well as ActRIIB ⁇ RII heteromultimers comprising the same comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the ActRIIB polypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 51, 52, 54, 55, or 109), wherein the ActRIIB polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation).
• the ActRIIB fusion polypeptide, as well as ActRIIB ⁇ RII heteromultimers comprising the same comprises an amino acid sequence that is at least 70%, 75%, 80%,
• linker portion of the fusion polypeptide comprises no more than 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or
• the ActRIIB fusion polypeptide as well as
• ActRIIB ⁇ RII heteromultimers comprising the same, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the heterologous portion sequences disclosed herein (e.g., SEQ ID NOs: 68, 69, 72, or 73), wherein the heterologous portion of the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation).
• the ActRIIB fusion polypeptide as well as ActRIIB:T RII heteromultimers comprising the same, comprises any of the ActRIIB polypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 51, 52, 54, 55, or 109), wherein the ActRIIB polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation).
• the ActRIIB fusion polypeptide as well as ActRIIB ⁇ RII heteromultimers comprising the same, comprises any of the linker sequences disclosed herein (e.g., SEQ ID NO: 6), wherein the linker portion of the fusion polypeptide comprises no more than 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation).
• heteromultimers comprising the same, comprises any of the heterologous portion sequences disclosed herein (e.g., SEQ ID NO: 68, 69, 72, or 73), wherein the heterologous portion of the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation).
• SEQ ID NO: 68, 69, 72, or 73 amino acids
• additional amino acids but which may include further post-translational modifications, such as PEGylation and/or glycosylation.
• the disclosure provides for a TbIIII fusion polypeptide, as well as ActRIIB :TbIBI heteromultimers comprising the same, wherein the fusion polypeptide consists or consists essentially of (and not necessarily in the following order): a) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the TbIIII polypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 18 or 27), wherein the TbIIII polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation); b) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
• ActRIIB :TbIIII heteromultimers comprising the same, wherein the fusion polypeptide consists or consists essentially of (and not necessarily in the following order): a) any of the TbMI polypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 18 or 27), wherein the TbMI polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post- translational modifications, such as PEGylation and/or glycosylation); b) any of the linker sequences disclosed herein (e.g., SEQ ID NO: 6), wherein the linker portion of the fusion polypeptide comprises no more than 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation); and c) any of the heterologous portion sequences disclosed herein (e.g., SEQ ID NO: 68, 69, 72,
• the disclosure provides for a ActRIIB fusion polypeptide, as well as ActRIIB :TbMI heteromultimers comprising the same, wherein the fusion polypeptide consists or consists essentially of (and not necessarily in the following order): a) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the ActRIIB polypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 51, 52, 54, 55, or 109), wherein the ActRIIB polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation); b) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%
• heterologous portion sequences disclosed herein e.g., SEQ ID NO: 68, 69, 72, or 73
• the heterologous portion of the fusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (but which may include further post-translational
• the disclosure provides for a ActRIIB fusion polypeptide, as well as ActRIIB :TbMI heteromultimers comprising the same, wherein the fusion polypeptide consists or consists essentially of (and not necessarily in the following order): a) any of the ActRIIB polypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 51, 52, 54, 55, or 109), wherein the ActRIIB polypeptide portion of the fusion polypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation); b) any of the linker sequences disclosed herein (e.g., SEQ ID NO: 6), wherein the linker portion of the fusion polypeptide comprises no more than
• the disclosure provides for a TbIIII fusion polypeptide, as well as ActRIIB :TbIBI heteromultimers comprising the same, consisting of or consisting essentially of (and not necessarily in the following order): a) a TbIIII polypeptide portion consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation); b) a linker portion consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6
• glycosylation and c) a heterologous portion consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 69 or 73 and no more than 25, 20, 15,
• the disclosure provides for a TbINI fusion polypeptide, as well as ActRIIB :TbIIII heteromultimers comprising the same, consisting or consisting essentially of (and not necessarily in the following order): a) a TbINI polypeptide portion consisting of the amino acid sequence of SEQ ID NO: 18 and no more than 10, 9, 8,
• the disclosure provides for an ActRIIB fusion polypeptide, as well as ActRIIB :TbMI heteromultimers comprising the same, consisting of or consisting essentially of (and not necessarily in the following order): a) a ActRIIB polypeptide portion consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 51 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and/or glycosylation); b) a linker portion consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6 and no
• glycosylation and c) a heterologous portion consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 68 or 72 and no more than 25, 20, 15,
• the disclosure provides for a ActRIIB fusion polypeptide, as well as ActRIIB :TbIBI heteromultimers comprising the same, consisting or consisting essentially of (and not necessarily in the following order): a) a ActRIIB polypeptide portion consisting of the amino acid sequence of SEQ ID NO: 51 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post- translational modifications, such as PEGylation and/or glycosylation); b) a linker portion consisting of the amino acid sequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as PEGylation and glycosylation); and c)
• PEGylation PEGylation
• a leader sequence e.g., SEQ ID NO: 23
• a heteromeric protein complex of the disclosure comprises an antigen-binding domain of antibody that binds to one or more of TGF l, TORb2.
• TORb3 and at least one ActRIIB polypeptide domain e.g. a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an ActRIIB polypeptide domain (e.g. a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an ActRIIB polypeptide domain (e.g. a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to an ActRIIB polypeptide domain (e.g. a polypeptide
• the first ActRIIB polypeptide is part of a fusion polypeptide that comprises a first member of an interaction pair (“Ci”), and further comprises an additional first member of an interaction pair (“Ai”).
• the second ActRIIB polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“Bi”).
• variable heavy chain (VH) polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“C2”), and further comprises a first member of an interaction pair (“A2”).
• variable light chain (VL) polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“B 2 ”).
• a linker may be positioned between the first or second ActRIIB polypeptide and the corresponding member of the interaction pair, between interaction pairs, and between the VH and VL polypeptides and a member of the interaction pair.
• Ai and A2 may be the same or different; Bi and B2 may be the same or different, and Ci and C2 may be the same or different.
• Figure 11A is an example of a heterodimer comprising a first and second ActRIIB extracellular domain.
• Figure 11B is an example of a heteromultimer comprising a single ActRIIB extracellular domain.
• the disclosure provides for a heteromultimer comprising an interaction pair, wherein one member of the interaction pair comprises a TGF -binding portion wherein the TGF -binding portion is an antibody or antigen-binding fragment thereof that binds any one or more of TGF l, TGF 2, or TORb3; and wherein the second member of the interaction pair comprises an ActRIIB polypeptide portion that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to any of the ActRIIB sequences disclosed herein (e.g., SEQ ID NO: 52).
• the antibody or antigen-binding fragment thereof binds to TGF 1 and TORb3 with significantly greater affinity than to TGF 1. In some embodiments, the antibody or antigen-binding fragment thereof binds to TGF 1 with significantly greater affinity than to TGF 1 or TGF 2. In some embodiments, the antibody or antigen-binding fragment thereof binds to TGF l. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to TGF 2 or does not bind to TGF 2 with appreciable affinity. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to TGF 2 or TGF 3. or does not bind to TGF 2 or TORb3 with appreciable affinity.
• the second member comprises a dimer of any two ActRIIB polypeptide portions disclosed herein.
• the ActRIIB polypeptide dimerizes with a TbIIII polypeptide portion that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to any of the TbIIII sequences disclosed herein (e.g., to SEQ ID NO: 18).
• the ActRIIB polypeptide portion is a monomeric or“single-arm” ActRIIB polypeptide portion.
• the interaction pair comprises a heterologous moiety that facilitates the interaction.
• the heterologous moiety is any of the Fc portions disclosed herein.
• the ActRIIB polypeptide portion is fused to a first heterologous moiety (e.g., a first Fc portion) and the antibody or antigen binding fragment thereof portion is fused to a second heterologous moiety (e.g., a first Fc portion).
• the ActRIIB polypeptide portion is fused to the N-terminus of the first Fc portion, and the antibody or antigen-binding fragment thereof portion is fused to the N-terminus of the second Fc portion.
• the ActRIIB polypeptide portion is fused to the N-terminus of the first Fc portion, and the antibody or antigen-binding fragment thereof portion is fused to the C-terminus of the second Fc portion. In some embodiments, the ActRIIB polypeptide portion is fused to the C-terminus of the first Fc portion, and the antibody or antigen-binding fragment thereof portion is fused to the N- terminus of the second Fc portion.
• the ActRIIB polypeptide portion is fused to the N-terminus of the first Fc portion, and the antibody or antigen-binding fragment thereof portion is fused to the N-terminus of the second Fc portion; and the ActRIIB polypeptide portion is a heterodimer with any of the TbMI polypeptides disclosed herein.
• the ActRIIB polypeptide portion is fused to the N-terminus of the first Fc portion, and the antibody or antigen-binding fragment thereof portion is fused to the C- terminus of the second Fc portion; and the ActRIIB polypeptide portion is a heterodimer with any of the TbIIII polypeptides disclosed herein.
• the ActRIIB polypeptide portion is fused to the C-terminus of the first Fc portion, and the antibody or antigen-binding fragment thereof portion is fused to the N-terminus of the second Fc portion; and the ActRIIB polypeptide portion is a heterodimer with any of the TbIIII polypeptides disclosed herein.
• the ActRIIB polypeptide may be fused to the Fc portion, or the TbIIII polypeptide may be fused to the Fc portion.
• the VL portion of the antibody or antigen-binding fragment thereof is fused to the Fc portion, and in some embodiments, the VH of the antibody or antigen-binding fragment thereof is fused to the Fc portion.
• the disclosure contemplates linkers to facilitate the fusion between any of the components in the interaction pair.
• the interaction pair comprises a second interaction pair that facilitates that interaction between the TbIIII polypeptide and the ActRIIB polypeptide.
• Figure 11 A provides an illustrative example of an interaction pair comprising an ActRIIB polypeptide portion that is fused to the N-terminus of the first Fc portion, and the antibody or antigen-binding fragment thereof portion is fused to the N- terminus of the second Fc portion; and wherein the ActRIIB polypeptide portion is a heterodimer with a TbIIII polypeptide.
• the disclosure provides for a fusion protein comprising any of the ActRIIB polypeptides disclosed herein (e.g ., an ActRIIB polypeptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 51 or 52) fused to any of the TbIIII polypeptides disclosd herein (e.g., a TbIIII polypeptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 170).
• an ActRIIB polypeptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%
• the ActRIIB polypeptide portion is N-terminal to the TbIIII polypeptide portion. In some embodiments, the ActRIIB polypeptide portion is C-terminal to the TbIIII polypeptide portion. In some embodiments, the ActRIIB polypeptide portion of the fusion protein is fused directly to the TbIIII polypeptide portion of the fusion protein. In some embodiments, a heterologous portion (e.g., any of the Fc portions disclosed herein) and/or one or more linker portions separate the ActRIIB and TbMI polypeptide portions in the fusion protein.
• a heterologous portion e.g., any of the Fc portions disclosed herein
• the heterologous portion is an Fc polypeptide portion comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 163.
• the heterologous portion is an Fc polypeptide portion comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 72 (which may optionally lack the C-terminal lysine residue).
• the heterologous portion is an Fc polypeptide portion comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 73 (which may optionally lack the C- terminal lysine residue).
• the TbINI polypeptide portion is fused to the Fc portion by means of a linker (e.g., any of the linkers disclosed herein).
• the TbMI polypeptide portion is fused to the Fc portion by means of a glycine- serine-rich linker, such as a linker comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 165.
• a linker e.g., any of the linkers disclosed herein.
• the ActRIIB polypeptide portion is fused to the Fc portion by means of a linker comprising a glycine linker, such as a linker comprising a GGG amino acid sequence.
• the fusion protein comprises any of the signal sequences disclosed herein.
• the signal sequence comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23.
• the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 183. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 195.
• any of the ActRIIB and TbMI polypeptides disclosed herein are encoded by a nucleic acid comprising a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 186.
• any of the ActRIIB and TbMI polypeptide fusion proteins disclosed herein multimerize with another protein.
• any of the ActRIIB and Tb ⁇ II polypeptide fusion proteins disclosed herein homomultimerize (e.g, homodimerize).
• the disclosure contemplates a homomultimer comprising two or more fusion proteins comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 183.
• the disclosure contemplates a homomultimer comprising two or more fusion proteins comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 195.
• any of the ActRIIB and TbIIII polypeptide fusion proteins disclosed herein heteromultimerize with one or more different proteins/polypeptides.
• any of the ActRIIB and TbIIII polypeptide fusion proteins disclosed herein heteromultimerize with a protein/polypeptide comprising an ActRIIB polypeptide portion but lacking a Tb ⁇ II polypeptide portion.
• the resulting fusion protein would comprise two ActRIIB polypeptide portion“arms,” but a single TbIIII polypeptide portion arm.
• each unit of the heteromultimer comprises a member of an interaction pair.
• the member of the interaction pair is any of the Fc portions disclosed herein.
• the Fc portions have been modified to promote heteromultimer formation and/or to inhibit homomultimer formation. In some embodiments, the Fc portions have been modified to promote heterodimer formation and/or to inhibit homodimer formation. In some embodiments, the Fc portions have been modified to include any of the“knob-in-hole” mutations disclosed herein.
• the heterologous portion is an Fc polypeptide portion comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical the amino acid sequence of SEQ ID NO: 72 (which may optionally lack the C-terminal lysine residue).
• the heterologous portion is an Fc polypeptide portion comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical the amino acid sequence of SEQ ID NO: 73 (which may optionally lack the C-terminal lysine residue).
• any of the ActRIIB and TbMI polypeptide fusion proteins disclosed herein heteromultimerize with a protein/polypeptide comprising a TbINI polypeptide portion but lacking an ActRIIB polypeptide portion.
• the resulting fusion protein would comprise two TbIIII polypeptide portion“arms,” but a single ActRIIB polypeptide portion arm.
• each unit of the heteromultimer comprises a member of an interaction pair.
• the member of the interaction pair is any of the Fc portions disclosed herein.
• the Fc portions have been modified to promote heteromultimer formation and/or to inhibit homomultimer formation.
• the Fc portions have been modified to promote heterodimer formation and/or to inhibit homodimer formation.
• the Fc portions have been modified to include any of the“knob-in-hole” mutations disclosed herein.
• the ActRIIB and TbIIII polypeptide fusion protein in such heteromultimers comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 184.
• the ActRIIB and TbIIII polypeptide fusion protein in such heteromultimers comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 184.
• the ActRIIB and TbIIII polypeptide fusion protein in such heteromultimers comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9
• the protein/polypeptide comprising the TbIIII polypeptide portion but lacking the ActRIIB polypeptide portion comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 185.
• the heteromultimer is a heterodimer comprising a first fusion protein comprising the amino acid sequence of SEQ ID NO: 184 and a second fusion protein comprising the amino acid sequence of SEQ ID NO: 185.
• the ActRIIB and TbIIII polypeptide fusion protein in such
• heteromultimers comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 196.
• the protein/polypeptide comprising the TbIIII polypeptide portion but lacking the ActRIIB polypeptide portion comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 197.
• the heteromultimer is a heterodimer comprising a first fusion protein comprising the amino acid sequence of SEQ ID NO: 196 and a second fusion protein comprising the amino acid sequence of SEQ ID NO: 197.
• any of the ActRIIB and TbMI polypeptides disclosed herein for use in any of the heteromultimers disclosed herein is encoded by a nucleic acid comprising a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 187.
• any of the proteins comprising the TbMI polypeptide portion but lacking the ActRIIB polypeptide portion is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 188.
• the heteromultimers disclosed herein do not bind with appreciable affinity to CD4, CD8, CD25, CTLA-4, IL-10, TGF Receptor, PD-l, PD-L1, PD-L2, RANK, RANKL, HER2/neu, EGFR1, CD20, VEGF, TNF-a, TNFR2, FoxP3, CD80, CD86, IFN-a, IFN-b, IFN-g, GITR, 4-1BB, OX-40, TLR1-10, ErbB-l, HER1, ErbB- 3/HER3, ErbB-4/HER4, IGFR, IGFBP, IGF-1R, PDGFR, FGFR, VEGFR, HGFR, TRK receptor, ephrin receptors, AXL receptors, LTK receptors, TIE receptors, angiopoietinl, 2, ROR receptor, DDR receptor, RET receptor, KLG receptor, RYK receptor, MuSK receptor, IL R,
• SSX-l 2, 3, 4, TRP2-1NT2, carcino-embryonic antigen (CEA), Kallikfein 4, mammaglobm-A, OA1, prostate specific antigen (PSA), prostate specific membrane antigen, TRP-l/, 75. TRP-2, AIM-2.
• BING-4 CPSF, cyclin Dl, Ep- CAM, EpbA3, FGF-5, gp250, iCE), AFP, M-CSF, mdm-2, MUCI, p53 (TP53), PBF, FRAME, PSMA, RAGE-l.
• HPV proteins HPV proteins, EBV proteins, Hepatitis B or C virus proteins, and/or HIV proteins.
• the disclosure provides for a Tb ⁇ HI fusion polypeptide wherein the polypeptide does not comprise an additional ligand binding domain in addition to the Tb ⁇ HI domain in the same linear sequence.
• the polypeptide comprises a linear amino acid sequence comprising a Tb ⁇ HI domain and a heterologous portion (e.g., an Fc portion), but the linear amino acid sequence does not comprise any additional ligand binding domains.
• the polypeptide comprises a linear amino acid sequence comprising a Tb ⁇ HI domain and an Fc portion, but the linear amino acid sequence does not comprise any additional ligand binding domains.
• the disclosure provides for a Tb ⁇ HI fusion polypeptide wherein the heterologous portion is an Fc domain, and wherein only one continuous linker is covalently bound to the Fc domain.
• the disclosure provides for a multispecific binder of TGF - superfamily ligands.
• the multispecific binder is capable of binding to a) at least one of TGF l and TGF 3. and b) at least one of activin A, activin B, activin AB, GDF11, and GDF8.
• the multispecific binder comprises: a) a first portion that is capable of binding to TGF l and/or TGF 3; and b) a second portion that is capable of binding to at least one of activin A, activin B, activin AB, GDF11, and GDF8.
• the multispecific binder comprises a TbIPI polypeptide and a follistatin or a follistatin-like protein domain. In some embodiments, the multispecific binder comprises a TbMI polypeptide and an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to one or more of activin A, activin B, activin AB, GDF11, and/or GDF8. In particular embodiments, the multispecific binder comprises a TbMI polypeptide and an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to GDF8.
• the disclosure provides for a multispecific binder comprising any of the TbMI polypeptides disclosed herein and a follistatin or follistatin-like polypeptide.
• follistatin refers to a family of follistatin (FST) proteins and follistatin-related proteins, derived from any species.
• Follistatin is an autocrine glycoprotein that is expressed in nearly all tissues of higher animals. It was initially isolated from follicular fluid and was identified as a protein fraction that inhibited follicle-stimulating hormone (FSH) secretion from the anterior pituitary, and therefore was designated as FSH- suppressing protein (FSP). Subsequently, its primary function has been determined to be the binding and neutralization of members of the TGF-b superfamily including, for example, activin, a paracrine hormone that enhances secretion of FSH in the anterior pituitary.
• follistatin polypeptide is used to refer to polypeptides comprising any naturally occurring polypeptide of the follistatin family as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity, including, for example, ligand binding (e.g., myostatin (GDF8), GDF11, activin A, activin B) or heparin binding.
• ligand binding e.g., myostatin (GDF8), GDF11, activin A, activin B
• heparin binding e.g., heparin binding.
• follistatin polypeptides include polypeptides comprising an amino acid sequence derived from the sequence of any known follistatin having a sequence at least about 80% identical to the sequence of a follistatin polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99% or greater identity.
• the term“follistatin polypeptide” may refer to fusion proteins that comprise any of the polypeptides mentioned above along with a heterologous (non-follistatin) portion.
• An amino acid sequence is understood to be heterologous to follistatin if it is not uniquely found in the long (315 amino acid) form of human follistatin, represented by SEQ ID NO: 112.
• heterologous portions may be immediately adjacent, by amino acid sequence, to the follistatin polypeptide portion of a fusion protein, or separated by intervening amino acid sequence, such as a linker or other sequence.
• Follistatin is a single-chain polypeptide with a range of molecular weights from 31 to 49 kDa based on alternative mRNA splicing and variable glycosylation of the protein.
• the alternatively spliced mRNAs encode two proteins of 315 amino acids (i.e., FST315) and 288 amino acids (i.e., FST288); follistatin 315 can be further proteolytically degraded to follistatin 303 (FST303).
• any of the follistatin polypeptides disclosed herein comprises any one or more of the follistatin polypeptide domains disclosed herein.
• the human follistatin-288 (FST288) precursor has the following amino acid sequence, with the signal peptide indicated in bold, the N-terminal domain (FSN) indicated by single underlining, and the follistatin domains I-III (FSI, FSII, FSIII) indicated by double underlining.
• the processed (mature) human follistatin variant FST(288) has the following amino acid sequence with the N-terminal domain indicated by single underlining, and the follistatin domains I-III indicated by double underlining. Moreover, it will be appreciated that any of the initial amino acids G or N, prior to the first cysteine may be removed by processing or intentionally eliminated without any consequence, and polypeptides comprising such slightly smaller polypeptides are further included.
• the human follistatin-315 (FST315) precursor has the following amino acid sequence, with the signal peptide indicated in bold, the N-terminal domain (FSN) indicated by single underlining, and the follistatin domains I-III (FSI, FSII, FSIII) indicated by double underlining (NCBI Accession Number AAH04107.1; 344 amino acids).
• the processed (mature) human FST(3l5) has the following amino acid sequence with the N-terminal domain indicated by single underlining, and the follistatin domains I-III indicated by double underlining. Moreover, it will be appreciated that any of the initial amino acids G or N, prior to the first cysteine may be removed by processing or intentionally eliminated without any consequence, and polypeptides comprising such slightly smaller polypeptides are further included.
• Follistatin proteins herein may be referred to as FST. If followed by a number, such as FST(288), this indicates that the protein is the 288 form of follistatin. If presented as FST(288)-Fc, this indicates a C-terminal Fc fusion to the FST(288), which may or may not include an intervening linker. The Fc in this instance may be any immunoglobulin Fc portion as that term is defined herein. If presented as FST(288)-IgG2, this indicates a C-terminal Fc fusion to the FST(288) of the Fc portion of human IgG2.
• biologically active in all its grammatical forms, when used in the context of a follistatin polypeptide or variant or fragment thereof, refers to a polypeptide with the ability to bind a ligand from at least one of the (1) activin or (2) bone morphogenic protein (BMP) class of ligands.
• BMP bone morphogenic protein
• the“biologically active” follistatin is capable of binding to GDF8.
• a biologically active polypeptide or fragment thereof inhibits the activity of a ligand from at least one of the (1) activin or (2) bone morphogenic protein (BMP) class of ligands.
• a biologically active follistatin polypeptide or variant or fragment thereof inhibits GDF8, activin A and/or GDF-l 1 in a cell-based reporter gene assay with a lower IC50 than the IC50 of a follistatin polypeptide comprising the amino acid sequence of SEQ ID NO: 111.
• a biologically active follistatin polypeptide or variant or fragment thereof inhibits GDF8, activin A and/or GDF-l 1 in a cell-based reporter gene assay with an equal IC50 as compared to the IC50 of a follistatin polypeptide comprising the amino acid sequence of SEQ ID NO: 111.
• a biologically active follistatin polypeptide or variant or fragment thereof binds to one or more ligands selected from the group consisting of: GDF8 (myostatin), GDF11, activin A and activin B with a KD less than 1 nM, 100 pM, 50 pM or 10 pM.
• a biologically active follistatin polypeptide or variant or fragment thereof binds heparin with a greater affinity as compared to a follistatin polypeptide comprising the amino acid sequence of SEQ ID NO: 113.
• a biologically active follistatin polypeptide or variant or fragment thereof binds heparin with an equal binding affinity to a follistatin polypeptide comprising the amino acid sequence of SEQ ID NO: 113.
• the follistatin proteins are truncated forms exemplified by polypeptides comprising SEQ ID NO: 111, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125, and variants thereof.
• any of the follistatin polypeptides, fragments, functional variants, and modified forms disclosed herein may have similar, the same or improved biological activities as compared to a wild-type follistatin polypeptide (e.g. , a polypeptide having the amino acid sequence of SEQ ID NO: 111 or 113).
• a follistatin variant of the disclosure may bind to and inhibit function of a follistatin ligand (e.g., activin A, activin AB, activin B, and GDF8).
• a follistatin polypeptide modulates growth of tissues, particularly muscle.
• follistatin polypeptides include polypeptides comprising, consisting essentially of or consisting of the amino acid sequences by any of SEQ ID NOs: 110-125, 135, 137-139, and 141-148 or biologically active fragments thereof, as well as polypeptides comprising, consisting essentially of or consisting of amino acid sequences that are at least 80%, 81%,
• the follistatin polypeptide comprises, consists or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 111. Variations on these polypeptides may be prepared according to the following guidance. Unless stated otherwise, the numbering of amino acids in the follistatin polypeptides is based on the sequence of SEQ ID NO: 110, regardless of whether the native leader sequence is used.
• follistatin is characterized by three cysteine-rich regions (i.e., FS domains I-III) that are believed to mediate follistatin-ligand binding.
• FS domains I-III cysteine-rich regions
• polypeptide constructs comprising only one of the three FS-binding domains (e.g., FSDI) retains strong affinity towards certain follistatin-ligands (e.g., myostatin) and is biologically active in vivo.
• FSDI polypeptide constructs comprising only one of the three FS-binding domains
• variant follistatin polypeptides of the disclosure may comprise one or more active portions of a follistatin protein.
• constructs of the disclosure may begin at a residue corresponding to amino acids 30-95 of
• SEQ ID NO: 112 and end at a position corresponding to amino acids 316-344 of SEQ ID NO: 112.
• Other examples include constructs that begin at a position from 30-95 of SEQ ID NO:
• Others may include any of SEQ ID Nos. 116-125. Further examples include constructs that end at a position corresponding to an amino acid selected from the group consisting of the amino acid corresponding to amino acid 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, and 305 of SEQ ID NO: 113.
• follistatin polypeptides and constructs of the disclosure may comprise follistatin polypeptides which do not include residues corresponding to the amino acids selected from the group consisting of amino acids 289-315, 290-315, 291-315, 292-315, 293-315, 294-315, 295-315, 296-315, 297- 315, 298-315, 299-315, 300-315, 301-315, 302-315, 303-315, 304-315, and 305-315 of SEQ ID NO: 113.
• Follistatin polypeptides of the disclosure may include any naturally occurring domain of a follistatin protein as well as variants thereof (e.g., mutants, fragments, and
• FST(3l5) and FST(288) have high affinity for both activin (activin A and activin B) and myostatin (and the closely related GDF11) and that the follistatin domains (e.g., FSN and FSD I-III) are thought to be involved in the binding of such TGF-b ligands.
• activin A and activin B activin A and activin B
• myostatin myostatin
• Follistatin domains e.g., FSN and FSD I-III
• each of these three domains may have a different affinity for these TGF-b ligands.
• polypeptide constructs comprising only the N-terminal domain (FSN) and two FSDI domains in tandem retained high affinity for myostatin, demonstrated little or no affinity for activin and promoted systemic muscle growth when introduced into a mouse by gene expression (Nakatani el al, The FASEB Journal, Vol. 22477-487 (2008)).
• the FSDI domain contains the heparin binding domain of human follistatin, which has the amino acid sequence of KKCRMNKKNKPR (SEQ ID NO: 114).
• This heparin binding domain can be represented as BBXBXXBBXBXB (SEQ ID NO: 115) wherein“B” means a basic amino acid, particularly lysine (K) or arginine (R).
• the present disclosure encompasses, in part, variant follistatin proteins that demonstrate selective binding and/or inhibition of a given TGF-b ligand relative to the naturally occurring FST protein (e.g., maintaining high-affinity for myostatin while having a significantly reduced affinity for activin).
• the disclosure includes polypeptides comprising the FSN domain, as set forth below, and, for example, one or more heterologous polypeptide, and moreover, it will be appreciated that any of the initial amino acids G or N, prior to the first cysteine may be deleted, as in the example shown below (SEQ ID NO: 117).
• the disclosure includes polypeptides comprising the FSDI domain which contains the minimal core activities of myostatin (and/or GDF11) binding along with heparin binding as set forth below, and, for example, one or more heterologous polypeptide.
• CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCK EQPELEVQYQGRC SEQ ID NO: 118
• FSDI sequence may be advantageously maintained in structural context by expression as a polypeptide further comprising the FSN domain. Accordingly, the disclosure includes polypeptides comprising the FSN-FSDI sequence, as set forth below (SEQ ID NO:
• polypeptides comprising such slightly smaller polypeptides are further included.
• an FSN-FSDI-FSDI construct is sufficient to confer systemic muscle growth when genetically expressed in a mouse, and accordingly the disclosure includes polypeptides comprising the amino acid sequences below and, for example, one or more heterologous polypeptide.
• the FSDI sequence confers myostatin and GDF11 binding. It has been demonstrated that activins, particularly activin A but also activin B, are also negative regulators of muscle, and therefore a follistatin polypeptide that inhibits both the myostatin/GDFl 1 group and the activin A/activin B group may provide a more potent muscle effect. Moreover, in view of the findings herein demonstrating the low systemic availability of certain follistatin polypeptides, particularly those comprising a heparin binding domain, and more particularly in a homodimeric form, such as an Fc fusion, safety concerns associated with the known effects of activin inhibition on the reproductive axis and other tissues are alleviated.
• FSDII confers activin A and B binding
• the disclosure provides polypeptides comprising FSDI and FSDII (SEQ ID NO: 121), as well as FSN-FSDI-FSDII constructs (SEQ ID NO: 122) and, for example, one or more heterologous polypeptide.
• ACSSGVLLEVKHSGSCNSIS (SEQ ID NO: 123) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAP NCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNEC ALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPA SSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCL WDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSG SCNSIS (SEQ ID NO: 124)
• the present invention relates to antagonizing a ligand of follistatin (also referred to as a follistatin ligand) with a subject follistatin polypeptide (e.g., an FST-IgG fusion polypeptide).
• a ligand of follistatin also referred to as a follistatin ligand
• a subject follistatin polypeptide e.g., an FST-IgG fusion polypeptide.
• ligands of follistatin include some TGF-b family members, such as activin A, activin B, myostatin (GDF8) and GDF11.
• variant follistatin proteins of the disclosure include polypeptides that comprise one or more FS domains selected from FSDI (amino acids 95-164 of SEQ ID NO: 110), FSDII (amino acids 168-239 of SEQ ID NO: 110), or FSDIII (amino acids 245-316 of SEQ ID NO: 110) as well as proteins that comprise one or more FS domains selected from a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to FSDI (amino acids 95-164 of SEQ ID NO: 110), FSDII (amino acids 168-239 of SEQ ID NO: 110), or FSDIII (amino acids 245- 316 of SEQ ID NO: 110).
• FSDI amino acids 95-164 of SEQ ID NO: 110
• FSDII amino acids 168-239 of SEQ ID NO: 110
• FSDIII amino acids 245- 316 of SEQ ID NO: 110
• any of the follistatin polypeptides disclosed herein comprises any of the FS domains disclosed herein. These FS domains may be combined in any order within a variant follistatin polypeptide of the disclosure provided that such recombinant proteins maintain the desired activity including, for example, follistatin ligand-binding activity (e.g., myostatin) and biological activity (e.g., inducing muscle mass and/or strength).
• follistatin ligand-binding activity e.g., myostatin
• biological activity e.g., inducing muscle mass and/or strength
• follistatin variant polypeptides include, for example, polypeptides having domain structures such as FSDI-FSDII-FSDIII, FSDI-FSDIII, FSDI- FSDI-FSDIII, FSDI-FSDII, FSDI-FSDI, FSN-FSDI-FSDII-FSDIII, FSN-FSDI-FSDII, FSN- FSDI-FSDI, FSN-FSDI-FSDIII, FSN-FSDI-FSDI-FSDIII, and polypeptides obtained by fusing other heterologous polypeptides to the N-termini or the C-termini of these
• polypeptides These domains may be directly linked or liked via a linker polypeptide.
• polypeptide linkers may be any sequence and may comprise 1-50, preferably 1- 10, and more preferably 1-5 amino acids. In certain aspects, preferred linkers contain no cysteine amino acids.
• follistatin variants includes follistatin polypeptides that are fragments and/or mutants/modified polypeptides as compared to a reference wildtype follistatin protein (e.g., a follistatin protein having the amino acid sequence of any of SEQ ID NOs: 110-113).
• follistatin variants of the disclosure have reduced or abolished binding affinity for one or more follistatin ligands as compared to a wildtype follistatin polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO:
• the disclosure provides follistatin variants that have reduced or abolished binding affinity for activin as compared to a wildtype follistatin polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 113). In certain aspects, the disclosure provides follistatin variants that have reduced or abolished binding affinity for activin but retain high affinity for myostatin as compared to a wildtype follistatin polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 113).
• the disclosure provides follistatin variants that have reduced or abolished binding affinity for GDF11 as compared to a wildtype follistatin polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 113).
• a wildtype follistatin polypeptide e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 113.
• follistatin fragments or variants of the disclosure have increased binding affinity for heparin. In some embodiments, follistatin fragments or variants of the disclosure have a binding affinity for heparin which is equivalent to the binding affinity of a follistatin polypeptide comprising SEQ ID NO: 111. In some embodiments, follistatin fragments or variants have a binding affinity for heparin that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the binding affinity for heparin of a follistatin polypeptide comprising SEQ ID NO: 111.
• follistatin fragments or variants of the disclosure have a binding affinity for heparin which is greater than the binding affinity of a follistatin polypeptide comprising SEQ ID NO: 111. In some embodiments, follistatin fragments or variants of the disclosure have a binding affinity for heparin which is greater than the binding affinity of a follistatin polypeptide comprising SEQ ID NO: 113. In some embodiments, follistatin fragments or variants of the disclosure have an unmasked heparin binding domain. In some embodiments, follistatin fragments or variants of the disclosure comprise a heparin binding domain which comprises the endogenous follistatin heparin binding sequence.
• follistatin fragments or variants of the disclosure comprise a heparin binding domain which comprises the endogenous follistatin heparin binding sequence (e.g., SEQ ID NO: 114). In some embodiments, follistatin fragments or variants of the disclosure comprise a heterologous heparin binding sequence.
• the disclosure provides follistatin fragments or variants that do not comprise a sequence corresponding to the FSDII domain or functionally active FSDII domain.
• follistatin polypeptides of the disclosure may include a variant obtained through partial or complete deletion of the FSDII domain.
• such follistatin variants include the deletion of one or more cysteine residues within the FSDII region or substitution with non-cysteine amino acids.
• the follistatin proteins of the disclosure may comprise a signal sequence.
• the signal sequence can be a native signal sequence of a follistatin protein (e.g., amino acids 1-29 of SEQ ID NO: 110) or a signal sequence from another protein, such as tissue plasminogen activator (TP A) signal sequence or a honey bee melatin (HBM) signal sequence.
• TP A tissue plasminogen activator
• HBM honey bee melatin
• the signal sequence is removed during processing of the follistatin protein.
• N-linked glycosylation sites may be added to a follistatin polypeptide, and may increase the serum half-life of an FST-Fc fusion protein.
• N-X-S/T sequences may be generally introduced at positions outside the ligand-binding pocket.
• N-X- S/T sequences may be introduced into the linker between the follistatin sequence and the Fc or other fusion component.
• Such a site may be introduced with minimal effort by introducing an N in the correct position with respect to a pre-existing S or T, or by introducing an S or T at a position corresponding to a pre-existing N.
• a follistatin variant may include one or more additional, non- endogenous N-linked glycosylation consensus sequences.
• the present disclosure contemplates making functional variants by modifying the structure of a follistatin polypeptide for such purposes as enhancing therapeutic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo).
• Modified follistatin polypeptides can also be produced, for instance, by amino acid substitution, deletion, or addition.
• Whether a change in the amino acid sequence of a follistatin polypeptide results in a functional homolog can be readily determined by assessing the ability of the variant follistatin polypeptide to produce a response in cells in a fashion similar to the wild-type follistatin polypeptide, or to bind to one or more ligands, such as activin or myostatin in a fashion similar to wild-type follistatin.
• the present invention contemplates specific mutations of the follistatin polypeptides so as to alter the glycosylation of the polypeptide.
• Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O- linked or N-linked glycosylation sites.
• Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine (where“X” is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes.
• the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type follistatin polypeptide (for O-linked glycosylation sites).
• a variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence.
• Another means of increasing the number of carbohydrate moieties on a follistatin polypeptide is by chemical or enzymatic coupling of glycosides to the follistatin polypeptide.
• the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine.
• Removal of one or more carbohydrate moieties present on an ActRIIB polypeptide may be accomplished chemically and/or enzymatically.
• Chemical deglycosylation may involve, for example, exposure of the follistatin polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N- acetylgalactosamine), while leaving the amino acid sequence intact.
• follistatin polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide.
• follistatin proteins for use in humans will be expressed in a cell line (e.g ., a mammalian cell line) that provides proper glycosylation, such as HEK293 or CHO cell lines, although other expression cell lines are expected to be useful as well.
• This disclosure further contemplates a method of generating variants, particularly sets of combinatorial variants of an follistatin polypeptide, including, optionally, truncation variants; pools of combinatorial mutants are especially useful for identifying functional variant sequences.
• the purpose of screening such combinatorial libraries may be to generate, for example, follistatin polypeptide variants that have altered properties, such as altered pharmacokinetics, or altered ligand binding as compared to a wildtype follistatin polypeptide (e.g. , a polypeptide having the amino acid sequence of SEQ ID NO: 111 or 113).
• a variety of screening assays are provided below, and such assays may be used to evaluate variants.
• a follistatin polypeptide variant may be screened for its ability to bind to a follistatin ligand, and/or to prevent binding of a follistatin ligand to a follistatin polypeptide.
• a follistatin polypeptide or its variants may also be tested in a cell- based or in vivo assay. For example, the effect of a follistatin polypeptide variant on the expression of genes involved in muscle production may be assessed. This may, as needed, be performed in the presence of one or more recombinant follistatin ligand proteins (e.g., activin A), and cells may be transfected so as to produce a follistatin polypeptide and/or variants thereof, and optionally, a follistatin ligand. Likewise, a follistatin polypeptide may be administered to a mouse or other animal, and one or more muscle properties, such as muscle mass or strength may be assessed.
• a follistatin polypeptide may be administered to a mouse or other animal, and one or more muscle properties, such as muscle mass or strength may be assessed.
• any of the follistatin polypeptides disclosed herein may be administered to an animal model of muscle contractures, and the effects of the follistatin polypeptide on the animal model may be assessed (see, e.g.. Example 8).
• Such assays are either described in the application or are well known and routine in the art.
• a responsive reporter gene may be used in such cell lines to monitor effects on downstream signaling.
• Combinatorially-derived variants can be generated which have a selective potency relative to a naturally occurring follistatin polypeptide.
• Such variant proteins when expressed from recombinant DNA constructs, can be used in gene therapy protocols.
• mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding a wild-type follistatin polypeptide.
• the altered protein can be rendered either more stable or less stable to proteolytic degradation or other processes which result in destruction of, or otherwise inactivation of a native follistatin polypeptide.
• variants, and the genes which encode them can be utilized to alter follistatin polypeptide levels by modulating the half-life of the follistatin polypeptides. For instance, a short half-life can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant follistatin polypeptide levels within the cell.
• the follistatin polypeptides of the disclosure may further comprise post-translational modifications in addition to any that are naturally present in the follistatin polypeptides.
• modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
• the modified follistatin polypeptides may contain non-amino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elements on the functionality of a follistatin polypeptide may be tested as described herein for other follistatin polypeptide variants.
• follistatin polypeptide When a follistatin polypeptide is produced in cells by cleaving a nascent form of the follistatin polypeptide, post-translational processing may also be important for correct folding and/or function of the protein.
• Different cells such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293 have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the follistatin polypeptides.
• follistatin polypeptides include fusion proteins having at least a portion of a follistatin polypeptide and one or more fusion domains.
• fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (e.g., an Fc), maltose binding protein (MBP), or human serum albumin.
• GST glutathione S transferase
• a fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography.
• relevant matrices for affinity chromatography such as glutathione-, amylase-, and nickel- or cobalt- conjugated resins are used.
• Many of such matrices are available in“kit” form, such as the Pharmacia GST purification system and the QIAexpressTM system (Qiagen) useful with (HIS6) fusion partners.
• a fusion domain may be selected so as to facilitate detection of the follistatin polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as“epitope tags,” which are usually short peptide sequences for which a specific antibody is available.
• fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.
• a follistatin polypeptide is fused with a domain that stabilizes the follistatin polypeptide in vivo (a“stabilizer” domain).
• stabilizing is meant anything that increases serum half-life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other
• Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins.
• fusions to human serum albumin can confer desirable properties.
• Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains (that confer an additional biological function, such as further stimulation of muscle growth).
• the present disclosure provides fusion proteins comprising follistatin polypeptides fused to a polypeptide comprising a heterologous moiety/domain.
• the heterologous moiety is serum albumin.
• the heterologous moiety is a constant domain of an immunoglobulin, such as a CH1, CH2 or CH3 domain of an immunoglobulin or an Fc. Fc domains derived from human IgGl and IgG2 are provided below (SEQ ID NO: 126 and SEQ ID NO: 127, respectively).
• an IgG2, IgG4 or IgG2/4 Fc domain is particularly advantageous for fusion with follistatin polypeptides that retain heparin binding activity because these Fc species have reduced CDC and/or ADCC activity which may be harmful to the cells to which these heparin binding polypeptides may adhere.
• Other mutations are known that decrease either CDC or ADCC activity, and collectively, any of these variants are included in the disclosure and may be used as advantageous components of a follistatin fusion protein.
• any of the follistatin polypeptides disclosed herein is conjugated to an Fc domain comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 126, or fragments thereof.
• any of the follistatin polypeptides disclosed herein is conjugated to an Fc domain comprising an amino acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 127, or fragments thereof.
• the Fc domain of SEQ ID NO: 126 (or variant or fragment thereof) has one or more mutations at residues such as Asp-265, Lys-322, and Asn-434 (numbered in accordance with the corresponding full-length IgGl).
• the mutant Fc domain having one or more of these mutations has reduced ability of binding to the Fey receptor relative to a wildtype Fc domain.
• the mutant Fc domain having one or more of these mutations e.g., Asn-434 mutation
• follistatin a follistatin
• polypeptide may be placed C-terminal to a heterologous moiety /domain, or, alternatively, a heterologous moiety/domain may be placed C-terminal to a follistatin polypeptide.
• the follistatin polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains.
• the follistatin polypeptide is conjugated directly to the heterologous moiety/domain.
• the follistatin polypeptide is conjugated to the heterologous moiety/domain by means of a linker.
• the linker is a glycine, threonine and/or serine rich linker. Other near neutral amino acids, such as, but not limited to, Asn, Pro and Ala, may also be used in the linker sequence.
• the linker comprises various permutations of amino acid sequences containing Gly and Thr.
• the linker comprises various permutations of amino acid sequences containing Gly and Ser.
• the linker has a length of at least 3, 4, 5, 7, 10, 12, 15, 20, 21, 25, 30, 35, 40, 45 or 50 amino acids.
• the linker comprises GlyGlyGly (GGG), or repetitions thereof.
• the linker comprises the amino acid sequence of Thr Gly Gly Gly (TGGG) (SEQ ID NO: 128) or repetitions thereof. In some embodiments, the linker is 1-5, 1-10 or 1-15 amino acids in length. In some embodiments, the linker consists of
• the linker is greater than 10 amino acids in length. In some embodiments, the linker comprises between 10-100, 10- 90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 10-15 amino acids. In some
• the linker comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 amino acids. In some embodiments, the linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to GAPGGGGGAAAAAGGGGGGAP (SEQ ID NO: 129) or fragments thereof. In some embodiments, the linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to
• the linker comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to
• the linker comprises the amino acid sequence of ALEVLFQGP (SEQ ID NO: 132). In some embodiments, the linker does not consist of or comprise the amino acid sequence of any one of SEQ ID NOs: 129-132.
• an immunoglobulin Fc domain or simply“Fc” is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof.
• an immunoglobulin Fc region may comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region.
• the immunoglobulin Fc region comprises at least an immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably lacks the CH1 domain. It is also understood that a follistatin polypeptide may comprise only a domain of an immunoglobulin, such as a CH1 domain, a CH2 domain or a CH3 domain. Many of these domains confer desirable pharmacokinetic properties as well as dimerization or higher order multimerization.
• the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igy) (g subclasses 1, 2, 3, or 4).
• IgG immunoglobulin
• IgE (Igs) and IgM (Igp) may be used.
• the choice of appropriate immunoglobulin heavy chain constant region is discussed in detail in U.S. Pat. Nos. 5,541,087 and 5,726,044.
• the choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a particular result is considered to be within the level of skill in the art.
• IgG immunoglobulin glycos 1, 2, 3, or 4
• IgE (Igs) and IgM (Igp) may be used.
• the constant domain of an IgG immunoglobulin has reduced or no substantial ADCC and/or CDC activity relative to native human IgGl.
• the portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of a hinge domain, and preferably at least a portion of a CEE domain of Fc gamma or the homologous domains in any of IgA, IgD, IgE, or IgM.
• substitution or deletion of amino acids within the immunoglobulin heavy chain constant regions may be useful in the practice of the methods and compositions disclosed herein.
• One example would be to introduce amino acid substitutions in the upper CH2 region to create an Fc variant with reduced affinity for Fc receptors (Cole et al. (1997) J. Immunol. 159:3613).
• the C- terminal lysine, or K will be removed and thus any of the polypeptides described herein may omit the C-terminal K that is found in an Fc domain, such as those shown in SEQ ID NO:
• the final (carboxy -terminal) lysine, or K, of the follistatin polypeptide is absent.
• the protein may comprise an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
• the follistatin polypeptides of the present disclosure contain one or more modifications that are capable of stabilizing the follistatin polypeptides.
• modifications enhance the in vitro half-life of the follistatin polypeptides, enhance circulatory half-life of the follistatin polypeptides or reducing proteolytic degradation of the follistatin polypeptides.
• Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising a follistatin polypeptide and a stabilizer domain), modifications of a glycosylation site (including, for example, addition of a glycosylation site to a follistatin polypeptide), and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from a follistatin polypeptide).
• fusion proteins including, for example, fusion proteins comprising a follistatin polypeptide and a stabilizer domain
• modifications of a glycosylation site including, for example, addition of a glycosylation site to a follistatin polypeptide
• modifications of carbohydrate moiety including, for example, removal of carbohydrate moieties from a follistatin polypeptide.
• a follistatin polypeptide is fused to a stabilizer domain such as an IgG molecule (e.g., an Fc domain).
• stabilizer domain not only refers to a fusion domain (e.g., Fc) as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproteinaceous polymer, such as polyethylene glycol.
• a representative follistatin-Fc fusion protein is FST(288)-IgG2 fusion has the unprocessed and mature amino acid sequences shown below.
• the initial“GN” sequence may be removed, yielding the following polypeptide. (SEQ ID NO: 138)
• a further representative follistatin-Fc fusion protein is FST(3l5)-IgG2 fusion, which has the unprocessed and mature amino acid sequences shown below.
• the initial“GN” sequence may be removed, yielding the following polypeptide. (SEQ ID NO: 142)
• a further representative follistatin-Fc fusion is the FST(29l)-IgGl fusion, which has the unprocessed and mature amino acid sequences shown below.
• the initial“GN” sequence may be removed, yielding the following polypeptide. (SEQ ID NO: 145)
• the FST(29l)-IgG2 fusion has the unprocessed and mature amino acid sequences shown below.
• the initial“GN” sequence may be removed, yielding the following polypeptide. (SEQ ID NO: 148)
• the present invention makes available isolated and/or purified forms of the follistatin polypeptides, which are isolated from, or otherwise substantially free of, other proteins.
• follistatin polypeptides (unmodified or modified) of the disclosure can be produced by a variety of art-known techniques.
• follistatin polypeptides can be synthesized using standard protein chemistry techniques such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992).
• automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600).
• the follistatin polypeptides, fragments or variants thereof may be recombinantly produced using various expression systems (e.g., E. coli, Chinese Hamster Ovary cells, COS cells, baculovirus) as is well known in the art (also see below).
• the modified or unmodified follistatin polypeptides may be produced by digestion of naturally occurring or recombinantly produced full-length follistatin polypeptides by using, for example, a protease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE).
• a protease e.g., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE).
• follistatin polypeptides may be produced from naturally occurring or recombinantly produced full-length follistatin polypeptides such as standard techniques known in the art, such as by chemical cleavage (e.g., cyanogen bromide, hydroxylamine).
• any of the follistatin or follistatin-like polypeptides disclosed herein is conjugated to any of the TbMI polypeptides disclosed herein.
• the follistatin or follistatin-like polypeptide is directly fused to the TbIHI polypeptide.
• the follistatin or follistatin-like polypeptide is fused to the TbMI polypeptide by means of a linker.
• the multi-specific binder is laid out in a format similar to that shown in Figure 15A.
• the multi- specific binder comprises a follistatin amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 111.
• the multi-specific binder comprises a heterologous domain.
• the heterologous domain is an Fc domain.
• the multispecific binder comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 163.
• the multispecific binder comprises a linker between the follistatin polypeptide portion and the Fc portion.
• the linker comprises the amino acid sequence of GGG or the amino acid sequence of SEQ ID NO: 3.
• the multi-specific binder comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 164.
• the multispecific binder comprises a TbINI polypeptide portion that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 170.
• the TbMI polypeptide portion is fused to the follistatin polypeptide portion or the Fc portion by means of a linker.
• the TbMI polypeptide portion is fused to the C- terminus of the Fc portion (e.g., the C-terminus of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 164) by means of a linker.
• the linker used to fuse the follistatin polypeptide portion or Fc portion to the TbMI polypeptide portion comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 165.
• a representative nucleotide encoding a portion of a multispecific binder may comprise a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 166.

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