ACTIVIN RECEPTOR TYPE IIB VARIANTS AND USES THEREOF
There are provided polypeptides that include an Activin receptor type IIB (ActRIIB) ectodomain (ECD) variant. In some embodiments, a polypeptide of the disclosure includes an ActRIIB-ECD variant fused to an Fc domain moiety. The disclosure also provides pharmaceutical compositions and methods of using the polypeptides to treat diseases and conditions associated with TGFβ superfamily ligand signaling, such as metabolic disorders, diabetes, obesity, cardiometabolic disease, pulmonary hypertension, fibrosis, muscle weakness and atrophy, bone damage, and/or low red blood cell levels (such as anemia).
1 . A polypeptide, comprising an amino acid sequence that is at least 90% identical to an Activin receptor type IIB (ActRIIB) ectodomain (ECD) variant, the ActRIIB ECD variant comprising a substitution of two or more amino acid residues corresponding to two or more of the following amino acid residues of SEQ ID NO. 2: L33, F58, G27, T69, L14, D30, K31, Q29, and R32.
2 . The polypeptide of claim 1 , wherein the ActRIIB ECD variant demonstrates reduced inhibition of BMP-9 and/or BMP-10, as compared to a wild-type ActRIIB ectodomain.
3 . The polypeptide of claim 1 or claim 2 , wherein the ActRIIB ECD variant demonstrates increased inhibition of activin A, as compared to a wild-type ActRIIB ectodomain.
4 . The polypeptide of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises an amino acid sequence having at least 90% identity to a wild-type ActRIIB ectodomain.
5 . The polypeptide of any one of claims 1-4 , wherein the ActRIIB ECD variant comprises an amino acid sequence having at least 90% identity to a wild-type ActRIIB ectodomain and comprises a substitution of two or more amino acid residues selected from: L33, F58, G27, T69, L14, D30, K31, Q29, and R32.
6 . The polypeptide of any one of claims 1-5 , wherein the ActRIIB ECD variant comprises a substitution of two amino acid residues corresponding to two of the following amino acid residues of SEQ ID NO: 2: L33, F58, G27, T69, L14, D30, K31, Q29, and R32.
7 . The polypeptide of any one of claims 2-5 , wherein the amino acid sequence of the wild-type ActRIIB ectodomain is SEQ ID NO: 2.
8 . The polypeptide of any one of claims 1-7 , wherein the ActRIIB ECD variant comprises an amino acid substitution of L33 and an amino acid substitution of F58.
9 . The polypeptide of claim 8 , wherein the ActRIIB ECD variant comprises the amino acid substitution of L33Y and an amino acid substitution selected from the following: F58Q, F58E, F58I, F58V, F58A, F58D, F58N, F58R, and F58H.
10 . The polypeptide of claim 8 , wherein the ActRIIB ECD variant comprises the amino acid substitution of L33Q and an amino acid substitution selected from the following: F58Q and F58E.
11 . The polypeptide of claim 8 , wherein the ActRIIB ECD variant comprises the amino acid substitution of L33W and an amino acid substitution selected from the following: F58V, F58A, F58E, F58Q, F58D, F58N, F58R and F58H.
12 . The polypeptide of claim 8 , wherein the ActRIIB ECD variant comprises the amino acid substitution of L33M and F58Q.
13 . The polypeptide of any one of claims 1-7 , wherein the ActRIIB ECD variant comprises an amino acid substitution of G27 and an amino acid substitution of F58.
14 . The polypeptide of claim 13 , wherein the ActRIIB ECD variant comprises the amino acid substitution of F58Q and an amino acid substitution selected from the following: G27D, G27N, G27T, G27H, G27E, G27S, G27Q, and G27M.
15 . The polypeptide of claim 13 , wherein the ActRIIB ECD variant comprises the amino acid substitution of F58E and an amino acid substitution selected from the following: G27D, G27E, G27N and G27Q.
16 . The polypeptide of any one of claims 1-7 , wherein the ActRIIB ECD variant comprises an amino acid substitution of T69 and an amino acid substitution of F58.
17 . The polypeptide of claim 16 , wherein the ActRIIB ECD variant comprises the amino acid substitution of T69R and F58Q.
18 . The polypeptide of any one of claims 1-7 , wherein the ActRIIB ECD variant comprises an amino acid substitution of L14 and an amino acid substitution of F58.
19 . The polypeptide of claim 18 , wherein the ActRIIB ECD variant comprises
(a) the amino acid substitution of L14E and an amino acid substitution selected from the following: F58Q, F58E, F58D, F58N, F58R and F58H;
(b) the ActRIIB ECD variant comprises the amino acid substitution of L14D and an amino acid substitution of L33W or L33Y; or
(c) the ActRIIB ECD variant comprises the amino acid substitution of L14N and an amino acid substitution of L33W or L33Y.
20 . The polypeptide of any one of claims 1-19 , wherein the ActRIIB ECD variant
(a) comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 5-56; or
(b) comprises or consists of the amino acid sequence of any one of SEQ ID NO: 5-56.
21 . The polypeptide of any one of claims 1-20 , wherein the ActRIIB ECD variant further comprises one or more additional amino acids at the N or C terminus.
22 . The polypeptide of claim 21 , wherein the ActRIIB ECD variant further comprises the following amino acids at the N terminus: GRGEA (SEQ ID NO: 63) and/or the following amino acids at the C-terminus: APT (SEQ ID NO: 64).
23 . The polypeptide of any one of claims 1-22 , further comprising an Fc domain monomer.
24 . The polypeptide of claim 23 , further comprising a peptide linker positioned between the ActRIIB ECD variant and the Fc domain monomer.
25 . The polypeptide of claim 24 , comprising the following structure, from N- to C-terminus: ActRIIB-ECD-peptide linker-Fc domain monomer.
26 . The polypeptide of claim 24 , comprising the following structure, from N- to C-terminus: a first ActRIIB-ECD—a first peptide linker—a second ActRIIB-ECD—a second peptide linker—Fc domain monomer.
27 . The polypeptide of claim 26 , wherein the first ActRIIB-ECD and the second ActRIIB-ECD are the same.
28 . The polypeptide of claim 26 , wherein the first ActRIIB-ECD and the second ActRIIB-ECD are different.
29 . The polypeptide of claim 26 , wherein each of the first ActRIIB-ECD and the second ActRIIB-ECD comprises the amino acid substitutions of L33Y and F58Q.
30 . The polypeptide of claim 26 , wherein the first peptide linker and the second peptide linker are the same.
31 . The polypeptide of claim 26 , wherein the first peptide linker and the second peptide linker are different.
32 . The polypeptide of claim 26 , wherein the first peptide linker is 9 amino acids long.
33 . The polypeptide of claim 26 , wherein the second peptide linker is 14 amino acids long or 3 amino acids long.
34 . The polypeptide of any one of claims 23-33 , wherein the Fc domain monomer is an IgG1, IgG2, IgG3 or IgG4 isotype.
35 . The polypeptide of any one of claims 23-34 , wherein the Fc domain monomer is a human Fc domain monomer or a murine Fc domain monomer.
36 . The polypeptide of any one of claims 23-35 , wherein the Fc domain monomer is engineered to reduce aggregation or to modulate stability of a dimer of the polypeptide.
37 . The polypeptide of any one of claims 23-36 , wherein the Fc domain monomer comprises the amino acid substitutions of M252Y, S254T, and T256E (YTE).
38 . The polypeptide of claim 37 , wherein the Fc domain monomer comprises the M252Y amino acid substitution.
39 . The polypeptide of any one of claims 23-38 , wherein the Fc domain monomer includes a D at position 356 and an L at position 358 (DL).
40 . The polypeptide of any one of claims 23-38 , wherein the Fc domain monomer includes an E at position 356 and an M at position 358 (EM).
41 . The polypeptide of any one of claims 23-40 , wherein the Fc domain monomer further comprises a Lysine residue (K) at the C terminus.
42 . The polypeptide of any one of claims 23-41 , wherein the Fc domain monomer comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 252-292.
43 . The polypeptide of claim 42 , wherein the Fc domain monomer comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 252-292.
44 . The polypeptide of any one of claims 23-43 , wherein the Fc domain monomer is an IgG1 isotype.
45 . The polypeptide of claim 44 , wherein the Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 253, SEQ ID NO: 255, or SEQ ID NO: 256.
46 . The polypeptide of any one of claims 23-45 , wherein the Fc domain monomer forms a dimer.
47 . The polypeptide of any one of claims 24-46 , wherein the peptide linker is glycine-rich.
48 . The polypeptide of claim 47 , wherein the peptide linker is between 10 and 40 amino acids long.
49 . The polypeptide of claim 48 , wherein the peptide linker is at least 9 amino acids long.
50 . The polypeptide of claim 48 or claim 49 , wherein the peptide linker is 9 amino acids long, 10 amino acids long, 12 amino acids long, 14 amino acids long, 16 amino acids long, 18 amino acids long, or 19 amino acids long.
51 . The polypeptide of any one of claims 47-50 , wherein the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 89, 90, 92, 94, 96, 98 or 99.
52 . The polypeptide of claim 51 , wherein the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 89.
53 . The polypeptide of claim 51 , wherein the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 98.
54 . The polypeptide of any one of claims 1-53 , wherein the polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 175-248.
55 . The polypeptide of any one of claims 1-54 , wherein the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 175-248.
56 . The polypeptide of any one of claims 1 to 55 , further comprising an albumin-binding domain, a fibronectin domain, or a human serum albumin domain fused to the N- or C-terminus of the ActRIIB-ECD via a linker.
57 . The polypeptide of any one of claims 1 to 56 , further comprising a signal peptide of SEQ ID NO: 1 at the N-terminus of the ActRIIB-ECD.
58 . The polypeptide of claim 57 , wherein the signal peptide is cleaved from the mature protein.
59 . The polypeptide of any one of claims 1 to 58 , wherein the polypeptide is conjugated with a targeting agent, a therapeutic moiety, a detectable moiety, or a diagnostic moiety.
60 . The polypeptide of claim 59 , wherein the targeting agent, the therapeutic moiety, the detectable moiety, or the diagnostic moiety comprises an antibody or antigen binding fragment thereof, a binding agent having affinity for another member of the TGFβ superfamily or for another therapeutic target, a radiotherapy agent, an imaging agent, a fluorescent moiety, a cytotoxic agent, an anti-mitotic drug, a nanoparticle-based carrier, a polymer-conjugated drug, a nanocarrier, an imaging agent, a stabilizing agent, a drug, a nanocarrier, or a dendrimer.
61 . The polypeptide of any one of claims 1 to 60 , wherein the polypeptide forms a dimer comprising a first and a second polypeptide linked by at least one disulfide bond between the Fc domain monomer of the first polypeptide and the Fc domain monomer of the second polypeptide.
62 . A TGFβ superfamily ligand binding agent comprising a first polypeptide of any one of claims 1 to 61 and a second polypeptide of any one of claims 1 to 61 , wherein the first and second polypeptides are linked by at least one disulfide bond between the Fc domain monomer of the first polypeptide and the Fc domain monomer of the second polypeptide.
63 . The binding agent of claim 62 , wherein the first polypeptide and the second polypeptide comprise or consist of an amino acid sequence selected from SEQ ID NOs: 175-231, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto.
64 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the polypeptide demonstrates similar or increased binding to human activin A, activin B, GDF-8, and/or GDF-11 and demonstrates reduced binding to human BMP-9 and/or BMP-10 compared to a polypeptide comprising a WT ActRIIB-ECD.
65 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the polypeptide does not substantially bind to human BMP-9.
66 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the polypeptide demonstrates reduced binding to human BMP-10 compared to a polypeptide comprising a WT ActRIIB-ECD.
67 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the polypeptide inhibits signaling of one or more of human activin A, activin B, GDF-8, and GDF-11.
68 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the polypeptide does not substantially inhibit human BMP-9 and/or BMP-10 signaling.
69 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the inhibition potency of the polypeptide for human BMP-9 and/or BMP-10 signaling is reduced by about 5-fold, about 10-fold or about 100-fold or more compared to the inhibition potency of human wild type ActRIIB-ECD for human BMP-9 and/or BMP-10 signaling.
70 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the inhibition potency of the polypeptide for human BMP-9 and/or BMP-10 signaling is reduced by about 200-fold, by about 300-fold, or more, compared to the inhibition potency of human wild type ActRIIB-ECD for human BMP-9 and/or BMP-10 signaling.
71 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the inhibition potency of the polypeptide for human BMP-9 and/or BMP-10 signaling is reduced by about 5-fold, about 10-fold or about 100-fold or more compared to the inhibition potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 for human BMP-9 and/or BMP-10 signaling.
72 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the inhibition potency of the polypeptide for human BMP-9 and/or BMP-10 signaling is reduced by about 200-fold, by about 300-fold, or more, compared to the inhibition potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171.
73 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the polypeptide's inhibition potency for one or more of human activin A, activin B, GDF-8, and GDF-11 is the same or substantially the same as the inhibition potency of human wild type ActRIIB-ECD for the same respective ligand(s).
74 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the relative inhibition potency for one or more of human activin A, activin B, GDF-8, and GDF-11 is increased compared to the inhibition potency of human wild type ActRIIB-ECD for the same respective ligand(s); and/or wherein the relative inhibition potency for BMP-9 and/or BMP-10 is reduced compared to the inhibition potency of human wild type ActRIIB-ECD for the same ligand.
75 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the relative inhibition potency for one or more of human activin A, activin B, GDF-8, and GDF-11 is increased compared to the inhibition potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 for the same respective ligand(s); and/or wherein the relative inhibition potency for BMP-9 and/or BMP-10 is reduced compared to the inhibition potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 for the same ligand.
76 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the relative inhibition potency for one or more of human activin A, activin B, GDF-8, and GDF-11 is increased by about 2-fold or more, about 3-fold or more, about 4-fold or more, or about 5-fold or more, compared to the inhibition potency of the human wild type ActRIIB-ECD or the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 for the same respective ligand(s).
77 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein the polypeptide's inhibition potency for BMP-9 and/or BMP-10 is at least about 10-fold lower than that of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171.
78 . The polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , wherein:
(a) the polypeptide's inhibition potency for activin A is at least about 5-fold higher than that of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171, and the polypeptide's inhibition potency for BMP-9 and/or BMP-10 is at least about 100-fold lower than that of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171;
(b) the polypeptide's inhibition potency for activin B is at least about 5-fold higher than that of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171, and the polypeptide's inhibition potency for BMP-9 and/or BMP-10 is at least about 100-fold lower than that of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171; or
(c) the polypeptide's inhibition potency for both activin A and activin B is at least about 5-fold higher than that of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171, and the polypeptide's inhibition potency for BMP-9 and/or BMP-10 is at least about 100-fold lower than that of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171.
79 . A nucleic acid molecule encoding the polypeptide of any one of claims 1 to 78 .
80 . The nucleic acid molecule of claim 79 , further comprising the sequence set forth in SEQ ID NO: 297 at the 5′ end of the nucleic acid molecule.
81 . A vector comprising the nucleic acid molecule of claim 79 or 80 .
82 . A host cell comprising the nucleic acid molecule of any one of claim 79 or 80 or the vector of claim 81 , wherein the nucleic acid molecule or the vector is expressed in the host cell.
83 . A method of preparing the polypeptide of any one of claims 1 to 61 comprising:
(a) providing a host cell comprising the nucleic acid molecule of any one of claim 79 or 80 or the vector of claim 81 , and
(b) culturing the host cell under conditions allowing expression of the polypeptide; and
(c) recovering the expressed polypeptide from the culture.
84 . A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 and a pharmaceutically acceptable carrier, diluent or excipient.
85 . The pharmaceutical composition of claim 84 , wherein the composition is formulated for administration by injection or infusion.
86 . The pharmaceutical composition of claim 84 , wherein the composition is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration.
87 . The pharmaceutical composition of any one of claims 84-86 , wherein the polypeptide or binding agent does not cause a vascular complication in a subject and/or does not increase vascular permeability or leakage in a subject.
88 . The pharmaceutical composition of any one of claims 84-87 , wherein the polypeptide or binding agent does not increase red blood cell mass, does not increase hemoglobin, does not cause thrombocytopenia, and/or does not cause a hematological complication in a subject.
89 . A kit comprising the polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , or the pharmaceutical composition of any one of claims 84-88 and, optionally, directions for use.
90 . A method of treating or preventing a disease or condition associated with TGFβ-superfamily ligand signaling in a subject in need thereof, the method comprising administering the polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , or the pharmaceutical composition of any one of claims 84-88 to the subject.
91 . The method of claim 90 , wherein the subject is a human.
92 . The method of claim 90 or 91 , wherein the TGFβ-superfamily ligand is one or more of activin A, activin B, GDF-8, and GDF-11.
93 . A method of treating or preventing a disease or condition mediated by activin A, activin B, GDF-8, and/or GDF-11 in a subject, the method comprising administering the polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , or the pharmaceutical composition of any one of claims 84-88 to the subject.
94 . The method of claim 93 , wherein the disease or condition is characterized by overexpression or overactivation of activin A and/or activin B and/or GDF-8 and/or GDF-11.
95 . The method of any one of claims 90-94 , wherein the disease or condition is selected from pulmonary hypertension (PH), fibrosis, muscle weakness or atrophy, metabolic disorders, cardiometabolic disease, bone damage, and low red blood cell levels.
96 . The method of claim 95 , wherein the PH is pulmonary arterial hypertension (PAH).
97 . The method of claim 96 , wherein the PAH is idiopathic PAH, heritable PAH, or PAH associated with an infection, a congenital heart abnormality, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, a connective tissue disorder, chronic obstructive pulmonary disease, an autoimmune disorder (e.g., scleroderma or lupus), or drug use (e.g., use of cocaine or methamphetamine)
98 . The method of claim 95 , wherein the fibrosis is pulmonary fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, liver fibrosis, lung fibrosis, kidney fibrosis, bone marrow fibrosis, systemic sclerosis, skin fibrosis, heart fibrosis, myelofibrosis, corneal fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarticular fibrosis, arthrofibrosis, tissue fibrosis, a fibroproliferative disorder or a connective tissue disorder.
99 . The method of claim 95 , wherein the muscle weakness or atrophy disease or condition is Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia.
100 . The method of claim 95 , wherein the metabolic disorder is obesity, Type 1 diabetes, Type 2 diabetes, or pre-diabetes.
101 . The method of claim 100 , wherein the metabolic disorder is obesity.
102 . The method of claim 95 , wherein the cardiometabolic disease or condition is heart failure with a reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF).
103 . The method of claim 95 , wherein the bone damage comprises bone demineralization, osteoporosis (e.g., primary or secondary), osteopenia, osteopetrosis, bone fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with the treatment of obesity, low gravity-related bone loss, or immobility-related bone loss.
104 . The method of claim 95 , wherein the low blood cell level disease or condition is anemia or blood loss.
105 . A method of reducing or inhibiting activin A, activin B, GDF-8, and/or GDF-11 signaling in a subject in need thereof without substantially reducing or inhibiting BMP-9 and/or BMP-10 signaling in the subject, the method comprising administering the polypeptide of any one of claims 1 to 61 or the binding agent of claim 62 or claim 63 , or the pharmaceutical composition of any one of claims 84-88 to the subject.
106 . The method of any one of claims 90-105 , wherein the subject is a mammal.
107 . The method of claim 106 , wherein the mammal is a human.
108 . The method of any one of claims 90-107 , wherein the method does not: cause a vascular complication in a subject; increase vascular permeability or leakage in a subject; increase red blood cell mass; increase hemoglobin; cause thrombocytopenia; and/or cause a hematological complication in a subject.