WO2018067874A1 - Variant actriib proteins and uses thereof - Google Patents

Variant actriib proteins and uses thereof Download PDF

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Publication number
WO2018067874A1
WO2018067874A1 PCT/US2017/055421 US2017055421W WO2018067874A1 WO 2018067874 A1 WO2018067874 A1 WO 2018067874A1 US 2017055421 W US2017055421 W US 2017055421W WO 2018067874 A1 WO2018067874 A1 WO 2018067874A1
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WIPO (PCT)
Prior art keywords
amino acid
seq
polypeptide
heteromultimer
actriib
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Ceased
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PCT/US2017/055421
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English (en)
French (fr)
Inventor
Ravindra Kumar
Asya Grinberg
Erik M. VOGAN
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Acceleron Pharma Inc
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Acceleron Pharma Inc
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Filing date
Publication date
Priority to JP2019518302A priority Critical patent/JP7280182B2/ja
Priority to CA3039525A priority patent/CA3039525A1/en
Priority to KR1020197012902A priority patent/KR102595559B1/ko
Priority to CN201780074935.6A priority patent/CN110036025B/zh
Priority to AU2017338916A priority patent/AU2017338916B2/en
Priority to EP17859219.2A priority patent/EP3523328A4/en
Priority to BR112019006918A priority patent/BR112019006918A2/pt
Priority to KR1020237036672A priority patent/KR102761094B1/ko
Priority to US16/340,048 priority patent/US11267865B2/en
Application filed by Acceleron Pharma Inc filed Critical Acceleron Pharma Inc
Publication of WO2018067874A1 publication Critical patent/WO2018067874A1/en
Anticipated expiration legal-status Critical
Priority to US17/687,934 priority patent/US12240887B2/en
Priority to AU2022201698A priority patent/AU2022201698A1/en
Priority to JP2023006458A priority patent/JP7617962B2/ja
Priority to AU2024200684A priority patent/AU2024200684A1/en
Ceased legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • TGF-beta The transforming growth factor-beta (TGF-beta) 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,
  • the family is divided into two general branches: the BMP/GDF and the TGF-beta/Activin/BMPlO branches, whose members have diverse, often complementary effects.
  • BMP/GDF the TGF-beta/Activin/BMPlO branches
  • TGF-beta/Activin/BMPlO branches whose members have diverse, often complementary effects.
  • the present disclosure provides variant ActRIIB polypeptides, particularly variant ActRIIB homomultimer proteins and variant ActRIIB heteromultimer proteins.
  • variant ActRIIB polypeptides have been identified that display altered binding affinity for one or more ActRIIB -binding ligands.
  • ActRIIB variants that decrease and increase ligand-binding activities were identified. Such variants may be particularly useful for increasing or decreasing ligand selectively compared to a corresponding unmodified ActRIIB polypeptide in a variety of applications.
  • variant ActRIIB polypeptides have various in vivo effects including, for example, the ability to increase body mass (e.g., muscle mass) as well as increasing red blood cell and hemoglobin levels. Therefore, variant ActRIIB polypeptides should be useful in a variety of therapeutic applications including, for example, those described herein.
  • the disclosure relate to variant ActRIIB polypeptides comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%), 99%), or 100% identical to an amino acid sequence that begins at any one of amino acid 20-29 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 2 and ends at any one of amino acid 109-134 (e.g., 109, 110, 11 1, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 2, and wherein the polypeptide comprises one or more amino acid substitutions at a position of SEQ ID NO: 2 selected from the group consisting of: K55, F82, L79, A24, K
  • the ActRIIB polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 of SEQ ID NO: 2. In some embodiments, the ActRIIB polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 25-131 of SEQ ID NO: 2.
  • the ActRIIB polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 20-134 of SEQ ID NO: 2. In some embodiments, the ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 53. In some
  • the ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 12.
  • the ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 5.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to K55 of SEQ ID NO: 2.
  • the substitution is K55A. In some embodiments, the substitution is K55E. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to L79 of SEQ ID NO: 2. For example, in some embodiments, the substitution is L79D. In some embodiments, the substitution is L79E. In some embodiments, the substitution is L79P. In some embodiments, the substitution is L79A. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to F82 of SEQ ID NO: 2. For example, in some embodiments, the substitution is F82I. In some embodiments, the substitution is F82K. In some embodiments, the substitution is F82A.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to A24 of SEQ ID NO: 2.
  • the substitution is A24N.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to K74 of SEQ ID NO: 2.
  • the substitution is K74A.
  • the substitution is K74A.
  • the substitution is K74F. In some embodiments, the substitution is K74A. In some embodiments, the substitution is K74I. In some embodiments, the substitution is K74A. In some embodiments, the substitution is K74Y.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to D80 of SEQ ID NO: 2. For example, in some embodiments, the substitution is D80A. In some embodiments, the substitution is D80F. In some embodiments, the substitution is D80K. In some embodiments, the substitution is D80G. In some embodiments, the substitution is D80M. In some embodiments, the substitution is D80I. In some embodiments,
  • the substitution is D80N. In some embodiments, the substitution is D80R. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to R64 of SEQ ID NO: 2. For example, in some embodiments, the substitution is R64K. In some embodiments, the substitution is R64N. In some
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to P129 of SEQ ID NO: 2.
  • the substitution is P129S.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to P130 of SEQ ID NO: 2.
  • the substitution is P130A.
  • the substitution is P130R.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to E37 of SEQ ID NO: 2.
  • the substitution is E37A.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to R40 of SEQ ID NO: 2.
  • the substitution is R40A.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to D54 of SEQ ID NO: 2.
  • the substitution is D54A.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to R56 of SEQ ID NO: 2.
  • the substitution is R56A.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to W78 of SEQ ID NO: 2.
  • the substitution is W78A.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 31.
  • the variant ActRIIB polypeptide comprises an alanine at the position corresponding to K55 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 33.
  • the variant ActRIIB polypeptide comprises an alanine at the position corresponding to K55 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 34.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to K55 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 36.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to K55 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 37.
  • the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 39.
  • the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 40.
  • the variant ActRIIB polypeptide comprises a lysine at the position
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 42.
  • the variant ActRIIB polypeptide comprises a lysine at the position
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 43.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 45.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO : 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 46.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 48.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 49.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 50.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 51.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO: 2.
  • the disclosure relates to a variant ActRIIB polypeptide comprises an amino acid sequence that is at least 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: 52.
  • the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO: 2.
  • variant ActRIIB polypeptide of the disclosure form homodimers. In some embodiments, variant ActRIIB polypeptides may from heterodimers through covalent interactions. In some embodiments, variant ActRIIB polypeptides may from heterodimers through non-covalent interactions. In some embodiments, variant ActRIIB polypeptides may from heterodimers through both covalent and non-covalent interactions.
  • a variant ActRIIB polypeptide binds to one or more TGF-beta superfamily ligands.
  • variant ActRIIB polypeptide, including homomultimers thereof binds to one or more TGF-beta superfamily ligands with a K D of at least 1 x 10 "7 M.
  • the one or more TGF-beta superfamily ligands is selected from the group consisting of: BMP6, BMP7, BMP9, BMP10, GDF3, GDF7, GDF8, GDFl l, GDF15, activin A, activin B, activin C, activin E, activin AB, activin AC, activin AE, activin BC, and activin BE.
  • a variant ActRIIB polypeptide including homomultimers thereof (e.g., homodimers), inhibits one or more TGF-beta super family ligands.
  • homomultimers thereof e.g., homodimers
  • variant ActRIIB polypeptide including homomultimers thereof, inhibits signaling of one or more TGF-beta super family ligands. In some embodiments, variant ActRIIB polypeptide, including homomultimers thereof, inhibits Smad signaling of one or more TGF-beta super family ligands. In some embodiments, variant ActRIIB polypeptide, including homomultimers thereof, inhibits signaling of one or more TGF-beta super family ligands in a cell-based assay.
  • variant ActRIIB polypeptide inhibits one or more TGF-beta super family ligands selected from the group consisting of: BMP6, BMP7, BMP9, BMP10, GDF3, GDF7, GDF8, GDFl l, GDF15, activin A, activin B, activin C, activin E, activin AB, activin AC, activin AE, activin BC, and activin BE.
  • TGF-beta super family ligands selected from the group consisting of: BMP6, BMP7, BMP9, BMP10, GDF3, GDF7, GDF8, GDFl l, GDF15, activin A, activin B, activin C, activin E, activin AB, activin AC, activin AE, activin BC, and activin BE.
  • a heteromultimer protein of the disclosure comprises a first ActRIIB polypeptide and a second ActRIIB polypeptide, wherein the first ActRIIB polypeptide comprises an amino acid sequence that is at least 75%, 80%>, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence that begins at any one of amino acid 20-29 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 2 and ends at any one of amino acid 109-134 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
  • amino acid 109-134 e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123
  • the first polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to amino acids 29-109 of SEQ ID NO: 2.
  • the second polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 of SEQ ID NO: 2.
  • the first polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 25-131 of SEQ ID NO: 2.
  • the second polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 25-131 of SEQ ID NO: 2.
  • the first polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 20-134 of SEQ ID NO: 2.
  • the second polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 25-131 of SEQ ID NO: 2.
  • the first polypeptide comprises an amino acid sequence that is at least 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: 53.
  • the second polypeptide comprises an amino acid sequence that is at least 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: 53.
  • the first polypeptide comprises an amino acid sequence that is at least 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: 5.
  • the second polypeptide comprises an amino acid sequence that is at least 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: 5.
  • the first polypeptide comprises an amino acid sequence that is at least 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: 12.
  • the second polypeptide comprises an amino acid sequence that is at least 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: 12.
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to K55 of SEQ ID NO: 2.
  • the substitution is K55A.
  • the substitution is K55E.
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to L79 of SEQ ID NO: 2.
  • the substitution is L79D.
  • the substitution is L79E.
  • the substitution is L79P.
  • the substitution is L79A.
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to F82 of SEQ ID NO: 2.
  • the substitution is F82I.
  • the substitution is F82K.
  • the substitution is F82A.
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to A24 of SEQ ID NO: 2.
  • the substitution is A24N.
  • the polypeptide comprises an amino acid substitution at the amino acid position corresponding to K74 of SEQ ID NO: 2.
  • the substitution is K74A.
  • the substitution is K74A.
  • the substitution is K74F.
  • the substitution is K74A.
  • the substitution is K74I.
  • the substitution is K74A.
  • the substitution is K74Y.
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to D80 of SEQ ID NO: 2.
  • the substitution is D80A.
  • the substitution is D80F.
  • the substitution is D80K.
  • the substitution is D80G. In some embodiments, the substitution is D80M. In some embodiments, the substitution is D80I. In some embodiments, the substitution is D80N. In some embodiments, the substitution is D80R. In some embodiments, the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to R64 of SEQ ID NO: 2. For example, in some embodiments, the substitution is R64K. In some embodiments, the substitution is R64N. In some embodiments, the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to P129 of SEQ ID NO: 2. For example, in some embodiments, the substitution is P129S. In some embodiments, the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to P130 of SEQ ID NO: 2. For example, in some embodiments, the substitution is P130A. In some embodiments, the substitution is P130R. In some
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to E37 of SEQ ID NO: 2.
  • the substitution is E37A.
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to R40 of SEQ ID NO: 2.
  • the substitution is R40A.
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to D54 of SEQ ID NO: 2.
  • the substitution is D54A.
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to R56 of SEQ ID NO: 2.
  • the substitution is R56A.
  • the first polypeptide comprises an amino acid substitution at the amino acid position corresponding to W78 of SEQ ID NO: 2.
  • the substitution is W78A.
  • the second ActRIIB polypeptide comprises one or more amino acid substitutions at a position of SEQ ID NO: 2 selected from the group consisting of: A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82.
  • the second ActRIIB polypeptide comprises one or more amino acid substitution with respect to the amino acid sequence of SEQ ID NO: 2 selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, K55A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modifications that promote heteromultimer formation. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modifications that inhibit heteromultimer formation. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modifications that promote heteromultimer formation and one or more amino acid modifications that inhibit
  • heteromultimers of the disclosure are heterodimers.
  • an ActRIIB polypeptides of the disclosure is fusion protein comprising an ActRIIB polypeptide domain and one or more heterologous domains.
  • an ActRIIB polypeptide is an
  • an ActRIIB-Fc fusion protein further comprises a linker domain positioned between the ActRIIB polypeptide domain and the one or more heterologous domains or Fc domain.
  • the linker domain is selected from: TGGG, TGGGG, SGGGG, GGGGS, GGG, GGGG, SGGG, and GGGGS.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 13, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 13.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 75%,
  • the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 14.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 15, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 15.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 16, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 16.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 17, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 17.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 75%,
  • the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 19.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 19, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 20, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 21.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 21, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 20.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 22, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 23.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 23, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 22.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 22, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 25.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 75%,
  • the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 24.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 26, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 27.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 27, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 26.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 28, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 29.
  • the first ActRIIB-Fc fusion protein Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217.
  • the second ActRIIB-Fc fusion protein Fc domain comprises a cysteine at amino acid position
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the second ActRIIB-Fc fusion protein, wherein the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 28, and the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 29.
  • the second ActRIIB-Fc fusion protein Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217.
  • the first ActRIIB-Fc fusion protein Fc domain comprises a cysteine at amino acid position 127, a serine at amino acid position 144, an alanine at position 146 an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the second ActRIIB-Fc fusion protein, wherein the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 30, and the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 23.
  • the first ActRIIB-Fc fusion protein Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435.
  • the second ActRIIB-Fc fusion protein Fc domain comprises cysteine at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.
  • the disclosure relates to ActRIIB heteromultimer proteins comprising a first ActRIIB-Fc fusion protein and a second ActRIIB-Fc fusion protein wherein the second ActRIIB-Fc fusion protein, wherein the second ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 30, and the first ActRIIB-Fc fusion protein comprises an Fc domain that is at least 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: 23.
  • the second ActRIIB-Fc fusion protein Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435.
  • the first ActRIIB-Fc fusion protein Fc domain comprises cysteine at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.
  • the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide that is at least 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: 33, and second ActRIIB polypeptide that is at least 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: 5, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide.
  • the first ActRIIB polypeptide comprises a alanine at the amino acid position corresponding to 55 of SEQ ID NO: 2.
  • the second ActRIIB polypeptide does not comprise a alanine at the amino acid position corresponding to 55 of SEQ ID NO: 2.
  • the second ActRIIB polypeptide comprises a lysine at the amino acid position corresponding to 55 of SEQ ID NO: 2.
  • the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.
  • the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that promote heteromultimer formation.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that inhibit heteromultimer formation.
  • the heteromultimer is a heterodimer.
  • the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide that is at least 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: 36, and second ActRIIB polypeptide that is at least 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: 5, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide.
  • the first ActRIIB polypeptide comprises a glutamic acid at the amino acid position corresponding to 55 of SEQ ID NO: 2.
  • the second ActRIIB polypeptide does not comprise a glutamic acid at the amino acid position corresponding to 55 of SEQ ID NO: 2.
  • the second ActRIIB polypeptide comprises a lysine at the amino acid position corresponding to 55 of SEQ ID NO: 2.
  • the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.
  • the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that promote heteromultimer formation.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that inhibit heteromultimer formation.
  • the heteromultimer is a heterodimer.
  • the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide that is at least 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: 39, and second ActRIIB polypeptide that is at least 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: 5, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide.
  • the first ActRIIB polypeptide comprises a isoleucine at the amino acid position corresponding to 82 of SEQ ID NO: 2.
  • the second ActRIIB polypeptide does not comprise a isoleucine acid at the amino acid position corresponding to 82 of SEQ ID NO: 2.
  • the second ActRIIB polypeptide comprises a phenylalanine at the amino acid position
  • the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, and D80 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, and D80R.
  • the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, and D80 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, and D80R.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that promote heteromultimer formation.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that inhibit heteromultimer formation.
  • the heteromultimer is a heterodimer.
  • the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide that is at least 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: 42, and second ActRIIB polypeptide that is at least 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: 5, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second
  • first ActRIIB polypeptide comprises a lysine at the amino acid position corresponding to 82 of SEQ ID NO: 2.
  • second ActRIIB polypeptide does not comprise a lysine acid at the amino acid position corresponding to 82 of SEQ ID NO: 2.
  • the second ActRIIB polypeptide comprises a phenylalanine at the amino acid position corresponding to 82 of
  • the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, and D80 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, and D80R.
  • the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, and D80 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, and D80R.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that promote heteromultimer formation.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that inhibit heteromultimer formation.
  • the heteromultimer is a heterodimer.
  • the disclosure relates to a heteromultimercomprising a first ActRIIB polypeptide that is at least 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: 45, and second ActRIIB polypeptide that is at least 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: 48, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide.
  • the first ActRIIB polypeptide comprises an acidic amino acid position corresponding to 79 of SEQ ID NO: 2.
  • the acidic amino acid is an aspartic acid.
  • the acidic amino acid is a glutamic acid.
  • the second ActRIIB polypeptide does not comprise an acidic acid (e.g., aspartic acid or glutamic acid) at the amino acid position corresponding to 79 of SEQ ID NO: 2.
  • the second ActRIIB polypeptide comprises a leucine at the amino acid position corresponding to 79 of SEQ ID NO: 2.
  • the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.
  • the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that promote heteromultimer formation.
  • the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that inhibit heteromultimer formation.
  • the heteromultimer is a heterodimer.
  • the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide that is at least 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: 50, and second ActRIIB polypeptide that is at least 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: 52, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide.
  • the first ActRIIB polypeptide comprises an acidic amino acid position corresponding to 79 of SEQ ID NO: 2.
  • the acidic amino acid is an aspartic acid.
  • the acidic amino acid is a glutamic acid.
  • the second ActRIIB polypeptide does not comprise an acidic acid (e.g., aspartic acid or glutamic acid) at the amino acid position corresponding to 79 of SEQ ID NO: 2.
  • the second ActRIIB polypeptide comprises a leucine at the amino acid position corresponding to 79 of SEQ ID NO: 2.
  • the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.
  • the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2.
  • the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.
  • the disclosure provides for a heteromultimer protein comprising any of the ActRIIB polypeptides disclosed herein and a second polypeptide selected from the group consisting of: ALKl, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, ActRIIA,
  • the second polypeptide is an ALKl polypeptide or a functional fragment thereof.
  • the ALKl polypeptide or functional fragment thereof comprises 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 SEQ ID NO: 54, or functional fragments thereof.
  • the ALK1 polypeptide or functional fragment thereof comprises 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 amino acid sequence of any one of SEQ ID Nos: 54, 55, 56, 57, 60, and 61, or functional fragments thereof.
  • the second polypeptide is an ALK2 polypeptide or a functional fragment thereof.
  • the ALK2 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64 or 65, or functional fragments thereof. In some embodiments, the ALK2 polypeptide or functional fragment thereof comprises 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 amino acid sequence of SEQ ID Nos: 64, 65, 66, 67, 70, and 71, or functional fragments thereof.
  • the second polypeptide is an ALK3 polypeptide or a functional fragment thereof.
  • the ALK3 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74, or functional fragments thereof.
  • the ALK3 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74, 75, 76, 77, 80, or 81, or functional fragments thereof.
  • the second polypeptide is an ALK4 polypeptide or a functional fragment thereof.
  • the ALK4 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 84 or 85, or functional fragments thereof.
  • the ALK4 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 84, 86, 85, 87, 88, 89, 92, and 93, or functional fragments thereof.
  • the second polypeptide is an ALK5 polypeptide or a functional fragment thereof.
  • the ALK5 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96 or 97, or functional fragments thereof. In some embodiments, the ALK5 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96, 98, 97, 99, 100, 101, 104, and 105, or functional fragments thereof.
  • the second polypeptide is an ALK6 polypeptide or a functional fragment thereof.
  • the ALK6 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 108 or 110, or functional fragments thereof.
  • the ALK6 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 108, 109, 110, 111, 112, 113, 116, and 117, or functional fragments thereof.
  • the second polypeptide is an ALK7 polypeptide or a functional fragment thereof.
  • the ALK7 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120, 121, or 122, or functional fragments thereof. In some embodiments, the ALK7 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120, 123, 124, 125, 121, 126, 122, 127, 128, 129, 130, 133, and 134, or functional fragments thereof.
  • the second polypeptide is an ActRIIA polypeptide or a functional fragment thereof.
  • the ActRIIA polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137, or functional fragments thereof.
  • the ActRIIA polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137, 138, 139, 140, 141, 144, and 145, or functional fragments thereof.
  • the second polypeptide is an TGFBRII polypeptide or a functional fragment thereof.
  • the TGFBRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 204, or functional fragments thereof.
  • the TGFBRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 161, 162, 160, 163, 164, 165, 166, 167, 172, 173, 174, and 175, or functional fragments thereof.
  • the second polypeptide is an BMPRII polypeptide or a functional fragment thereof.
  • the BMPRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 148 or 149, or functional fragments thereof. In some embodiments, the BMPRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 148, 150, 149, 151, 152, 153, 156, and 157, or functional fragments thereof.
  • the second polypeptide is an MISRII polypeptide or a functional fragment thereof.
  • the MISRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 180, 181, or 182, or functional fragments thereof.
  • the MISRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 180, 183, 181, 184, 182, and 185, or functional fragments thereof.
  • heteromultimers of the disclosure bind to one or more TGF-beta superfamily ligands. In some embodiments, heteromultimers of the disclosure binds to one or more TGF-beta superfamily ligands with a K D of at least 1 x 10 "7 M. In some
  • the one or more TGF-beta superfamily ligands is selected from the group consisting of: BMP6, BMP7, BMP9, BMP10, GDF3, GDF7, GDF8, GDF11, GDF15, activin A, activin B, activin C, activin E, activin AB, activin AC, activin AE, activin BC, and activin BE.
  • heteromultimers of the disclosure inhibits one or more TGF-beta super family ligands. In some embodiments, heteromultimers of the disclosure inhibits signaling of one or more TGF-beta super family ligands. In some embodiments,
  • heteromultimers of the disclosure inhibits Smad signaling of one or more TGF-beta super family ligands. In some embodiments, heteromultimers of the disclosure inhibits signaling of one or more TGF-beta super family ligands in a cell-based assay. In some embodiments, heteromultimers of the disclosure inhibits one or more TGF-beta super family ligands selected from the group consisting of: BMP6, BMP7, BMP9, BMP10, GDF3, GDF7, GDF8, GDF11, GDF15, activin A, activin B, activin C, activin E, activin AB, activin AC, activin AE, activin BC, and activin BE.
  • the disclosure relates to ActRIIB polypeptides, including variant ActRIIB polypeptides as well as homomultimer and heteromultimers comprising the same, that comprises one or more amino acid modifications selected from the group consisting of: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, and an amino acid conjugated to a lipid moiety.
  • ActRIIB polypeptides of the disclosure are glycosylated and has a glycosylation pattern obtainable from of the polypeptide in a CHO cell.
  • the disclosure provides for a heteromultimer protein comprising any of the ActRIIB polypeptides disclosued herein and a second polypeptide selected from the group consisting of: ALKl, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, ActRIIA, TGFBRII, BMPRII, and MISRII polypeptide, or functional fragments thereof.
  • the second polypeptide is an ALKl polypeptide or a functional fragment thereof.
  • the ALKl polypeptide or functional fragment thereof comprises 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 SEQ ID NO: 54, or functional fragments thereof.
  • the ALKl polypeptide or functional fragment thereof comprises 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 amino acid sequence of any one of SEQ ID Nos: 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63, or functional fragments thereof.
  • the second polypeptide is an ALK2 polypeptide or a functional fragment thereof.
  • the ALK2 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64 or 65, or functional fragments thereof.
  • the ALK2 polypeptide or functional fragment thereof comprises 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 amino acid sequence of SEQ ID Nos: 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73, or functional fragments thereof.
  • the second polypeptide is an ALK3 polypeptide or a functional fragment thereof.
  • the ALK3 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74, or functional fragments thereof. In some embodiments, the ALK3 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74, 75, 76, 77, 80, or 81, or functional fragments thereof. In some embodiments, the second polypeptide is an ALK4 polypeptide or a functional fragment thereof.
  • the ALK4 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 84 or 85, or functional fragments thereof. In some embodiments, the ALK4 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 84, 86, 85, 87, 88, 89, 90, 91, 92, 93, 94, and 95, or functional fragments thereof.
  • the second polypeptide is an ALK5 polypeptide or a functional fragment thereof.
  • the ALK5 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96 or 97, or functional fragments thereof.
  • the ALK5 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96, 98, 97, 99, 100, 101, 102, 103, 104, 105, 106, and 107, or functional fragments thereof.
  • the second polypeptide is an ALK6 polypeptide or a functional fragment thereof.
  • the ALK6 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 108 or 110, or functional fragments thereof. In some embodiments, the ALK6 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 108, 109, 1 10, 111, 112, 113, 114, 115, 116, 117, 118, and 119, or functional fragments thereof.
  • the second polypeptide is an ALK7 polypeptide or a functional fragment thereof.
  • the ALK7 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120, 121, or 122, or functional fragments thereof.
  • the ALK7 polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120, 123, 124, 125, 121, 126, 122, 127, 128, 129, 130, 131, 132, 133, 134, 135, and 136, or functional fragments thereof.
  • the second polypeptide is an ActRIIA polypeptide or a functional fragment thereof.
  • the ActRIIA polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137, or functional fragments thereof. In some embodiments, the ActRIIA polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147, or functional fragments thereof. In some embodiments, the second polypeptide is an TGFBRII polypeptide or a functional fragment thereof. In some embodiments,
  • the TGFBRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 204, or functional fragments thereof.
  • the TGFBRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 161, 162, 160, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, and 179, or functional fragments thereof.
  • the second polypeptide is an BMPRII polypeptide or a functional fragment thereof.
  • the BMPRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 148 or 149, or functional fragments thereof. In some embodiments, the BMPRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 148, 150, 149, 151, 152, 153, 154, 155, 156, 157, 158, and 159, or functional fragments thereof.
  • the second polypeptide is an MISRII polypeptide or a functional fragment thereof.
  • the MISRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 180, 181, or 182, or functional fragments thereof.
  • the MISRII polypeptide or functional fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 180, 183, 181, 184, 182, 185, 186, 187, 188, 189, 190, 191, 192, and 193, or functional fragments thereof.
  • the disclosure relates to pharmaceutical preparations comprising a ActRIIB polypeptide, including variant ActRIIB polypeptides as well as homomultimer and heteromultimers comprising the same, a pharmaceutically acceptable carrier.
  • pharmaceutical preparations comprising one or more ActRIIB heteromulitmers comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% horn reminder timers.
  • the disclosure relates to isolated and/or recombinant nucleic acids comprising a coding sequence for one or more of the ActRIIB polypeptide(s) as described herein.
  • the disclosure relates to an isolated and/or recombinant nucleic acid that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%), 97%), 98%), 99%), or 100% identical to the nucleic acid sequence corresponding to any one of SEQ ID Nos: 3, 10, 31, 35, 38, 41, 44, or 47.
  • an isolated and/or recombinant polynucleotide sequence of the disclosure comprises a promoter sequence operably linked to a coding sequence described herein (e.g., a nucleic acid that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence corresponding to any one of SEQ ID Nos: 3, 10, 31, 35, 38, 41, 44, or 47).
  • the disclosure relates to vectors comprising an isolated and/or recombinant nucleic acid described herein (e.g., a nucleic acid that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence corresponding to any one of SEQ ID Nos: 3, 10, 31, 35, 38, 41, 44, or 47).
  • an isolated and/or recombinant nucleic acid described herein e.g., a nucleic acid that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence corresponding to any one of SEQ ID Nos: 3, 10, 31, 35, 38, 41, 44, or 47).
  • the disclosure relates to a cell comprising an isolated and/or recombinant polynucleotide sequence described herein (e.g., a nucleic acid that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence corresponding to any one of SEQ ID Nos: 3, 10, 31, 35, 38, 41, 44, or 47).
  • the cell is a CHO cell.
  • the cell is a COS cell.
  • the disclosure relates to methods of making ActRIIB polypeptides, including variant ActRIIB polypeptides as well as homomultimer and heteromultimers comprising the same, as described herein.
  • a method may include expressing any of the nucleic acids) disclosed herein in a suitable cell (e.g., a CHO cell or COS cell).
  • a suitable cell e.g., a CHO cell or COS cell.
  • Such a method may comprise: a) culturing a cell under conditions suitable for expression of the soluble ActRIIB polypeptide, wherein said cell comprise with an ActRIIB polypeptide expression construct.
  • the method further comprises recovering the expressed ActRIIB polypeptide.
  • ActRIIB polypeptides may be recovered as crude, partially purified or highly purified fractions using any of the well-known techniques for obtaining protein from cell cultures.
  • the disclosure relates to methods for increasing red blood cell levels or hemoglobin levels in a patient, comprising administering a patient in need thereof an ActRIIB polypeptide, including variant ActRIIB polypeptides as well as homomultimer and heteromultimers comprising the same, as described herein.
  • the disclosure relates to methods for treating anemia or a disorder associated with anemia (e.g., those described herein) in a patient, comprising administering a patient in need thereof an ActRIIB polypeptide, including variant ActRIIB polypeptides as well as homomultimer and heteromultimers comprising the same, as described herein.
  • the disclosure relates to methods for increasing muscle mass and/or muscle strength in a patient, comprising administering a patient in need thereof an ActRIIB polypeptide, including variant ActRIIB polypeptides as well as homomultimer and heteromultimers comprising the same, as described herein.
  • the disclosure relate to methods for treating a muscle-related disorder in a patient, comprising administering a patient in need thereof an ActRIIB, including variant ActRIIB polypeptides as well as homomultimer and heteromultimers comprising the same, as described herein.
  • the disorder is associated with undesirably low muscle growth and/or muscle weakness.
  • Such disorders include muscle atrophy, muscular dystrophy, amyotrophic lateral sclerosis (ALS), and a muscle wasting disorder (e.g., cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophies, neuromuscular diseases, motor neuron diseases, diseases of the neuromuscular junction, and inflammatory myopathies).
  • the disclosure relate to methods for decreasing the body fat content or reducing the rate of increase in body fat content, and for treating a disorder associated with undesirable body weight gain, such as obesity, non-insulin dependent diabetes mellitus (NIDDM), cardiovascular disease, cancer, hypertension, osteoarthritis, stroke, respiratory problems, and gall bladder disease, comprising administering a patient in need thereof an ActRIIB, including variant ActRIIB polypeptides as well as homomultimer and heteromultimers comprising the same, as described herein.
  • NIDDM non-insulin dependent diabetes mellitus
  • Figure 1 shows schematic examples of heteromeric protein complexes comprising a first variant ActRIIB polypeptide (indicated as "X") and either a second variant ActRIIB polypeptide (indicated as "Y") or an unmodified ActRIIB polypeptide (indicated as " Y")
  • the first variant ActRIIB polypeptide is part of a fusion polypeptide that comprises a first member of an interaction pair ("Ci")
  • either a second variant ActRIIB polypeptide or an unmodified ActRIIB polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair ("C 2 ").
  • Suitable interaction pairs include, 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 first variant ActRIIB polypeptide, second variant ActRIIB polypeptide, or unmodified 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 1 A.
  • 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 IB.
  • Figure 2 shows an alignment of extracellular domains of human ActRIIA and human ActRIIB with the residues that are deduced herein to directly contact ligand (indicated by boxes) based on composite analysis of multiple ActRIIB and ActRIIA crystal structures.
  • Figure 3 shows a multiple sequence alignment of various vertebrate ActRIIB precursor proteins without their intracellular domains, human ActRIIA precursor protein without its intracellular domain, and a consensus ActRII precursor protein.
  • Figure 4 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 (SEQ ID NO: 13) Fc to promote asymmetric chain pairing and the corresponding positions with respect to other isotypes IgG4 (SEQ ID NO: 17), IgG2 (SEQ ID NO: 14), and IgG3 (SEQ ID NO: 15).
  • Figure 5 shows the amino acid sequence of a human ActRIIB extracellular domain polypeptide (SEQ ID NO: 1) in which numbering is based on the native human ActRIIB precursor sequence (see SEQ ID NO: 2).
  • Figure 6 shows the amino acid sequence of human ActRIIB precursor protein (SEQ ID NO: 2; NCBI Reference Sequence NP 001097.2). The signal peptide is underlined, the extracellular domain is in bold (also referred to as SEQ ID NO: 1), and the potential N-linked glycosylation sites are boxed.
  • Figure 7 shows a nucleic acid sequence encoding a human ActRIIB(20-134) extracellular domain polypeptide.
  • Figure 8 shows a nucleic acid sequence encoding human ActRIIB precursor protein.
  • SEQ ID NO: 4 consists of nucleotides 25-1560 of NCBI Reference Sequence NM_001106.
  • Figure 9 shows values for ligand binding kinetics of homodimeric Fc-fusion proteins comprising variant or unmodified ActRIIB domains, as determined by surface plasmon resonance at 37°C. Amino acid numbering is based on SEQ ID NO: 2.
  • Figure 10 shows values for ligand binding kinetics of homodimeric Fc-fusion proteins comprising variant or unmodified ActRIIB domains, as determined by surface plasmon resonance at 25°C. Amino acid numbering is based on SEQ ID NO: 2.
  • Figure 11 shows changes in body weight from baseline for wild-type mice treated with vehicle or homodimeric Fc-fusion proteins comprising variant or unmodified ActRIIB domains.
  • Figure 12 shows hemoglobin concentrations in wild-type mice treated with vehicle or homodimeric Fc-fusion proteins comprising variant or unmodified ActRIIB domains.
  • Figure 13 shows red blood cell counts in cynomolgus monkeys treated with ActRIIB-
  • ActRIIB(F82I)-Fc at 9 mg/kg (s.c.) on days 1 and 15.
  • ActRIIB(F82I)-Fc treatment increased RBC counts compared to ActRIIB-Fc (negative control) by an amount similar to that of ActRIIA-Fc (postitive control).
  • the present invention relates to ActRIIB polypeptides.
  • ActRIIB refers to a family of activin receptor type IIB (ActRIIB) proteins and ActRIIB -related proteins, derived from any species.
  • ActRIIB activin receptor type IIB
  • ActRIIB -related proteins derived from any species.
  • Members of the ActRIIB family are generally all transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase specificity.
  • the amino acid sequence of human ActRIIB precursor protein is shown in Figure 6 (SEQ ID NO: 2).
  • ActRIIB polypeptide is used to refer to polypeptides comprising any naturally occurring polypeptide of an ActRIIB family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • ActRIIB polypeptides include polypeptides derived from the sequence of any known ActRIIB having a sequence at least about 80% identical to the sequence of an ActRIIB polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99% or greater identity.
  • the invention relates to soluble ActRIIB polypeptides.
  • soluble ActRIIB polypeptide generally refers to polypeptides comprising an extracellular domain of an ActRIIB protein.
  • soluble ActRIIB polypeptide includes any naturally occurring extracellular domain of an ActRIIB protein as well as any variants thereof (including mutants, fragments and
  • the extracellular domain of an ActRIIB protein binds to a ligand and is generally soluble.
  • soluble ActRIIB polypeptides include an ActRIIB extracellular domain (SEQ ID NO: 1) shown in Figure 5 as well as SEQ ID NO: 53.
  • Other examples of soluble ActRIIB polypeptides comprise a signal sequence in addition to the extracellular domain of an ActRIIB protein (see Example 1).
  • the signal sequence can be a native signal sequence of an ActRIIB, or a signal sequence from another protein, such as a tissue plasminogen activator (TP A) signal sequence or a honey bee melatin signal sequence.
  • TP A tissue plasminogen activator
  • TGF- ⁇ signals are mediated by heteromeric complexes of type I and type II serine/ threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand stimulation (Massague, 2000, Nat. Rev. Mol. Cell Biol. 1 : 169-178).
  • type I and type II receptors are all transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity.
  • Type I receptors are essential for signaling, and type II receptors are required for binding ligands.
  • Type I and type II activin receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors.
  • Two related type II receptors, ActRIIA and ActRIIB have been identified as the type II receptors for activins (Mathews and Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68: 97-108).
  • ActRIIA and ActRIIB can biochemically interact with several other TGF- ⁇ family proteins, including BMP7, Nodal, GDF8, and GDFl 1 (Yamashita et al., 1995, J. Cell Biol.
  • the present invention relates to antagonizing a ligand of
  • ActRIIB receptors also referred to as an ActRIIB ligand
  • a subject ActRIIB polypeptide e.g., a soluble ActRIIB polypeptide
  • compositions and methods of the present invention are useful for treating disorders associated with abnormal activity of one or more ligands of ActRIIB receptors.
  • ligands of ActRIIB receptors include some TGF- ⁇ family members, such as activin, Nodal, GDF8, GDFl 1, and BMP7.
  • Activins are dimeric polypeptide growth factors and belong to the TGF-beta superfamily. There are three activins (A, B, and AB) that are homo/heterodimers of two closely related ⁇ subunits (PAPA, PBPB, and PAPB)- In the TGF-beta superfamily, 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 SocEp Biol Med. 198:500-512; Dyson et al., 1997, Curr Biol.
  • erythroid differentiation factor isolated from the stimulated human monocytic leukemic cells was found to be identical to activin A (Murata et al., 1988, PNAS, 85:2434). It was suggested that activin A acts as a natural regulator of erythropoiesis in the bone marrow. In several tissues, activin signaling is antagonized by its related heterodimer, inhibin. For example, during the release of follicle-stimulating hormone (FSH) from the pituitary, activin promotes FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis.
  • FSH follicle-stimulating hormone
  • follistatin FS
  • FSRP follistatin-related protein
  • Cerberus a 2 -macroglobulin
  • endoglin a 2 -macroglobulin, Cerberus, and endoglin.
  • Nodal proteins have functions in mesoderm and endoderm induction and formation, as well as subsequent organization of axial structures such as heart and stomach in early embryogenesis. It has been demonstrated that dorsal tissue in a developing vertebrate embryo contributes predominantly to the axial structures of the notochord and pre-chordal plate while it recruits surrounding cells to form non-axial embryonic structures. Nodal appears to signal through both type I and type II receptors and intracellular effectors known as Smad proteins.
  • Nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins.
  • Recent results provide direct biochemical evidence that Nodal signaling is mediated by both activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidence has shown that the extracellular cripto protein is required for Nodal signaling, making it distinct from activin or TGF-beta signaling.
  • GDF8 Growth and differentiation factor-8
  • GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highly expressed in the developing and adult skeletal muscle. The GDF8 null mutation in transgenic mice is characterized by a marked hypertrophy and hyperplasia of the 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 (Ashmore et al., 1974, Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994, 38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci.
  • GDF8 can modulate the production of muscle-specific enzymes (e.g., creatine kinase) and modulate myoblast cell proliferation (WO 00/43781).
  • the GDF8 propeptide can be modulate the production of muscle-specific enzymes (e.g., creatine kinase) and modulate myoblast cell proliferation (WO 00/43781).
  • the GDF8 propeptide can be modulate the production of muscle-specific enzymes (e.g., creatine kinase) and modulate myoblast cell proliferation (WO 00/43781).
  • GDFl 1 Growth and differentiation factor-11 (GDFl 1), also known as BMP11, is a secreted protein (McPherron et al., 1999, Nat. Genet. 22: 260-264). GDFl 1 is expressed in the tail bud, limb bud, maxillary and mandibular arches, and dorsal root ganglia during mouse development (Nakashima et al., 1999, Mech. Dev. 80: 185-189). GDFl 1 plays a unique role in patterning both mesodermal and neural tissues (Gamer et al., 1999, Dev Biol., 208:222- 32).
  • GDFl 1 was shown to be a negative regulator of chondrogenesis and myogenesis in developing chick limb (Gamer et al., 2001, Dev Biol. 229:407-20).
  • the expression of GDFl 1 in muscle also suggests its role in regulating muscle growth in a similar way to GDF8.
  • the expression of GDFl 1 in brain suggests that GDFl 1 may also possess activities that relate to the function of the nervous system.
  • GDFl 1 was found to inhibit neurogenesis in the olfactory epithelium (Wu et al., 2003, Neuron. 37: 197-207).
  • GDFl 1 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).
  • diseases such as muscle diseases and neurodegenerative diseases (e.g., amyotrophic lateral sclerosis).
  • Bone morphogenetic protein also called osteogenic protein-1 (OP-1)
  • BMP7 also called osteogenic protein-1 (OP-1)
  • 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 IIB.
  • 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).
  • the present invention relates to the use of certain ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) to antagonize the signaling of ActRIIB ligands generally, in any process associated with ActRIIB activity.
  • ActRIIB polypeptides of the invention may antagonize one or more ligands of ActRIIB receptors, such as activin, Nodal, GDF8, and GDFl 1, and may therefore be useful in the treatment of additional disorders. Therefore, the present invention contemplates using ActRIIB polypeptides in treating or preventing diseases or conditions that are associated with abnormal activity of an ActRIIB or an ActRIIB ligand.
  • ActRIIB or ActRIIB ligands are involved in the regulation of many critical biological processes. Due to their key functions in these processes, they may be desirable targets for therapeutic intervention.
  • ActRIIB polypeptides e.g., soluble ActRIIB polypeptides
  • Such disorders or conditions include, but are not limited to, metabolic disorders such as type 2 diabetes, impaired glucose tolerance, metabolic syndrome (e.g., syndrome X), and insulin resistance induced by trauma (e.g., burns or nitrogen imbalance); adipose tissue disorders (e.g., obesity); muscle and neuromuscular disorders such as muscular dystrophy (including Duchenne muscular dystrophy); amyotrophic lateral sclerosis (ALS); muscle atrophy; organ atrophy; frailty; carpal tunnel syndrome; congestive obstructive pulmonary disease; and sarcopenia, cachexia and other muscle wasting syndromes.
  • metabolic disorders such as type 2 diabetes, impaired glucose tolerance, metabolic syndrome (e.g., syndrome X), and insulin resistance induced by trauma (e.g., burns or nitrogen imbalance); adipose tissue disorders (e.g., obesity); muscle and neuromuscular disorders such as muscular dystrophy (including Duchenne muscular dystrophy); amyotrophic lateral sclerosis (ALS); muscle atrophy;
  • osteoporosis especially in the elderly and/or postmenopausal women; glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis; and osteoporosis-related fractures.
  • glucocorticoid-induced osteoporosis especially in the elderly and/or postmenopausal women
  • osteopenia especially in the elderly and/or postmenopausal women
  • osteopenia especially in the elderly and/or postmenopausal women
  • osteopenia osteoporosis
  • osteoarthritis osteoarthritis
  • osteoporosis-related fractures include low bone mass due to chronic glucocorticoid therapy, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary
  • “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about” and
  • “approximately” may mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • the methods of the invention may include steps of comparing sequences to each other, including an unmodified (wild-type) sequence to one or more mutants (sequence variants).
  • Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
  • sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
  • 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.
  • Alternize 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.
  • ActRIIB Polypeptides In certain aspects, the invention relates to ActRIIB variant polypeptides (e.g., soluble
  • ActRIIB polypeptides the fragments, functional variants, and modified forms have similar or the same biological activities of their corresponding wild-type ActRIIB polypeptides.
  • an ActRIIB variant of the invention may bind to and inhibit function of an ActRIIB ligand (e.g., activin A, activin AB, activin B, Nodal, GDF8, GDFl 1 or BMP7).
  • an ActRIIB polypeptide modulates growth of tissues such as bone, cartilage, muscle or fat.
  • Examples of ActRIIB polypeptides include human ActRIIB precursor polypeptide (SEQ ID NO: 2), and soluble human ActRIIB polypeptides (e.g., SEQ ID NOs: 1, 5, 6 and 12).
  • the disclosure identifies functionally active portions and variants of ActRIIB.
  • an Fc fusion protein having the sequence disclosed by Hilden et al. (Blood. 1994 Apr 15;83(8):2163-70), which has an alanine at the position corresponding to amino acid 64 of SEQ ID NO: 2 (A64), has a relatively low affinity for activin and GDFl 1.
  • the same Fc fusion protein with an arginine at position 64 (R64) has an affinity for activin and GDF-11 in the low nanomolar to high picomolar range. Therefore, a sequence with an R64 is used as the wild-type reference sequence for human ActRIIB in this disclosure.
  • Attisano et al. Cell.
  • 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. Therefore, an ActRIIB-Fc fusion protein may end as early as amino acid 109 (the final cysteine), however, forms ending at or between 109 and 119 are expected to have reduced ligand binding.
  • Amino acid 119 is poorly conserved and so is readily altered or truncated. Forms ending at 128 or later retain ligand binding activity. Forms ending at or between 119 and 127 will have an intermediate binding ability. Any of these forms may be desirable to use, depending on the clinical or experimental setting.
  • constructs beginning at position 20, 21, 22, 23 and 24 will retain activity, and constructs beginning at positions 25, 26, 27, 28 and 29 are also expected to retain activity.
  • Data are shown in WO2008097541 demonstrates that, surprisingly, a construct beginning at 22, 23, 24 or 25 will have the most activity.
  • an active portion of ActRIIB comprises amino acids 29-109 of SEQ ID NO:2, and constructs may, for example, begin at a residue corresponding to amino acids 20-29 and end at a position corresponding to amino acids 109-134.
  • constructs that begin at a position from 20-29 or 21-29 and end at a position from 119-134, 119-133 or 129-134, 129-133.
  • constructs that begin at a position from 20-24 (or 21-24, or 22-25) and end at a position from 109-134 (or 109-133), 119-134 (or 119-133) or 129-134 (or 129-133).
  • Variants within these ranges are also contemplated, particularly those having at least 80%, 85%, 90%, 95% or 99% identity to the corresponding portion of SEQ ID NO:4.
  • the disclosure includes the results of an analysis of composite ActRIIB structures, shown in Figure 2, demonstrating that the ligand binding pocket is defined 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.
  • R40 is a K in Xenopus, indicating that basic amino acids at this position will be tolerated.
  • Q53 is R in bovine ActRIIB and K in Xenopus ActRIIB, and therefore amino acids including R, K, Q, N and H will be tolerated at this position.
  • a general formula for an active ActRIIB variant protein is one that comprises amino acids 29-109, but optionally beginning at a position ranging from 20-24 or 22-25 and ending at a position ranging from 129-134, and comprising no more than 1, 2, 5, 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.
  • Such a protein may retain greater than 80%, 90%, 95% or 99% sequence identity to the sequence of amino acids 29-109 of SEQ ID NO: 2.
  • Sites outside the binding pocket include the amino and carboxy termini of the extracellular domain (as noted above), and positions 42-46 and 65-73.
  • An asparagine-to- alanine alteration at position 65 actually improves ligand binding in the A64 background and is thus expected to have no detrimental effect on ligand binding in the R64 background.
  • This change probably eliminates glycosylation at N65 in the A64 background, thus demonstrating that a significant change in this region is likely to be tolerated.
  • R64A change is poorly tolerated, R64K is well-tolerated, and thus another basic residue such as H may be tolerated at position 64.
  • an active, human ActRIIB variant 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 sequence. The following examples illustrate this approach to defining an active ActRIIB variant.
  • L46 is a valine in Xenopus ActRIIB, 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 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 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.
  • F 108 is a Y in Xenopus, and therefore Y or other hydrophobic group, such as I, V or L should be tolerated.
  • El 11 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 is K in Xenopus, indicating that basic residues are tolerated at this position, including R and H.
  • a at position 119 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.
  • N-X-S/T N-linked glycosylation site
  • Other NX(T/S) sequences are found at 42-44 (NQS) and 65-67 (NSS), although the latter may not be efficiently glycosylated with the R at position 64.
  • N-X-S/T sequences may be generally introduced at positions outside the ligand binding pocket defined in Figure 2. Particularly suitable sites for the introduction of non- endogenous N-X-S/T sequences include amino acids 20-29, 20-24, 22-25, 109-134, 120-134 or 129-134.
  • N-X-S/T sequences may also be introduced into the linker between the ActRIIB 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.
  • desirable alterations that would create an N-linked glycosylation site are: A24N, R64N, S67N (possibly combined with an N65A alteration), E106N, Rl 12N, G120N, E123N, P129N, A132N, Rl 12S and Rl 12T. Any S that is predicted to be glycosylated may be altered to a T without creating an immunogenic site, because of the protection afforded by the glycosylation.
  • an ActRIIB variant may include one or more additional, non-endogenous N-linked glycosylation consensus sequences.
  • Position L79 may be altered to confer altered activin - myostatin (GDF-11) binding properties.
  • L79A or L79P reduces GDF-11 binding to a greater extent than activin binding.
  • L79E or L79D retains GDF-11 binding.
  • the L79E and L79D variants have greatly reduced activin binding. In vivo experiments indicate that these non-activin receptors retain significant ability to increase muscle mass but show decreased effects on other tissues.
  • the results of mutagenesis program described herein indicate that there are amino acid positions in ActRIIB that are often beneficial to conserve. These include position 64 (basic amino acid), 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).
  • the disclosure provides a framework of amino acids that may be conserved.
  • Other 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).
  • isolated fragments of the ActRIIB polypeptides can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an ActRIIB polypeptide (e.g., SEQ ID NOs: 3 and 4).
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function, for example, as antagonists (inhibitors) or agonists (activators) of an ActRIIB protein or an ActRIIB ligand.
  • a functional variant of the ActRIIB polypeptides has an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, and 53. In certain cases, the functional variant has an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, and 53.
  • the present invention contemplates making functional variants by modifying the structure of an ActRIIB polypeptide for such purposes as enhancing therapeutic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo).
  • Modified ActRIIB polypeptides can also be produced, for instance, by amino acid substitution, deletion, or addition. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of an ActRIIB polypeptide results in a functional homolog can be readily determined by assessing the ability of the variant ActRIIB polypeptide to produce a response in cells in a fashion similar to the wild-type ActRIIB polypeptide, or to bind to one or more ligands, such as activin, GDF11, or GDF8, in a fashion similar to wild type-
  • the present invention contemplates making mutations in the extracellular domain (also referred to as ligand-binding domain) of an ActRIIB polypeptide such that the variant (or mutant) ActRIIB polypeptide has altered ligand-binding activities (e.g., binding affinity or binding selectivity). In certain cases, such variant ActRIIB polypeptides have altered (elevated or reduced) binding affinity for a specific ligand. In other cases, the variant ActRIIB polypeptides
  • the present invention contemplates specific mutations of the ActRIIB polypeptides so as to alter the glycosylation of the polypeptide.
  • Exemplary glycosylation sites in ActRIIB polypeptides are illustrated in Figure 6 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 ActRIIB 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 an ActRIIB polypeptide is by chemical or enzymatic coupling of glycosides to the ActRIIB 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 ActRIIB 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.
  • Chemical 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 ActRIIB 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 an ActRIIB 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.
  • ActRIIB proteins 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 are expected to be useful as well.
  • This disclosure further contemplates a method of generating variants, particularly sets of combinatorial variants of an ActRIIB 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, ActRIIB polypeptide variants which have altered properties, such as altered pharmacokinetics, or altered ligand binding.
  • a variety of screening assays are provided below, and such assays may be used to evaluate variants.
  • an ActRIIB polypeptide variant may be screened for ability to bind to an ActRIIB polypeptide, to prevent binding of an ActRIIB ligand to an ActRIIB polypeptide.
  • an ActRIIB polypeptide or its variants may also be tested in a cell- based or in vivo assay.
  • the effect of an ActRIIB polypeptide variant on the expression of genes involved in bone production in an osteoblast or precursor may be assessed. This may, as needed, be performed in the presence of one or more recombinant ActRIIB ligand protein (e.g., BMP7), and cells may be transfected so as to produce an ActRIIB polypeptide or its variants.
  • a recombinant ActRIIB ligand protein e.g., BMP7
  • ActRIIB polypeptide and/or variants thereof, and optionally, an ActRIIB ligand may be administered to a mouse or other animal, and one or more bone properties, such as density or volume may be assessed. The healing rate for bone fractures may also be evaluated.
  • the activity of an ActRIIB polypeptide or its variants may be tested in muscle cells, adipocytes, and neuronal cells for any effect on growth of these cells, for example, by the assays as described below. Such assays are well known and routine in the art.
  • a SMAD-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 ActRIIB 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 ActRIIB 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 ActRIIB polypeptide.
  • ActRIIB polypeptides of the invention may further comprise post-translational modifications in addition to any that are naturally present in the ActRIIB polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • the modified ActRIIB 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 ActRIIB polypeptide may be tested as described herein for other ActRIIB polypeptide variants.
  • 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 ActRIIB polypeptide may be tested as described herein for other ActRIIB polypeptide variants.
  • post-translational processing may also be important for correct folding and/or function of the protein.
  • 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.
  • a fusion domain may be selected so as to confer a desired property.
  • 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.
  • fusion domain may be selected so as to facilitate detection of the ActRIIB polypeptides.
  • 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.
  • epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags.
  • the 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.
  • an ActRIIB polypeptide is fused with a domain that stabilizes the 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
  • 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).
  • polypeptides disclosed herein may form homomeric variant ActRIIB polypeptides, meaning that each fusion polypeptide chain in the protein complex comprises the same ActRIIB variant as any other such chain in the complex.
  • polypeptides disclosed herein may form heteromultimers comprising at least one variant ActRIIB polypeptide associated, covalently or non-covalently, with at least one unmodified ActRIIB polypeptide or at least one variant ActRIIB polypeptide different from the first ActRIIB variant.
  • polypeptides disclosed herein may form heteromultimers comprising at least one variant ActRIIB polypeptide associated, covalently or non-covalently, with at least one TGF-beta superfamily type I serine/threonine kinase receptor polypeptide (e.g., an ALKl, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 polypeptide), including fragments and variants thereof.
  • TGF-beta superfamily type I serine/threonine kinase receptor polypeptide e.g., an ALKl, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 polypeptide
  • the polypeptides disclosed herein may form heteromultimers comprising at least one variant ActRIIB polypeptide associated, covalently or non-covalently, with at least one TGF-beta superfamily type II serine/threonine kinase receptor polypeptide (e.g., ActRIIA, TGFBRII, BMPRII, and MISRII), including fragments and variants thereof.
  • TGF-beta superfamily type II serine/threonine kinase receptor polypeptide e.g., ActRIIA, TGFBRII, BMPRII, and MISRII
  • heteromeric polypeptides disclosed herein form heterodimers, although higher order heteromultimers are also included such as, but not limited to, heterotrimers, heterotetramers, and further oligomeric structures.
  • variant ActRIIB polypeptides of the present disclosure 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.
  • Variant ActRIIB 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., variant ActRIIB polypeptide) and a second polypeptide (e.g., an unmodified ActRIIB polypeptide or a variant ActRIIB polypeptide different from that present in the first polypeptide) to promote heteromultimer formation (e.g., heterodimer formation), and optionally hinders or otherwise disfavors homomultimer formation (e.g., homodimer formation), thereby increasing the yield of desired heteromultimer (see, e.g., Figure 1).
  • a first polypeptide e.g., variant ActRIIB polypeptide
  • a second polypeptide e.g., an unmodified ActRIIB polypeptide or a variant ActRIIB polypeptide different from that present in the first polypeptide
  • heteromultimer formation e.g., heterodimer formation
  • optionally hinders or otherwise disfavors homomultimer formation e.g., homo
  • the disclosure relates to a heteromultimer comprising at least one ALKl-Fc fusion protein and at least one ActRIIB-Fc fusion protein.
  • an ALKl-Fc:ActRIIB-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ALKl- Fc: ActRIIB-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ALKl-Fc: ActRIIB-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ALK2-FC fusion protein and at least one ActRIIB-Fc fusion protein.
  • an ALK2-Fc: ActRIIB-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ALKl- Fc: ActRIIB-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK2-Fc: ActRIIB-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ALK3-FC fusion protein and at least one ActRIIB-Fc fusion protein.
  • an ALK3-Fc:ActRIIB-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK3- Fc: ActRIIB-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK3-Fc: ActRIIB-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ALK4-Fc fusion protein and at least one ActRIIB-Fc fusion protein.
  • an ALK4-Fc: ActRIIB-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK4- Fc: ActRIIB-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK4-Fc: ActRIIB-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ALK5-FC fusion protein and at least one ActRIIB-Fc fusion protein.
  • an ALK5-Fc: ActRIIB-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK5- Fc:ActRIIB-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK5-Fc: ActRIIB-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ALK6-FC fusion protein and at least one ActRIIB-Fc fusion protein.
  • an ALK6-Fc: ActRIIB-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK6- Fc: ActRIIB-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK6-Fc: ActRIIB-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ALK7-FC fusion protein and at least one ActRIIB-Fc fusion protein.
  • an ALK7-Fc: ActRIIB-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK7- Fc: ActRIIB-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ALK7-Fc: ActRIIB-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ActRIIB-Fc fusion protein and at least one ActRIIA-Fc fusion protein.
  • an ActRIIB-Fc: ActRIIA-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ActRIIB- Fc: ActRIIA-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ActRIIB-Fc: ActRIIA-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ActRIIB-Fc fusion protein and at least one BMPRII-Fc fusion protein.
  • an ActRIIB-Fc :BMPRII-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ActRIIB- Fc:BMPRII-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ActRIIB-Fc: BMPRII-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ActRIIB-Fc fusion protein and at least one TGFBRII-Fc fusion protein.
  • an ActRIIB-Fc:TGFBRII-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ActRIIB- Fc:TGFBRII-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ActRIIB-Fc:TGFBRII-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer comprising at least one ActRIIB-Fc fusion protein and at least one MISRII-Fc fusion protein.
  • an ActRIIB-Fc:MISRII-Fc heteromultimers binds to one or more TGF-beta superfamily ligands such as those described herein.
  • an ActRIIB- Fc:MISRII-Fc heteromultimers inhibit signaling of one or more TGF-beta superfamily ligands such as those described herein.
  • an ActRIIB-Fc:MISRII-Fc heteromultimers is a heterodimer.
  • the disclosure relates to a heteromultimer that comprises an ALK1- Fc fusion protein.
  • the ALKl-Fc fusion protein comprises an ALK1 domain comprising 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 an amino acid sequence that begins at any one of amino acids of 22-34 (e.g., amino acid residues 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34) SEQ ID NO: 54, ends at any one of amino acids 95-118 (e.g., amino acid residues 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, and 118) of SEQ ID NO: 54, ends at
  • the ALKl-Fc fusion protein comprises an ALK1 domain comprising 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 amino acids 22-118 of SEQ ID NO: 54.
  • the ALKl-Fc fusion protein comprises an ALKl domain comprising 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 amino acids 34-95 of SEQ ID NO: 54.
  • the ALKl-Fc fusion protein comprises an ALKl domain comprising 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 amino acid sequence of any one of SEQ ID Nos: 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63.
  • a representative ALKl-Fc fusion polypeptide (SEQ ID NO: 60) is as follows:
  • the leader sequence and linker sequence are underlined.
  • the mature ALKl-Fc fusion protein sequence (SEQ ID NO: 61) is as follows and may optionally be provided with the lysine removed from the C-terminus.
  • the ALKl-Fc fusion polypeptide (SEQ ID NO: 56) is as follows:
  • the leader sequence and linker sequence are underlined.
  • two amino acid substitutions can be introduced into the Fc domain of the ALKl-Fc fusion polypeptide as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 56 may optionally be provided with a lysine added at the C-terminus.
  • This ALKl-Fc fusion protein is encoded by the following nucleic acid (SEQ ID NO:
  • the mature ALKl-Fc fusion protein sequence (SEQ ID NO: 57) is as follows and may optionally be provided with a lysine added at the C-terminus.
  • the present disclosure relates to protein complexes that comprise an ALK2 polypeptide.
  • ALK2 refers to a family of activin receptorlike kinase-2 proteins from any species and variants derived from such ALK2 proteins by mutagenesis or other modification. Reference to ALK2 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK2 family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine- rich region, a transmembrane domain, and a cytoplasmic domain with predicted
  • ALK2 polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ALK2 family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • a human ALK2 precursor protein sequence (NCBI Ref Seq NP_001096.1) is as follows:
  • the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.
  • a processed extracellular ALK2 polypeptide sequence is as follows:
  • a nucleic acid sequence encoding human ALK2 precursor protein is shown in SEQ ID NO: 217, corresponding to nucleotides 431 -1957 of Genbank Reference Sequence NM_001 105.4.
  • a nucleic acid sequence encoding the extracellular ALK2 polypeptide is as in SEQ ID NO: 218.
  • the disclosure relates to heteromultimers that comprise at least one ALK2 polypeptide, which includes fragments, functional variants, and modified forms thereof.
  • ALK2 polypeptides for use in accordance with inventions of the disclosure e.g., heteromultimers comprising an ALK2 polypeptide and uses thereof
  • ALK2 polypeptides for use in accordance with the inventions of the disclosure bind to and/or inhibit (antagonize) activity (e.g., induction of Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamily ligands.
  • heteromultimers of the disclosure comprise at least one ALK2 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64 or 65.
  • heteromultimer complexes of the disclosure consist or consist essentially of at least one ALK2 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64 or 65.
  • the disclosure relates to a heteromultimer that comprises an ALK2- Fc fusion protein.
  • the ALK2-Fc fusion protein comprises an ALK2 domain comprising 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 an amino acid sequence that begins at any one of amino acids 21-35 (e.g., amino acid residues 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35) SEQ ID NO: 64, and ends at any one of amino acids 99-123 (e.g., amino acid residues 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 1 1, 1 12, 1 13, 1 14, 1 15, 1 16, 1 17, 1 18, 1 19, 120, 121, 122, and 123) of SEQ ID NO: 64.
  • the ALK2-Fc fusion protein comprises an ALK2 domain comprising 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 amino acids 35-99 of SEQ ID NO: 64.
  • the ALK2-Fc fusion protein comprises an ALK2 domain comprising 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 amino acids 21-123 of SEQ ID NO: 64.
  • the ALK2-Fc fusion protein comprises an ALK2 domain comprising 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 amino acid sequence of SEQ ID Nos: 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
  • the ALK2-Fc fusion protein employs the TPA leader and is as follows (SEQ ID NO: 66):
  • the signal sequence and linker sequence are underlined.
  • two amino acid substitutions can be introduced into the Fc domain of the fusion protein as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 66 may optionally be provided with a lysine added at the C-terminus.
  • This ALK2-Fc fusion protein is encoded by the following nucleic acid (SEQ ID NO:
  • the mature ALK2-Fc fusion protein sequence (SEQ ID NO: 67) is as follows and may optionally be provided with a lysine added at the C-terminus.
  • the Fc domains are altered to introduce complementary hydrophobic interactions and an additional intermolecular disulfide bond.
  • the ALK2-Fc fusion polypeptide (SEQ ID NO: 70) is as follows:
  • SLSPGK SEQ ID NO: 70
  • the leader sequence and linker sequence are underlined.
  • four amino acid substitutions can be introduced into the Fc domain of the ALK2 fusion polypeptide as indicated by double underline above.
  • the C-terminal lysine residue of the Fc domain can be deleted.
  • the amino acid sequence of SEQ ID NO: 70 may optionally be provided with the lysine removed from the C-terminus.
  • the mature ALK2-Fc fusion protein sequence (SEQ ID NO: 71) is as follows and may optionally be provided with the lysine removed from the C-terminus.
  • the present disclosure relates to protein complexes that comprise an ALK2 polypeptide.
  • ALK2 refers to a family of activin receptorlike kinase-2 proteins from any species and variants derived from such ALK2 proteins by mutagenesis or other modification. Reference to ALK2 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK2 family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine- rich region, a transmembrane domain, and a cytoplasmic domain with predicted
  • ALK2 polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ALK2 family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.
  • a processed extracellular ALK2 polypeptide sequence is as follows:
  • a nucleic acid sequence encoding the extracellular ALK2 polypeptide is as in SEQ ID NO: 218.
  • the disclosure relates to heteromultimers that comprise at least one ALK2 polypeptide, which includes fragments, functional variants, and modified forms thereof.
  • ALK2 polypeptides for use in accordance with inventions of the disclosure e.g., heteromultimers comprising an ALK2 polypeptide and uses thereof
  • are soluble e.g., an extracellular domain of ALK2.
  • ALK2 polypeptides for use in accordance with the inventions of the disclosure bind to and/or inhibit (antagonize) activity (e.g., induction of Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamily ligands.
  • heteromultimer complexes of the disclosure consist or consist essentially of at least one ALK2 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64 or 65.
  • heteromultimer complexes of the disclosure consist or consist essentially of at least one ALK2 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64 or 65.
  • the present disclosure relates to protein complexes that comprise an ALK3 polypeptide.
  • ALK3 refers to a family of activin receptor- like kinase-3 proteins from any species and variants derived from such ALK3 proteins by mutagenesis or other modification. Reference to ALK3 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK3 family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine- rich region, a transmembrane domain, and a cytoplasmic domain with predicted
  • ALK3 polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ALK3 family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • a human ALK3 precursor protein sequence (NCBI Ref Seq NP_004320.2) is as follows:
  • the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.
  • a processed extracellular ALK3 polypeptide sequence is as follows:
  • a nucleic acid sequence encoding the extracelluar human ALK3 polypeptide is shown in SEQ ID NO: 220.
  • a general formula for an active (e.g., ligand binding) ALK3 polypeptide is one that comprises a polypeptide that begins at any amino acid position 25-31 (i.e., position 25, 26, 27, 28, 29, 30, or 31) of SEQ ID NO: 74 and ends at any amino acid position 140-152 of SEQ ID NO: 74 (i.e., 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, or 152). See U.S. Patent 8,338,377, the teachings of which are incorporated herein by reference in their entirety.
  • the disclosure relates to heteromultimers that comprise at least one ALK3 polypeptide, which includes fragments, functional variants, and modified forms thereof.
  • ALK3 polypeptides for use in accordance with inventions of the disclosure e.g., heteromultimers comprising an ALK3 polypeptide and uses thereof
  • ALK3 polypeptides for use in accordance with the inventions of the disclosure bind to and/or inhibit (antagonize) activity (e.g., induction of Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamily ligands.
  • heteromultimers of the disclosure comprise at least one ALK3 polypeptide that comprises an amino acid beginning at any amino acid position 25-31 (i.e., position 25, 26, 27, 28, 29, 30, or 31) of SEQ ID NO: 74 and ending at any amino acid position 140-153 of SEQ ID NO: 74 (i.e., 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, or 152) of SEQ ID NO: 74.
  • ALK3 polypeptide that comprises an amino acid beginning at any amino acid position 25-31 (i.e., position 25, 26, 27, 28, 29, 30, or 31) of SEQ ID NO: 74 and ending at any amino acid position 140-153 of SEQ ID NO: 74 (i.e., 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, or 152) of SEQ ID NO: 74.
  • heteromultimer complexes of the disclosure comprise at least one ALK3 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74, 75, 76, 77, 80, or 81.
  • heteromultimer complexes of the disclosure consist or consist essentially of at least one ALK3 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 74, 75, 76, 77, 80, or 81.
  • the disclosure relates to a heteromultimer that comprises an ALK3- Fc fusion protein.
  • the ALK3-Fc fusion protein comprises an ALK3 domain comprising 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 an amino acid sequence that begins at any one of amino acids 24-61 (e.g., amino acid residues 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, and 61) SEQ ID NO: 74, and ends at any one of amino acids 130-152 (e.g., amino acid residues 130, 131, 132, 133, 134, 13
  • amino acids 24-61
  • the ALK3-Fc fusion protein comprises an ALK3 domain comprising 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 amino acids 61-130 of SEQ ID NO: 74.
  • the ALK3-Fc fusion protein comprises an ALK3 domain comprising 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 amino acids 24-152 of SEQ ID NO: 74.
  • the ALK3-Fc fusion protein comprises an ALK3 domain comprising 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 amino acid sequence of any one of SEQ ID Nos: 74, 75, 76, 77, 78, 79, 80, 81, 82, and 83.
  • the ALK3-Fc fusion protein employs the TPA leader and is as follows:
  • the leader and linker sequences are underlined. To promote formation of the
  • ActRIIB-Fc:ALK3-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing lysines with aspartic acids) can be introduced into the Fc domain of the fusion protein as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 76 may optionally be provided with a lysine added at the C- terminus.
  • This ALK3-FC fusion protein is encoded by the following nucleic acid (SEQ ID NO:
  • the mature ALK3-Fc fusion protein sequence is as follows (SEQ ID NO: 77) and may optionally be provided with a lysine added at the C-terminus.
  • ALK3-Fc fusion polypeptide SEQ ID NO: 80.
  • the leader sequence and linker are underlined.
  • four amino acid substitutions can be introduced into the Fc domain of the ALK3 fusion polypeptide as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 80 may optionally be provided with the lysine removed from the C-terminus.
  • the mature ALK3-Fc fusion protein sequence (SEQ ID NO: 81) is as follows and may optionally be provided with the lysine (K) removed from the C-terminus.
  • the present disclosure relates to protein complexes that comprise an ALK4 polypeptide.
  • ALK4 refers to a family of activin receptorlike kinase-4 proteins from any species and variants derived from such ALK4 proteins by mutagenesis or other modification. Reference to ALK4 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK4 family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine- rich region, a transmembrane domain, and a cytoplasmic domain with predicted
  • ALK4 polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ALK4 family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • a human ALK4 precursor protein sequence (NCBI Ref Seq P 004293) is as follows:
  • the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.
  • a processed extracellular human ALK4 polypeptide sequence is as follows:
  • a nucleic acid sequence encoding the extracellular ALK4 polypeptide is shown in SEQ ID NO: 222.
  • An alternative isoform of human ALK4 precursor protein sequence, isoform C (NCBI Ref Seq P_064733.3), is as follows:
  • a processed extracellular ALK4 polypeptide sequence is as follows:
  • a nucleic acid sequence encoding an ALK4 precursor protein (isoform C) is shown in SEQ ID NO: 223, corresponding to nucleotides 78-1715 of Genbank Reference Sequence NM_020328.3.
  • a nucleic acid sequence encoding the extracelluar ALK4 polypeptide (isoform C) is shown in SEQ ID NO: 224.
  • the disclosure relates to heteromultimers that comprise at least one ALK4 polypeptide, which includes fragments, functional variants, and modified forms thereof.
  • ALK4 polypeptides for use in accordance with inventions of the disclosure e.g., heteromultimers comprising an ALK4 polypeptide and uses thereof
  • ALK4 polypeptides for use in accordance with the inventions of the disclosure bind to and/or inhibit (antagonize) activity (e.g., induction of Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamily ligands.
  • heteromultimers of the disclosure comprise at least one ALK4 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 84, 86, 85, 87, 88, 89, 92, or 93.
  • heteromultimers of the disclosure consist or consist essentially of at least one ALK4 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 84, 86, 85, 87, 88, 89, 92, or 93.
  • the disclosure relates to a heteromultimer that comprises an ALK4- Fc fusion protein.
  • the ALK4-Fc fusion protein comprises an ALK4 domain comprising 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 an amino acid sequence that begins at any one of amino acids 23-34 (e.g., amino acid residues 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) SEQ ID NO: 84 or 85, and ends at any one of amino acids 101-126 (e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125
  • the ALK4-Fc fusion protein comprises an ALK4 domain comprising 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 amino acids 34-101 of SEQ ID NOs: 84 or 85.
  • the ALK4- Fc fusion protein comprises an ALK4 domain comprising 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 amino acids 23-126 of SEQ ID Nos: 84 or 85.
  • the ALK4-Fc fusion protein comprises an ALK4 domain comprising 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 amino acid sequence of any one of SEQ ID Nos: 84, 86, 85, 87, 88, 89, 90, 91, 92, 93, 94, and 95.
  • polypeptide comprises an ALK4-Fc fusion polypeptide (SEQ ID NO: 88) as follows:
  • the leader sequence and linker sequence are underlined.
  • two amino acid substitutions can be introduced into the Fc domain of the ALK4-Fc fusion polypeptide as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 88 may optionally be provided with lysine added at the C-terminus.
  • This ALK4-FC fusion protein is encoded by the following nucleic acid (SEQ ID NO:
  • the mature ALK4-Fc fusion protein sequence (SEQ ID NO: 89) is as follows and may optionally be provided with lysine added at the C-terminus.
  • the ALK4-Fc fusion polypeptide (or any Fc fusion polypeptide disclosed herein) employs the tissue plasminogen activator (TP A) leader:
  • MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 246) .
  • the ALK4-Fc fusion polypeptide (SEQ ID NO: 92) is as follows and may optionally be provided with lysine removed from the C-terminus.
  • the leader sequence and the linker are underlined.
  • four amino acid substitutions can be introduced into the Fc domain of the ALK4 fusion polypeptide as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 92 may optionally be provided with lysine removed from the C-terminus.
  • the mature ALK4-Fc fusion protein sequence is as follows and may optionally be provided with lysine removed from the C-terminus.
  • ALK4-Fc complexes could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • the ALK4-Fc fusion polypeptide (SEQ ID NO: 247) is as follows and may optionally be provided with lysine removed from the C-terminus.
  • the leader sequence and the linker are underlined.
  • four amino acid substitutions can be introduced into the Fc domain of the ALK4 fusion polypeptide as indicated by double underline above.
  • ALK4-Fc ActRIIB-Fc heterodimer
  • two amino acid substitutions can also be introduced into the Fc domain of the ALK4-Fc fusion polypeptide as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 247 may optionally be provided with lysine removed from the C-terminus.
  • This ALK4-FC fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO:
  • the mature ALK4-Fc fusion polypeptide sequence is as follows (SEQ ID NO: 249) and may optionally be provided with lysine removed from the C-terminus.
  • This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 250):
  • the ALK4-Fc fusion polypeptide is SEQ ID NO: 92 (shown above), which contains four amino acid substitutions to guide heterodimer formation certain Fc fusion polypeptides disclosed herein, and may optionally be provided with lysine removed from the C-terminus.
  • This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 251):
  • the mature ALK4-Fc fusion polypeptide sequence is SEQ ID NO: 93 (shown above) and may optionally be provided with lysine removed from the C-terminus.
  • This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 252):
  • ActRIIB-Fc complexes could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography and epitope-based affinity chromatography (e.g., with an antibody or functionally equivalent ligand directed against an epitope on ALK4 or ActRIIB), and multimodal chromatography (e.g., with resin containing both electrostatic and hydrophobic ligands).
  • the purification could be completed with viral filtration and buffer exchange.
  • the present disclosure relates to protein complexes that comprise an ALK5 polypeptide.
  • ALK5 refers to a family of activin receptorlike kinase-5 proteins from any species and variants derived from such ALK4 proteins by mutagenesis or other modification. Reference to ALK5 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK5 family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine- rich region, a transmembrane domain, and a cytoplasmic domain with predicted
  • ALK5 polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ALK5 family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • a human ALK5 precursor protein sequence (NCBI Ref Seq P 004603.1) is as follows:
  • the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.
  • a processed extracellular ALK5 polypeptide sequence is as follows:
  • a nucleic acid sequence encoding the ALK5 precursor protein is shown in SEQ ID NO: 225, corresponding to nucleotides 77-1585 of Genbank Reference Sequence M 004612.2.
  • a nucleic acid sequence encoding an extracellular human ALK5 polypeptide is shown in SEQ ID NO: 226.
  • isoform 2 (NCBI Ref Seq XP_005252207.1), is as follows:
  • the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.
  • a processed extracellular ALK5 polypeptide sequence (isoform 2) is as follows:
  • a nucleic acid sequence encoding human ALK5 precursor protein is shown in SEQ ID NO: 227, corresponding to nucleotides 77-1597 of Genbank Reference Sequence XM_005252150.1.
  • a nucleic acid sequence encoding a processed extracellular ALK5 polypeptide is shown in SEQ ID NO: 228.
  • the disclosure relates to heteromultimers that comprise at least one ALK5 polypeptide, which includes fragments, functional variants, and modified forms thereof.
  • ALK5 polypeptides for use in accordance with inventions of the disclosure e.g., heteromultimers comprising an ALK5 polypeptide and uses thereof
  • ALK5 polypeptides for use in accordance with the inventions of the disclosure bind to and/or inhibit (antagonize) activity (e.g., induction of Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamily ligands.
  • heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96, 98, 97, or 99.
  • heteromultimer complexes of the disclosure consist or consist essentially of at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:
  • the disclosure relates to a heteromultimer that comprises an ALK5- Fc fusion protein.
  • the ALK5-Fc fusion protein comprises an ALK5 domain comprising 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 an amino acid sequence that begins at any one of amino acids 25-36 (e.g., amino acid residues 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36) SEQ ID NO: 96 or 97, and ends at any one of amino acids 106-126 (e.g., amino acid residues 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and 126) of SEQ ID NO
  • the ALK5-Fc fusion protein comprises an ALK5 domain comprising 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 amino acids 36-106 of SEQ ID NOs: 96 or 97.
  • the ALK5-Fc fusion protein comprises an ALK5 domain comprising 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 amino acids 25-126 of SEQ ID NOs: 96 or 97.
  • the ALK5-FC fusion protein comprises an ALK5 domain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
  • the complementary ALK5-Fc fusion protein employs the TPA leader and is as follows (SEQ ID NO: 100):
  • 351 PG SEQ I D NO : 1 00
  • the signal sequence and linker sequence are underlined.
  • two amino acid substitutions can be introduced into the Fc domain of the fusion protein as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 100 may optionally be provided with a lysine added at the C- terminus.
  • This ALK5-FC fusion protein is encoded by the following nucleic acid (SEQ ID NO:
  • the mature ALK5-Fc fusion protein sequence (SEQ ID NO: 101) is as follows and may optionally be provided with a lysine added at the C-terminus.
  • the Fc domains are altered to introduce complementary hydrophobic interactions and an additional intermolecular disulfide bond.
  • the ALK5-Fc fusion polypeptide (SEQ ID NO: 104) is as follows:
  • 351 PGK (SEQ ID NO: 104)
  • the leader sequence and linker sequence are underlined.
  • four amino acid substitutions can be introduced into the Fc domain of the ALK5 fusion polypeptide as indicated by double underline above.
  • the C-terminal lysine residue of the Fc domain can be deleted.
  • the amino acid sequence of SEQ ID NO: 104 may optionally be provided with the lysine removed from the C-terminus.
  • the mature ALK5-Fc fusion protein sequence (SEQ ID NO: 105) is as follows and may optionally be provided with the lysine removed from the C-terminus.
  • the present disclosure relates to protein complexes that comprise an ALK6 polypeptide.
  • ALK6 refers to a family of activin receptorlike kinase-6 proteins from any species and variants derived from such ALK6 proteins by mutagenesis or other modification. Reference to ALK6 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK6 family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine- rich region, a transmembrane domain, and a cytoplasmic domain with predicted
  • ALK6 polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ALK6 family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • a human ALK6 precursor protein sequence (NCBI Ref Seq P 001194.1) is as follows:
  • the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.
  • the processed extracellular ALK6 polypeptide sequence is as follows:
  • a nucleic acid sequence encoding the ALK6 precursor protein is shown in SEQ ID NO: 229, corresponding to nucleotides 275-1780 of Genbank Reference Sequence NM 001203.2.
  • a nucleic acid sequence encoding processed extracellular ALK6 polypeptide is shown in SEQ ID NO: 230.
  • isoform 2 (NCBI Ref Seq NP_001243722.1) is as follows:
  • the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.
  • a processed extracellular ALK6 polypeptide sequence (isoform 2) is as follows:
  • a nucleic acid sequence encoding human ALK6 precursor protein is shown in SEQ ID NO: 231, corresponding to nucleotides 22-1617 of Genbank Reference Sequence NM_001256793.1.
  • a nucleic acid sequence encoding a processed extracellular ALK6 polypeptide is shown in SEQ ID NO: 232.
  • the disclosure relates to heteromultimers that comprise at least one ALK6 polypeptide, which includes fragments, functional variants, and modified forms thereof.
  • ALK6 polypeptides for use in accordance with inventions of the disclosure e.g., heteromultimers comprising an ALK6 polypeptide and uses thereof
  • are soluble e.g., an extracellular domain of ALK6.
  • ALK6 polypeptides for use in accordance with the inventions of the disclosure bind to and/or inhibit (antagonize) activity (e.g., induction of Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamily ligands.
  • heteromultimers of the disclosure comprise at least one ALK6 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 108, 109, 110, or 111.
  • heteromultimers of the disclosure consist or consist essentially of at least one ALK6 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 108, 109, 110, or 111.
  • the disclosure relates to a heteromultimer that comprises an ALK6-
  • the ALK6-Fc fusion protein comprises an ALK6 domain comprising 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 an amino acid sequence that begins at any one of amino acids 14-32 (e.g., amino acid residues 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32) SEQ ID NO: 108, and ends at any one of amino acids 102-126 (e.g., amino acid residues 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and 126) of SEQ ID NO: 108.
  • amino acids 14-32 e.g., amino acid residue
  • the ALK6-Fc fusion protein comprises an ALK6 domain comprising 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 amino acids 32-102 of SEQ ID NO: 108.
  • the ALK6-Fc fusion protein comprises an ALK6 domain comprising 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 amino acids 14-126 of SEQ ID NO: 108.
  • the ALK6-Fc fusion protein comprises an ALK6 domain comprising 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 amino acid sequence of any one of SEQ ID Nos: 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, and 119.
  • the ALK6-Fc fusion protein comprises an ALK6 domain comprising 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 an amino acid sequence that begins at any one of amino acids 26-62 (e.g., amino acid residues 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62) SEQ ID NO: 110, and ends at any one of amino acids 132-156 (e.g., amino acid residues 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,
  • the ALK6-Fc fusion protein comprises an ALK6 domain comprising 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 amino acids 62-132 of SEQ ID NO: 110.
  • the ALK6-Fc fusion protein comprises an ALK6 domain comprising 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 amino acids 26-156 of SEQ ID NO: 110.
  • the complementary ALK6-Fc fusion protein employs the TPA leader and is as follows (SEQ ID NO: 112):
  • the signal sequence and linker sequence are underlined.
  • two amino acid substitutions can be introduced into the Fc domain of the fusion protein as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 112 may optionally be provided with a lysine added at the C- terminus.
  • This ALK6-FC fusion protein is encoded by the following nucleic acid (SEQ ID NO:
  • the mature ALK6-Fc fusion protein sequence (SEQ ID NO: 113) is as follows and may optionally be provided with a lysine added at the C-terminus.
  • the Fc domains can be altered to introduce complementary hydrophobic interactions and an additional intermolecular disulfide bond.
  • ALK6-Fc fusion polypeptide SEQ ID NO: 1166
  • VEVHNAKTKP REEQYNS TYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP 251 IEKT I SKAKG QPREPQVgTL PPSREEMTKN QVSL CAyKG FYPSDIAVEW 301 ESNGQPENNY KTTPPVLDSD GS FFL SKLT VDKSRWQQGN VFSCSVMHEA 351 LHNHYTQKSL SLS PGK ( SEQ I D NO : 1 1 6 )
  • the leader sequence and linker sequence are underlined.
  • four amino acid substitutions can be introduced into the Fc domain of the ALK6 fusion polypeptide as indicated by double underline above.
  • the C-terminal lysine residue of the Fc domain can be deleted.
  • the amino acid sequence of SEQ ID NO: 116 may optionally be provided with the lysine removed from the C-terminus.
  • the mature ALK6-Fc fusion protein sequence (SEQ ID NO: 117) can be as follows and may optionally be provided with the lysine removed from the C-terminus.
  • the present disclosure relates to protein complexes that comprise an ALK7 polypeptide.
  • ALK7 refers to a family of activin receptorlike kinase-7 proteins from any species and variants derived from such ALK7 proteins by mutagenesis or other modification. Reference to ALK7 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK7 family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine- rich region, a transmembrane domain, and a cytoplasmic domain with predicted
  • ALK7 polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ALK7 family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • isoforms of human ALK7 Four naturally occurring isoforms of human ALK7 have been described.
  • sequence of human ALK7 isoform 1 precursor protein (NCBI Ref Seq NP 660302.2) is as follows:
  • the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.
  • a processed extracellular ALK7 isoform 1 polypeptide sequence is as follows:
  • a nucleic acid sequence encoding human ALK7 isoform 1 precursor protein is shown below in SEQ ID NO: 233, corresponding to nucleotides 244-1722 of Genbank Reference Sequence NM_145259.2.
  • a nucleic acid sequence encoding the processed extracellular ALK7 polypeptide (isoform 1) is show in in SEQ ID NO: 234.
  • An amino acid sequence of the extracellular ALK7 polypeptide is as follows: MLTNGKEQVn SCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHLPTASPNAPK LGPME (SEQ ID NO: 125).
  • a nucleic acid sequence encoding the processed ALK7 polypeptide (isoform 2) is shown below in SEQ ID NO: 235, corresponding to nucleotides 279-1607 of NCBI
  • a nucleic acid sequence encoding an extracellular ALK7 polypeptide is shown in SEQ ID NO: 236.
  • amino acid sequence of an alternative human ALK7 precursor protein, isoform 3 (NCBI Ref Seq NP_001104502.1), is shown as follows (SEQ ID NO: 121), where the signal peptide is indicated by a single underline.
  • the amino acid sequence of a processed ALK7 polypeptide is as follows (SEQ ID NO: 126). This isoform lacks a transmembrane domain and is therefore proposed to be soluble in its entirety (Roberts et al., 2003, Biol Reprod 68: 1719-1726). N-terminal variants of SEQ ID NO: 126 are predicted as described below.
  • a nucleic acid sequence encoding an unprocessed ALK7 polypeptide precursor protein is shown in SEQ ID NO: 237, corresponding to nucleotides 244-1482 of NCBI Reference Sequence NM_001111032.1.
  • a nucleic acid sequence encoding a processed ALK7 polypeptide is shown in SEQ ID NO: 238.
  • An amino acid sequence of an alternative human ALK7 precursor protein, isoform 4 is shown as follows (SEQ ID NO: 122), where the signal peptide is indicated by a single underline.
  • isoform 4 An amino acid sequence of a processed ALK7 polypeptide (isoform 4) is as follows (SEQ ID NO: 127). Like ALK7 isoform 3, isoform 4 lacks a transmembrane domain and is therefore proposed to be soluble in its entirety (Roberts et al., 2003, Biol Reprod 68: 1719- 1726). N-terminal variants of SEQ ID NO: 127 are predicted as described below.
  • a nucleic acid sequence encoding the unprocessed ALK7 polypeptide precursor protein (isoform 4) is shown in SEQ ID NO: 239, corresponding to nucleotides 244-1244 of NCBI Reference Sequence NM OOl 111033.1.
  • a nucleic acid sequence encoding the processed ALK7 polypeptide (isoform 4) is shown in SEQ ID NO: 240.
  • Active variants of processed ALK7 isoform 1 are predicted in which SEQ ID NO: 123 is truncated by 1, 2, 3, 4, 5, 6, or 7 amino acids at the N-terminus and SEQ ID NO: 128 is truncated by 1 or 2 amino acids at the N-terminus. Consistent with SEQ ID NO: 128, it is further expected that leucine is the N-terminal amino acid in the processed forms of human ALK7 isoform 3 (SEQ ID NO: 126) and human ALK7 isoform 4 (SEQ ID NO: 127).
  • the disclosure relates to heteromultimers that comprise at least one ALK7 polypeptide, which includes fragments, functional variants, and modified forms thereof.
  • ALK7 polypeptides for use in accordance with inventions of the disclosure are soluble (e.g., an extracellular domain of ALK7).
  • ALK7 polypeptides for use in accordance with the inventions of the disclosure bind to and/or inhibit (antagonize) activity (e.g., induction of Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamily ligands.
  • heteromultimers of the disclosure comprise at least one ALK7 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120, 123, 129, 130, 124, 125, 121, 126, 122, 127, 128, 133, or 134.
  • heteromultimers of the disclosure consist or consist essentially of at least one ALK7 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120, 123, 129, 130, 124, 125, 121, 126, 122, 127, 128, 133, or 134.
  • the disclosure relates to a heteromultimer that comprises an ALK7- Fc fusion protein.
  • the ALK7-Fc fusion protein comprises an ALK7 domain comprising 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 an amino acid sequence that begins at any one of amino acids 21-28 (e.g., amino acid residues 21, 22, 23, 24, 25, 26, 27, and 28) SEQ ID NO: 120, 121, or 122, and ends at any one of amino acids 92-1 13 (e.g., amino acid residues 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 1 1, 1 12, and 1 13) of SEQ ID NO: 120, 121
  • the ALK7-Fc fusion protein comprises an ALK7 domain comprising 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 amino acids 28-92 of SEQ ID NOs: 120, 121, or 122.
  • the ALK7-Fc fusion protein comprises an ALK7 domain comprising 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 amino acids 21-1 13 of SEQ ID NOs: 120, 121, or 122.
  • the ALK7-Fc fusion protein comprises an ALK7 domain comprising 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 amino acid sequence of any one of SEQ ID Nos: 120, 123, 124, 125, 121, 126, 122, 127, 128, 129, 130, 131, 132, 133, 134, 135, and 136.
  • the ALK7-Fc fusion protein employs the TPA leader and is as follows (SEQ ID NO: 129):
  • the signal sequence and linker sequence are underlined.
  • two amino acid substitutions can be introduced into the Fc domain of the fusion protein as indicated by double underline above.
  • the amino acid sequence of SEQ ID NO: 129 may optionally be provided with a lysine added at the C- terminus.
  • This ALK7-FC fusion protein is encoded by the following nucleic acid (SEQ ID NO: 255):
  • the mature ALK7-Fc fusion protein sequence (SEQ ID NO: 130) is expected to be as follows and may optionally be provided with a lysine added at the C-terminus.
  • ALK7-Fc fusion polypeptide (SEQ ID NO: 133) is as follows:
  • the leader sequence and linker sequence are underlined.
  • four amino acid substitutions can be introduced into the Fc domain of the ALK7 fusion polypeptide as indicated by double underline above.
  • the C-terminal lysine residue of the Fc domain can be deleted.
  • the amino acid sequence of SEQ ID NO: 133 may optionally be provided with the lysine removed from the C-terminus.
  • the mature ALK7-Fc fusion protein sequence (SEQ ID NO: 134) is expected to be as follows and may optionally be provided with the lysine removed from the C-terminus.
  • the present disclosure relates to a protein complex comprising an ActRIIA polypeptide.
  • ActRIIA refers to a family of activin receptor type IIA (ActRIIA) proteins from any species and variants derived from such ActRIIA proteins by mutagenesis or other modification. Reference to ActRIIA herein is understood to be a reference to any one of the currently identified forms.
  • ActRIIA Members of the ActRIIA 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.
  • ActRIIA polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ActRIIA family member as well as any variants thereof
  • the human ActRIIA precursor protein sequence is as follows:
  • the signal peptide is indicated by a single underline; the extracellular domain is indicated in bold font; and the potential, endogenous N-linked glycosylation sites are indicated by a double underline.
  • the processed extracellular human ActRIIA polypeptide sequence is as follows:
  • the C-terminal "tail” of the extracellular domain is indicated by a single underline.
  • the sequence with the "tail” deleted is as follows:
  • a nucleic acid sequence encoding the human ActRIIA precursor protein is shown in SEQ ID NO: 241, corresponding to nucleotides 159-1700 of Genbank Reference Sequence NM 001616.4.
  • a nucleic acid sequence encoding a processed extracellular ActRIIA polypeptide is as shown in SEQ ID NO: 242.
  • ActRIIA polypeptide is one that comprises a polypeptide that starts at amino acid 30 and ends at amino acid 1 10 of SEQ ID NO: 137. Accordingly, ActRIIA polypeptides of the present disclosure may comprise a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-1 10 of SEQ ID NO: 137.
  • ActRIIA polypeptides of the present disclosure comprise a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids amino acids 12-82 of SEQ ID NO: 137 optionally beginning at a position ranging from 1-5 (e.g., 1, 2, 3, 4, or 5) or 3-5 (e.g., 3, 4, or 5) and ending at a position ranging from 1 10-1 16 (e.g., 1 10, 1 1 1, 1 12, 1 13, 1 14, 1 15, or 1 16) or 1 10- 1 15 (e.g., 1 10, 1 1 1, 1 12, 1 13, 1 14, or 1 15), respectively, and comprising no more than 1, 2, 5, 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 with respect to SEQ ID NO: 137.
  • 1-5 e.g., 1, 2, 3,
  • the disclosure relates to heteromultimers that comprise at least one ActRIIA polypeptide, which includes fragments, functional variants, and modified forms thereof.
  • ActRIIA polypeptides for use in accordance with inventions of the disclosure e.g., heteromultimers comprising an ActRIIA polypeptide and uses thereof
  • are soluble e.g., an extracellular domain of ActRIIA.
  • ActRIIA polypeptides for use in accordance with the inventions of the disclosure bind to and/or inhibit (antagonize) activity (e.g., induction of Smad 2/3 and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamily ligands.
  • heteromultimers of the disclosure comprise at least one ActRIIA polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 137, 138, 139, 140, 141, 144, or 145. In some embodiments, heteromultimers of the disclosure comprise at least one ActRIIA polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 137, 138, 139, 140, 141, 144, or 145.
  • the disclosure relates to a heteromultimer that comprises an ActRIIA-Fc fusion protein.
  • the ActRIIA-Fc fusion protein comprises an ActRIIA domain comprising 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 an amino acid sequence that begins at any one of amino acids 21-30 (e.g., amino acid residues 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) SEQ ID NO: 137, and ends at any one of amino acids 1 10-135 (e.g., 1 10, 1 1 1, 1 12, 1 13, 1 14, 1 15, 1 16, 1 17, 1 18, 1 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135) of SEQ ID NO: 137.
  • amino acids 21-30 e
  • the ActRIIA-Fc fusion protein comprises an ActRIIA domain comprising 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 amino acids 30-1 10 of SEQ ID NO: 137.
  • the ActRIIA-Fc fusion protein comprises an ActRIIA domain comprising 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 amino acids 21-135 of SEQ ID NO: 137.
  • the ActRIIA-Fc fusion protein comprises an ActRIIA domain comprising 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 amino acid sequence of any one of SEQ ID Nos: 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147.
  • the ActRIIA-Fc polypeptide sequence (SEQ ID NO: 140) is shown below:

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Cited By (19)

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CN109293763A (zh) * 2018-10-26 2019-02-01 中国农业科学院特产研究所 水貂激活素b蛋白及其制备与应用
WO2019213446A1 (en) * 2018-05-03 2019-11-07 Acceleron Pharma Inc. NOVEL BINDERS OF TGFβ-SUPERFAMILY LIGANDS AND USES THEREOF
WO2019213442A1 (en) * 2018-05-03 2019-11-07 Acceleron Pharma Inc. MULTISPECIFIC BINDERS OF TGFβ-SUPERFAMILY LIGANDS AND USES THEREOF
WO2019241625A1 (en) * 2018-06-15 2019-12-19 Acceleron Pharma Inc. Bi-and tri-functional fusion proteins and uses thereof
US10913782B2 (en) 2015-04-22 2021-02-09 Biogen Ma Inc. Hybrid ActRIIB ligand trap proteins for treating muscle wasting diseases
US11013785B2 (en) 2016-11-10 2021-05-25 Keros Therapeutics, Inc. Activin receptor type IIA variants and methods of use thereof
JP2021523148A (ja) * 2018-05-09 2021-09-02 ケロス セラピューティクス インコーポレイテッドＫｅｒｏｓ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． アクチビンｉｉａ型受容体変異体およびそれらの使用方法
AU2017345435B2 (en) * 2016-10-20 2021-11-11 Alivegen Inc. Methods for treating muscle wasting and bone disease using novel hybrid ActRIIB ligand trap proteins
WO2022150590A1 (en) * 2021-01-08 2022-07-14 Acceleron Pharma Inc. Compositions and methods for treating pulmonary hypertension
US11471510B2 (en) 2014-12-03 2022-10-18 Celgene Corporation Activin-ActRII antagonists and uses for treating anemia
US11484573B2 (en) 2017-11-09 2022-11-01 Keros Therapeutics, Inc. Activin receptor type IIa variants and methods of use thereof
US20230183319A1 (en) * 2020-02-03 2023-06-15 Acceleron Pharma Inc. Variant actriib proteins and uses thereof
US11813308B2 (en) 2014-10-09 2023-11-14 Celgene Corporation Treatment of cardiovascular disease using ActRII ligand traps
US11884715B2 (en) 2018-01-12 2024-01-30 Keros Therapeutics, Inc. Activin receptor type IIB variants and methods of use thereof
US11945856B2 (en) 2022-01-28 2024-04-02 35Pharma Inc. Activin receptor type IIB variants and uses thereof
US12024563B2 (en) 2016-04-06 2024-07-02 Acceleron Pharma Inc. ALK7 antagonists and uses thereof
US12186370B1 (en) 2020-11-05 2025-01-07 Celgene Corporation ACTRIIB ligand trap compositions and uses thereof
US12240887B2 (en) 2016-10-05 2025-03-04 Acceleron Pharma Inc. Variant ActRIIB proteins and uses thereof
US12440539B2 (en) 2020-03-20 2025-10-14 Keros Therapeutics, Inc. Methods of using activin receptor type IIB variants

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220088699A (ko) 2019-09-27 2022-06-28 디스크 메디슨, 인크. 골수섬유증 및 관련 상태의 치료 방법
WO2021189006A1 (en) * 2020-03-20 2021-09-23 Keros Therapeutics, Inc. Methods of using activin receptor type iia variants
KR20230012539A (ko) 2020-05-13 2023-01-26 디스크 메디슨, 인크. 골수섬유증을 치료하기 위한 항-헤모주벨린 (hjv) 항체
EP4171603A4 (en) * 2020-06-25 2024-06-26 Gliknik Inc. ACE2-FC FUSION PROTEINS AND METHODS OF USE

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984001106A1 (en) 1982-09-24 1984-03-29 Us Health Repair of tissue in animals
EP0148155A2 (en) 1984-01-04 1985-07-10 International Genetic Engineering, Inc. (Ingene) Osteogenic factors
EP0169016A2 (en) 1984-07-16 1986-01-22 Celtrix Pharmaceuticals, Inc. Polypeptide cartilage-inducing factors found in bone
WO1987005330A1 (en) 1986-03-07 1987-09-11 Michel Louis Eugene Bergh Method for enhancing glycoprotein stability
WO1993011162A1 (en) 1991-11-29 1993-06-10 Protein Design Labs, Inc. Bispecific antibody heterodimers
US5283317A (en) 1987-08-03 1994-02-01 Ddi Pharmaceuticals, Inc. Intermediates for conjugation of polypeptides with high molecular weight polyalkylene glycols
US5525490A (en) 1994-03-29 1996-06-11 Onyx Pharmaceuticals, Inc. Reverse two-hybrid method
WO1996026432A1 (en) 1995-02-23 1996-08-29 University Of Utah Research Foundation Composite waveguide for solid phase binding assays
US5677196A (en) 1993-05-18 1997-10-14 University Of Utah Research Foundation Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5955280A (en) 1995-04-11 1999-09-21 The General Hospital Corporation Reverse two-hybrid system
WO2000043781A2 (en) 1999-01-21 2000-07-27 Metamorphix, Inc. Growth differentiation factor inhibitors and uses therefor
US6521750B2 (en) 1997-07-30 2003-02-18 Univ Emory Bone mineralization proteins, DNA, vectors, expression systems
WO2006012627A2 (en) 2004-07-23 2006-02-02 Acceleron Pharma Inc. Actrii receptor polypeptides, methods and compositions
US7183076B2 (en) 1997-05-02 2007-02-27 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
WO2007147901A1 (en) 2006-06-22 2007-12-27 Novo Nordisk A/S Production of bispecific antibodies
WO2008097541A2 (en) 2007-02-02 2008-08-14 Acceleron Pharma Inc. Variants derived from actriib and uses therefor
WO2009089004A1 (en) 2008-01-07 2009-07-16 Amgen Inc. Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
WO2010019261A1 (en) 2008-08-14 2010-02-18 Acceleron Pharma Inc. Use of gdf traps to increase red blood cell levels
WO2010151426A1 (en) 2009-06-12 2010-12-29 Acceleron Pharma Inc. Truncated actriib-fc fusion proteins
WO2011034605A2 (en) 2009-09-16 2011-03-24 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
US8338377B2 (en) 2009-03-30 2012-12-25 Acceleron Pharma Inc. BMP-ALK3 antagonists and uses for promoting bone growth
WO2013059347A1 (en) * 2011-10-17 2013-04-25 Acceleron Pharma, Inc. Methods and compositions for treating ineffective erythropoiesis
WO2015143403A1 (en) * 2014-03-21 2015-09-24 Acceleron Pharma, Inc. Methods for increasing red blood cell levels and treating ineffective erythropoiesis by inhibiting activin b and/or gdf11
WO2015161220A1 (en) * 2014-04-18 2015-10-22 Acceleron Pharma, Inc. Methods for increasing red blood cell levels and treating sickle-cell disease
WO2015192111A1 (en) * 2014-06-13 2015-12-17 Acceleron Pharma, Inc. Methods and compositions for treating ulcers

Family Cites Families (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050186593A1 (en) * 1991-05-10 2005-08-25 The Salk Institute For Biological Studies Cloning and recombinant production of CRF receptor(s)
EP1729793B1 (en) 2004-03-02 2015-07-22 Acceleron Pharma Inc. Alk7 and myostatin inhibitors for muscle wasting disorders
US20100028332A1 (en) 2006-12-18 2010-02-04 Acceleron Pharma Inc. Antagonists of actriib and uses for increasing red blood cell levels
EP2468291B1 (en) 2006-12-18 2017-11-22 Acceleron Pharma, Inc. Activin-actrii antagonists and uses for increasing red blood cell levels
TWI586364B (zh) 2008-05-02 2017-06-11 艾西利羅製藥公司 調節血管新生與周圍細胞組成的方法與組合物
NZ590326A (en) 2008-06-26 2013-05-31 Acceleron Pharma Inc Antagonists of activin-actriia and uses for increasing red blood cell levels
US8216997B2 (en) 2008-08-14 2012-07-10 Acceleron Pharma, Inc. Methods for increasing red blood cell levels and treating anemia using a combination of GDF traps and erythropoietin receptor activators
EP2387412A4 (en) 2009-01-13 2013-04-03 Acceleron Pharma Inc PROCESS FOR INCREASING THE ADIPONECTIN MIRROR
ES2796121T3 (es) 2009-08-13 2020-11-25 Acceleron Pharma Inc Uso combinado de trampas de GDF y activadores del receptor de la eritropoyetina para aumentar los niveles de glóbulos rojos
CA2773494A1 (en) * 2009-09-09 2011-03-17 Acceleron Pharma Inc. Actriib antagonists and dosing and uses thereof
WO2012027065A2 (en) 2010-08-27 2012-03-01 Celgene Corporation Combination therapy for treatment of disease
CN102850458B (zh) 2011-06-28 2014-10-01 华博生物医药技术（上海）有限公司 新型重组双功能融合蛋白及其制法和用途
WO2013063536A1 (en) 2011-10-27 2013-05-02 Acceleron Pharma, Inc. Actriib binding agents and uses thereof
CN105658672A (zh) 2013-08-22 2016-06-08 阿塞勒隆制药公司 TGF-β受体II型变体及其用途
SMT202300166T1 (it) 2014-12-03 2023-07-20 Celgene Corp Antagonisti di attivina- actrii e usi per il trattamento di sindrome mielodisplastica
MA41919A (fr) 2015-04-06 2018-02-13 Acceleron Pharma Inc Hétéromultimères alk4:actriib et leurs utilisations
BR112017021510A2 (pt) 2015-04-06 2018-07-03 Acceleron Pharma Inc heteromultímeros do receptor tipo i e tipo ii da superfamília tgf-beta e sua utilização
JP6810702B2 (ja) 2015-04-06 2021-01-06 アクセルロン ファーマ インコーポレイテッド シングルアームｉ型およびｉｉ型受容体融合タンパク質およびその使用
RS61881B1 (sr) 2015-04-22 2021-06-30 Biogen Ma Inc Novi hibridni actriib proteini zamke za ligand za lečenje bolesti gubljenja mišića
WO2016205370A1 (en) 2015-06-15 2016-12-22 Santa Maria Biotherapeutics, Inc. Methods of using activin receptor iib-based proteins
EP3344660B1 (en) 2015-08-31 2025-03-05 National Research Council of Canada Tgf-beta-receptor ectodomain fusion molecules and uses thereof
EP3439741A4 (en) 2016-04-06 2020-05-06 Acceleron Pharma Inc. ALK7 ANTAGONISTS AND USES THEREOF
AU2017293778B2 (en) 2016-07-07 2022-03-24 Acceleron Pharma Inc. TGF-beta superfamily heteromultimers and uses thereof
WO2018067874A1 (en) 2016-10-05 2018-04-12 Acceleron Pharma Inc. Variant actriib proteins and uses thereof
EP3522913A4 (en) 2016-10-05 2020-10-28 Acceleron Pharma Inc. ALK4 HETEROMULTIMERS: ACTRIIB AND THEIR USES
AU2017338915B2 (en) 2016-10-05 2024-11-21 Acceleron Pharma Inc. TGF-beta superfamily type I and type II receptor heteromultimers and uses thereof
JOP20190085A1 (ar) 2016-10-20 2019-04-17 Biogen Ma Inc طرق علاج الضمور العضلي ومرض العظام باستخدام بروتينات احتجاز مركب ترابطي actriib هجين حديثة
EP3538123A4 (en) 2016-11-10 2020-10-14 Keros Therapeutics, Inc. ACTIVIN RECEPTOR TYPE IIA VARIANTS AND THEIR METHODS OF USE

Patent Citations (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984001106A1 (en) 1982-09-24 1984-03-29 Us Health Repair of tissue in animals
EP0148155A2 (en) 1984-01-04 1985-07-10 International Genetic Engineering, Inc. (Ingene) Osteogenic factors
EP0169016A2 (en) 1984-07-16 1986-01-22 Celtrix Pharmaceuticals, Inc. Polypeptide cartilage-inducing factors found in bone
WO1987005330A1 (en) 1986-03-07 1987-09-11 Michel Louis Eugene Bergh Method for enhancing glycoprotein stability
US5283317A (en) 1987-08-03 1994-02-01 Ddi Pharmaceuticals, Inc. Intermediates for conjugation of polypeptides with high molecular weight polyalkylene glycols
WO1993011162A1 (en) 1991-11-29 1993-06-10 Protein Design Labs, Inc. Bispecific antibody heterodimers
US5932448A (en) 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
US5677196A (en) 1993-05-18 1997-10-14 University Of Utah Research Foundation Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays
US5525490A (en) 1994-03-29 1996-06-11 Onyx Pharmaceuticals, Inc. Reverse two-hybrid method
WO1996026432A1 (en) 1995-02-23 1996-08-29 University Of Utah Research Foundation Composite waveguide for solid phase binding assays
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5955280A (en) 1995-04-11 1999-09-21 The General Hospital Corporation Reverse two-hybrid system
US5965368A (en) 1995-04-11 1999-10-12 The General Hospital Corporation Reverse two-hybrid systems
US7183076B2 (en) 1997-05-02 2007-02-27 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
US6521750B2 (en) 1997-07-30 2003-02-18 Univ Emory Bone mineralization proteins, DNA, vectors, expression systems
WO2000043781A2 (en) 1999-01-21 2000-07-27 Metamorphix, Inc. Growth differentiation factor inhibitors and uses therefor
WO2006012627A2 (en) 2004-07-23 2006-02-02 Acceleron Pharma Inc. Actrii receptor polypeptides, methods and compositions
WO2007147901A1 (en) 2006-06-22 2007-12-27 Novo Nordisk A/S Production of bispecific antibodies
WO2008097541A2 (en) 2007-02-02 2008-08-14 Acceleron Pharma Inc. Variants derived from actriib and uses therefor
WO2009089004A1 (en) 2008-01-07 2009-07-16 Amgen Inc. Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
US8592562B2 (en) 2008-01-07 2013-11-26 Amgen Inc. Method for making antibody Fc-heterodimeric molecules using electrostatic steering effects
WO2010019261A1 (en) 2008-08-14 2010-02-18 Acceleron Pharma Inc. Use of gdf traps to increase red blood cell levels
US8338377B2 (en) 2009-03-30 2012-12-25 Acceleron Pharma Inc. BMP-ALK3 antagonists and uses for promoting bone growth
WO2010151426A1 (en) 2009-06-12 2010-12-29 Acceleron Pharma Inc. Truncated actriib-fc fusion proteins
US20120302737A1 (en) 2009-09-16 2012-11-29 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
WO2011034605A2 (en) 2009-09-16 2011-03-24 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
WO2013059347A1 (en) * 2011-10-17 2013-04-25 Acceleron Pharma, Inc. Methods and compositions for treating ineffective erythropoiesis
WO2015143403A1 (en) * 2014-03-21 2015-09-24 Acceleron Pharma, Inc. Methods for increasing red blood cell levels and treating ineffective erythropoiesis by inhibiting activin b and/or gdf11
WO2015161220A1 (en) * 2014-04-18 2015-10-22 Acceleron Pharma, Inc. Methods for increasing red blood cell levels and treating sickle-cell disease
WO2015192111A1 (en) * 2014-06-13 2015-12-17 Acceleron Pharma, Inc. Methods and compositions for treating ulcers

Non-Patent Citations (103)

* Cited by examiner, † Cited by third party
Title
"Gene Expression Technology: Methods in Enzymology", 1990, ACADEMIC PRESS
"Laboratory Manual", 1989, COLD SPRING HARBOR
AIZAWA ET AL., AM J HEMATOL, vol. 74, 2003, pages 68 - 72
AMTHOR ET AL., DEV BIOL., vol. 251, 2002, pages 241 - 57
APLINWRISTON, CRC CRIT. REV. BIOCHEM., 1981, pages 259 - 306
ASHMORE ET AL., GROWTH, vol. 38, 1974, pages 501 - 507
ATTISANO ET AL., CELL, vol. 68, 1992, pages 97 - 108
ATTISANO ET AL., CELL, vol. 68, no. 1, 10 January 1992 (1992-01-10), pages 97 - 108
BARTEL ET AL., BIOTECHNIQUES, vol. 14, 1993, pages 920 - 924
BELL ET AL., OBES RES., vol. 8, 2000, pages 249 - 54
BLOOD REV, vol. 26, 2012, pages S16 - S19
BRON ET AL., SEMIN ONCOL, vol. 28, no. 8, 2001, pages 1 - 6
BROWN ET AL., GROWTH FACTORS, vol. 3, 1990, pages 35 - 43
BUNN, N ENGL J MED, vol. 346, no. 7, 2002, pages 522 - 523
CAO ET AL., PEDIATR REP, vol. 3, no. 2, 2011, pages e17
CHENG ET AL., J BONE JOINT SURG AM., vol. 85, no. 8, 2003, pages 1544 - 52
DALUISKI ET AL., NAT GENET., vol. 27, no. 1, 2001, pages 84 - 8
DAVIS ET AL., PROTEIN ENG DES SEL, vol. 23, 2010, pages 195 - 202
DAVIS ET AL., PROTEIN ENG DESIGN SEL, vol. 23, 2010, pages 195 - 202
DELANTY ET AL., NEUROLOGY, vol. 49, 1997, pages 686 - 689
DENG ET AL., ENDOCRINOLOGY, vol. 141, 2000, pages 2370 - 6
DENNLER ET AL., EMBO, vol. 17, 1998, pages 3091 - 3100
DEPAOLO ET AL., PROC SOCEP BIOL MED., vol. 198, 1991, pages 500 - 512
DI MATTEO ET AL., J BIOL REGUL HOMEOST AGENTS, vol. 22, 2008, pages 211 - 216
DYSON ET AL., CURR BIOL., vol. 7, 1997, pages 81 - 84
EDGE ET AL., ANAL. BIOCHEM., vol. 118, 1981, pages 131
ESPOSITO ET AL., BLOOD, vol. 102, 2003, pages 2670 - 2677
FRALEY ET AL., TRENDS BIOCHEM. SCI., vol. 6, 1981, pages 77
GAFTER ET AL., KIDNEY INT, vol. 45, 1994, pages 224 - 231
GALANELLO ET AL., ANN NY ACAD SCI, vol. 1202, 2010, pages 79 - 86
GALANELLO ET AL., ORPHANET J RARE DIS, vol. 5, 2010, pages 11
GAMER ET AL., DEV BIOL., vol. 208, 1999, pages 222 - 32
GAMER ET AL., DEV BIOL., vol. 229, 2001, pages 407 - 20
GAMER ET AL., DEV. BIOL., vol. 208, 1999, pages 222 - 232
GANZ, J AM SOC NEPHROL, vol. 18, 2007, pages 394 - 400
GARDENGHI ET AL., J CLIN INVEST, vol. 120, 2010, pages 4466 - 4477
GONZALEZ-CADAVID ET AL., PNAS, vol. 95, 1998, pages 14938 - 43
GROBET ET AL., NAT GENET., vol. 17, no. 1, 1997, pages 71 - 4
GROOPMAN ET AL., J NATL CANCER INST, vol. 91, 1999, pages 1616 - 1634
HAIDAR ET AL., BONE, vol. 48, 2011, pages 425 - 432
HAKIMUDDIN ET AL., ARCH. BIOCHEM. BIOPHYS., vol. 259, 1987, pages 52
HERSHKO, HAEMATOLOGICA, vol. 91, 2006, pages 1307 - 1312
HILDEN ET AL., BLOOD, vol. 83, no. 8, 15 April 1994 (1994-04-15), pages 2163 - 70
HINO ET AL., FRONT BIOSCI., vol. 9, 2004, pages 1520 - 9
HOCHULI ET AL., J. CHROMATOGRAPHY, vol. 411, 1987, pages 177
IWABUCHI ET AL., ONCOGENE, vol. 8, 1993, pages 1693 - 1696
JACOBS ET AL., NEPHROL DIAL TRANSPLANT, vol. 15, no. 4, 2000, pages 51 - 56
JAKOBY WB ET AL., METHODS IN ENZYMOLOGY, vol. 46, 1974, pages 1
JANKNECHT ET AL., PNAS USA, vol. 88, pages 8972
KALINOWSKI ET AL., PHARMACOL REV, vol. 57, 2005, pages 547 - 583
KAMBADUR ET AL., GENOME RES., vol. 7, 1997, pages 910 - 915
KLEIN ET AL., MABS, vol. 4, 2012, pages 653 - 663
KUBO ET AL., STEROID BIOCHEMISTRY & MOLECULAR BIOLOGY, vol. 68, 1999, pages 197 - 202
LEEMCPHERRON, PROC. NATL. ACAD. SCI., vol. 98, 2001, pages 9306 - 9311
LEKAWANVIJIT ET AL., CAN J CARDIOL, vol. 25, 2009, pages 213 - 218
LEVIN ET AL., AM J KIDNEY DIS, vol. 27, 1999, pages 347 - 354
MACIAS-SILVA ET AL., J BIOL CHEM., vol. 273, 1998, pages 25628 - 36
MADURA ET AL., J BIOL CHEM, vol. 268, 1993, pages 12046 - 12054
MANNINO ET AL., BIOTECHNIQUES, vol. 6, 1988, pages 682
MASSAGU6, NAT. REV. MOL. CELL BIOL., vol. 1, 2000, pages 169 - 178
MASUNO ET AL., TOXICOL SCI., vol. 75, 2003, pages 314 - 20
MATHEWSVALE, CELL, vol. 65, 1991, pages 973 - 982
MCPHERRON ET AL., NAT. GENET., vol. 22, 1999, pages 260 - 264
MCPHERRON ET AL., NATURE, vol. 387, 1997, pages 83 - 90
MCPHERRONLEE, PROC. NATL. ACAD. SCI. USA, vol. 94, 1997, pages 12457 - 12461
MERCHANT ET AL., NAT BIOTECH, vol. 16, 1998, pages 677 - 681
MURATA ET AL., PNAS, vol. 85, 1988, pages 2434
MUSALLAM ET AL., COLD SPRING HARB PERSPECT MED, vol. 2, 2012, pages a013482
NAKASHIMA ET AL., MECH. DEV., vol. 80, 1999, pages 185 - 189
NAMKUNG-MATTHAI ET AL., BONE, vol. 28, 2001, pages 80 - 86
NISSENSON, AM J KIDNEY DIS, vol. 20, no. 1, 1992, pages 21 - 24
OH ET AL., GENES DEV., vol. 16, 2002, pages 2749 - 54
PACK & PLUECKTHUN, BIOCHEMISTRY, vol. 31, 1992, pages 1579 - 1584
PACK ET AL., BIO/TECHNOLOGY, vol. 11, 1993, pages 1271 - 1277
PICO ET AL., MOL CELL BIOCHEM., vol. 189, 1998, pages 1 - 7
PIPPARD ET AL., LANCET, vol. 2, 1979, pages 819 - 821
REUSCH ET AL., MOL CELL BIOL., vol. 20, 2000, pages 1008 - 20
REVICKI ET AL., AM J KIDNEY DIS, vol. 25, 1995, pages 548 - 554
RICKETTS ET AL., CLIN NUCL MED, vol. 3, 1978, pages 159 - 164
RIDGWAY ET AL., PROTEIN ENG, vol. 9, 1996, pages 617 - 621
ROBERTS ET AL., BIOL REPROD, vol. 68, 2003, pages 1719 - 1726
RUND ET AL., N ENGL J MED, vol. 353, 2005, pages 1135 - 1146
SAKUMA ET AL., GENES CELLS., vol. 7, 2002, pages 401 - 12
SCHRIER, CURR OPIN HEMATOL, vol. 9, 2002, pages 123 - 126
SCHUELKE ET AL., N ENGL J MED, vol. 350, 2004, pages 2682 - 8
See also references of EP3523328A4
SINGIBARTI, J. CLIN INVESTIG, vol. 72, 1994, pages S36 - S43
SPIESS ET AL., MOLECULAR IMMUNOLOGY, vol. 67, 2015, pages 95 - 106
SWATLANDKIEFFER, J. ANIM. SCI., vol. 38, 1994, pages 752 - 757
TANNO ET AL., ADV HEMATOL, 2010, pages 750643
THOMAS ET AL., J BIOL CHEM., vol. 275, 2000, pages 40235 - 43
THOTAKURA ET AL., METH. ENZYMOL., vol. 138, 1987, pages 350
VICHINSKY, ANN NY ACAD SCI, vol. 1054, 2005, pages 18 - 24
VIDALLEGRAIN, NUCLEIC ACIDS RES, vol. 27, 1999, pages 919 - 29
VIDALLEGRAIN, TRENDS BIOTECHNOL, vol. 17, 1999, pages 374 - 81
WAKEFIELD ET AL., J. BIOL. CHEM., vol. 263, 1988, pages 7646 - 7654
WHITTEMORE ET AL., BIOCHEM BIOPHYS RES COMMUN., vol. 300, 2003, pages 965 - 71
WOODRUFF, BIOCHEM PHARMACOL., vol. 55, 1998, pages 953 - 963
WRANIK ET AL., J BIOL CHEM, vol. 287, 2012, pages 43331 - 43339
WU ET AL., NEURON., vol. 37, 2003, pages 197 - 207
YAMASHITA ET AL., J. CELL BIOL., vol. 130, 1995, pages 217 - 226
YEOWHITMAN, MOL. CELL, vol. 7, 2001, pages 949 - 957
ZERVOS ET AL., CELL, vol. 72, 1993, pages 223 - 232

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11813308B2 (en) 2014-10-09 2023-11-14 Celgene Corporation Treatment of cardiovascular disease using ActRII ligand traps
US11471510B2 (en) 2014-12-03 2022-10-18 Celgene Corporation Activin-ActRII antagonists and uses for treating anemia
US11292826B2 (en) 2015-04-22 2022-04-05 Biogen Ma Inc. Hybrid ActRIIB ligand trap proteins for treating muscle wasting diseases
US11932677B2 (en) 2015-04-22 2024-03-19 Alivegen Inc. Hybrid ActRIIB ligand trap proteins for treating muscle wasting diseases
US10913782B2 (en) 2015-04-22 2021-02-09 Biogen Ma Inc. Hybrid ActRIIB ligand trap proteins for treating muscle wasting diseases
US12024563B2 (en) 2016-04-06 2024-07-02 Acceleron Pharma Inc. ALK7 antagonists and uses thereof
US12240887B2 (en) 2016-10-05 2025-03-04 Acceleron Pharma Inc. Variant ActRIIB proteins and uses thereof
AU2017345435B2 (en) * 2016-10-20 2021-11-11 Alivegen Inc. Methods for treating muscle wasting and bone disease using novel hybrid ActRIIB ligand trap proteins
US12187783B2 (en) 2016-10-20 2025-01-07 Alivegen Inc Methods for treating muscle wasting and bone disease using novel hybrid ActRIIB ligand trap proteins
US11013785B2 (en) 2016-11-10 2021-05-25 Keros Therapeutics, Inc. Activin receptor type IIA variants and methods of use thereof
US11090361B2 (en) 2016-11-10 2021-08-17 Keros Therapeutics, Inc. Activin receptor type IIa variants and methods of use thereof
US12350313B2 (en) 2016-11-10 2025-07-08 Keros Therapeutics, Inc. Activin receptor type IIa variants and methods of use thereof
US11717558B2 (en) 2016-11-10 2023-08-08 Keros Therapeutics, Inc. Nucleic acid molecules encoding activin receptor type IIA variants
US11484573B2 (en) 2017-11-09 2022-11-01 Keros Therapeutics, Inc. Activin receptor type IIa variants and methods of use thereof
US11884715B2 (en) 2018-01-12 2024-01-30 Keros Therapeutics, Inc. Activin receptor type IIB variants and methods of use thereof
US12269858B2 (en) 2018-01-12 2025-04-08 Keros Therapeutics, Inc. Activin receptor type IIB variants and methods of use thereof
WO2019213442A1 (en) * 2018-05-03 2019-11-07 Acceleron Pharma Inc. MULTISPECIFIC BINDERS OF TGFβ-SUPERFAMILY LIGANDS AND USES THEREOF
US12103959B2 (en) 2018-05-03 2024-10-01 Acceleron Pharma Inc. Multispecific binders of TGFBeta-superfamily ligands and uses thereof
WO2019213446A1 (en) * 2018-05-03 2019-11-07 Acceleron Pharma Inc. NOVEL BINDERS OF TGFβ-SUPERFAMILY LIGANDS AND USES THEREOF
US12168683B2 (en) 2018-05-03 2024-12-17 Acceleron Pharma Inc. Binders of TGFβ-superfamily ligands and uses thereof
JP2021523148A (ja) * 2018-05-09 2021-09-02 ケロス セラピューティクス インコーポレイテッドＫｅｒｏｓ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． アクチビンｉｉａ型受容体変異体およびそれらの使用方法
EP3790572A4 (en) * 2018-05-09 2022-03-16 Keros Therapeutics, Inc. ACTIVIN RECEPTOR TYPE IIA VARIANTS AND METHODS OF USE THEREOF
JP7405772B2 (ja) 2018-05-09 2023-12-26 ケロス セラピューティクス インコーポレイテッド アクチビンｉｉａ型受容体変異体および同変異体を含む医薬組成物
US12364737B2 (en) 2018-05-09 2025-07-22 Keros Therapeutics, Inc. Activin receptor type IIA variants and methods of use thereof
JP7725551B2 (ja) 2018-05-09 2025-08-19 ケロス セラピューティクス インコーポレイテッド アクチビンｉｉａ型受容体変異体を含む医薬組成物
JP2024023673A (ja) * 2018-05-09 2024-02-21 ケロス セラピューティクス インコーポレイテッド アクチビンｉｉａ型受容体変異体およびそれらの使用方法
WO2019241625A1 (en) * 2018-06-15 2019-12-19 Acceleron Pharma Inc. Bi-and tri-functional fusion proteins and uses thereof
CN109293763A (zh) * 2018-10-26 2019-02-01 中国农业科学院特产研究所 水貂激活素b蛋白及其制备与应用
EP4100431A4 (en) * 2020-02-03 2024-10-16 Acceleron Pharma Inc. Variant actriib proteins and uses thereof
US20230183319A1 (en) * 2020-02-03 2023-06-15 Acceleron Pharma Inc. Variant actriib proteins and uses thereof
US12440539B2 (en) 2020-03-20 2025-10-14 Keros Therapeutics, Inc. Methods of using activin receptor type IIB variants
US12186370B1 (en) 2020-11-05 2025-01-07 Celgene Corporation ACTRIIB ligand trap compositions and uses thereof
WO2022150590A1 (en) * 2021-01-08 2022-07-14 Acceleron Pharma Inc. Compositions and methods for treating pulmonary hypertension
US11945856B2 (en) 2022-01-28 2024-04-02 35Pharma Inc. Activin receptor type IIB variants and uses thereof
US12421296B2 (en) 2022-01-28 2025-09-23 35Pharma Inc. Activin receptor type IIB variants and uses thereof

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