COMBINATION THERAPY WITH ACTRIIB RECEPTOR VARIANTS AND GLP-1 AGONISTS

1 . A combination comprising

(a) a TGFβ superfamily ligand binding agent comprising a first polypeptide and a second polypeptide, wherein the first and second polypeptides each comprise:

i. 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 an amino acid substitution at the position corresponding to position 33 of SEQ ID NO: 2;

ii. an Fc domain monomer; and

iii. a peptide linker joining the polypeptide to the Fc domain monomer; and

(b) a Glucagon-like peptide 1 (GLP-1) agonist.

2 . The combination of claim 1 , wherein the ActRIIB ECD variant demonstrates reduced inhibition of BMP-9 and/or BMP-10 compared to a wild-type ActRIIB ectodomain.

3 . The combination of claim 1 or claim 2 , wherein the amino acid substitution is selected from L33F, L33Q, L33Y, L33W, L33H, L33R, L33E, L33K, and L33M.

4 . The combination of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises the amino acid substitution L33F.

5 . The combination of claim 4 , 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 SEQ ID NO: 10; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 10.

6 . The combination of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises the amino acid substitution L33Q.

7 . The combination of claim 6 , 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 SEQ ID NO: 11; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 11.

8 . The combination of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises the amino acid substitution L33Y.

9 . The combination of claim 8 , 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 SEQ ID NO: 12; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 12.

10 . The combination of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises the amino acid substitution L33W.

11 . The combination of claim 10 , 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 SEQ ID NO: 13; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 13.

12 . The combination of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises the amino acid substitution L33H.

13 . The combination of claim 12 , 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 SEQ ID NO: 14; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 14.

14 . The combination of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises the amino acid substitution L33R.

15 . The combination of claim 14 , 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 SEQ ID NO: 15; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 15.

16 . The combination of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises the amino acid substitution L33E.

17 . The combination of claim 16 , 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 SEQ ID NO: 16; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 16.

18 . The combination of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises the amino acid substitution L33K.

19 . The combination of claim 18 , 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 SEQ ID NO: 17; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 17.

20 . The combination of any one of claims 1-3 , wherein the ActRIIB ECD variant comprises the amino acid substitution L33M.

21 . The combination of claim 20 , 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 SEQ ID NO: 18; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 18.

22 . The combination of claim 21 , wherein the ActRIIB ECD variant further comprises an amino acid substitution at position 27 of SEQ ID NO: 2.

23 . The combination of claim 21 or 22 , wherein the ActRIIB ECD variant further comprises an amino acid substitution at position 69 of SEQ ID NO: 2.

24 . The combination of any one of claims 22-23 , wherein the ActRIIB ECD variant further comprises one or more amino acid substitutions which is G27D, G27E, T69E, T69Q, or T69H.

25 . The combination of any one of claims 1-24 , wherein the ActRIIB ECD variant further comprises one or more additional amino acids at the N or C terminus.

26 . The combination of claim 25 , 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.

27 . The combination of any one of claims 1-26 , wherein the first and the second polypeptides comprise the following structure, from N- to C-terminus: ActRIIB-ECD-peptide linker-Fc domain monomer.

28 . The combination of any one of claims 1-27 , wherein the Fc domain monomer is an IgG1, IgG2, IgG3 or IgG4 isotype.

29 . The combination of any one of claims 1-28 , wherein the Fc domain monomer is a human Fc domain monomer or a murine Fc domain monomer.

30 . The combination of any one of claims 1-29 , wherein the Fc domain monomer is engineered to reduce aggregation or to modulate stability of a dimer of the polypeptide.

31 . The combination of claim 30 , wherein the Fc domain monomer comprises the amino acid substitutions of M252Y, S254T, and T256E (YTE).

32 . The combination of claim 30 , wherein the Fc domain monomer comprises the M252Y amino acid substitution.

33 . The combination of any one of claims 28-32 , wherein the Fc domain monomer includes a D at position 356 and an L at position 358 (DL).

34 . The combination of any one of claims 28-32 , wherein the Fc domain monomer includes an E at position 356 and an M at position 358 (EM).

35 . The combination of any one of claims 28-34 , wherein the Fc domain monomer further comprises a Lysine residue (K) at the C terminus.

36 . The combination of any one of claims 1-35 , wherein the Fc domain monomer

(a) 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; or

(b) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 252-292.

37 . The combination of any one of claims 28-36 , wherein the Fc domain monomer is an IgG1 isotype

38 . The combination of claim 37 , 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.

39 . The combination of any one of claims 1-38 , wherein the Fc domain monomer in the first polypeptide forms a dimer with the Fc domain monomer in the second polypeptide.

40 . The combination of any one of claims 1-39 , wherein the peptide linker is Glycine-rich.

41 . The combination of any one of claims 1-40 , wherein the peptide linker is between 10 and 40 amino acids long.

42 . The combination of claim 41 , wherein the linker is at least 10 amino acids long, at least 14 amino acids long, at least 19 amino acids long, or at least 39 amino acids long.

43 . The combination of claim 42 , wherein the linker is 10 amino acids long, 14 amino acids long, 19 amino acids long, or 39 amino acids long.

44 . The combination of any one of claims 40-43 , wherein the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 89, 94, or 98.

45 . The combination of any one of claims 1-44 , wherein the ActRIIB-ECD

(a) comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:4-62; or

(b) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 4-62.

46 . The combination of any one of claims 1-45 , wherein the ActRIIB-ECD comprises or consists of the amino acid sequence of SEQ ID NO: 10-18, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

47 . The combination of any one of claims 1-46 , wherein the ActRIIB-ECD comprises or consists of the amino acid sequence of SEQ ID NO: 13 or a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

48 . The combination of any one of claims 1-47 , wherein the first and/or second polypeptide:

(a) comprise 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: 174-251; or

(b) comprise or consist of an amino acid sequence selected from SEQ ID NOs: 174-251.

49 . The combination of any one of claims 1-48 , wherein the first and/or second polypeptide:

(a) 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, 176, 180, 204-214, and 230-234; or

(b) comprises or consists of an amino acid sequence selected from SEQ ID NOs: 175, 176, 180, 204-214, and 230-234.

50 . The combination of any one of claims 1-48 , wherein the first and/or second polypeptide:

(a) 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: 211 and 230-234; or

(b) comprises or consists of an amino acid sequence selected from SEQ ID NOs: 211 and 230-234.

51 . The combination of any one of claims 1-48 , wherein the first and/or second polypeptide:

(a) comprises or consists of the amino acid sequence of SEQ ID NO: 231, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 231.

52 . The combination of any one of claims 1-48 , wherein the first and/or second polypeptide (a) comprises or consists of the amino acid sequence of SEQ ID NO: 234, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto; or

(b) comprises or consists of the amino acid sequence of SEQ ID NO: 234.

53 . The combination of any one of claims 1-52 , wherein the first and/or second polypeptide further comprise 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.

54 . The combination of any one of claims 1-53 , wherein the first and/or second polypeptide further comprises a signal peptide of SEQ ID NO: 1 at the N-terminus of the ActRIIB-ECD.

55 . The combination of claim 54 , wherein the signal peptide is cleaved from the mature protein.

56 . The combination of any one of claims 1-55 , wherein the binding agent is conjugated with a targeting agent, a therapeutic moiety, a detectable moiety, or a diagnostic moiety.

57 . The combination of claim 56 , 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.

58 . The combination of any one of claims 1-57 , wherein the first and second polypeptides form a dimer 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.

59 . The combination of any one of claims 1-58 , wherein the binding agent:

(a) 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;

(b) does not substantially bind to human BMP-9;

(c) demonstrates reduced binding to human BMP-10 compared to a polypeptide comprising a WT ActRIIB-ECD;

(d) inhibits signaling of one or more of human activin A, activin B, GDF-8, and GDF-11; and/or

(e) does not substantially inhibit human BMP-9 and/or BMP-10 signaling.

60 . The combination of any one of claims 1-59 , wherein the GLP-1 agonist is an antibody, small molecule, peptide, or aptamer.

61 . The combination of any one of claims 1-60 , wherein the GLP-1 agonist is selected from the group consisting of exenatide, exenatide extended-release, efpeglenatide, dulaglutide, liraglutide, lixisenatide, lotiglipron, semaglutide, tirzepatide, cotadutide, noiiglutide, oxyntomodulin, orforglipron, retatrutide, albiglutide, beinaglutide and PEG-loxenatide, pemvidutide, and danuglipron.

62 . The combination of any one of claims 1-59 , wherein the GLP-1 agonist is selected from semaglutide and tirzepatide.

63 . The combination of any one of claims 1-59 , wherein the GLP-1 agonist is a dual GLP-1 agonist and GIP agonist, a dual GLP-1 agonist and GCG agonist, or a triagonist of GIP/GLP-1/glucagon receptors.

64 . The combination of any one of claims 1-63 , wherein the binding agent and GLP-1 agonist are formulated in separate pharmaceutical compositions.

65 . The combination of any one of claims 1-63 , wherein the binding agent and GLP-1 agonist are formulated in the same pharmaceutical composition.

66 . The combination of any one of claims 64-65 , wherein the pharmaceutical composition is formulated for administration by injection or infusion.

67 . The combination of claim 66 , wherein the composition is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

68 . A kit comprising the combination of any one of claims 1-67 , and optionally, directions for use.

69 . A method of treating or preventing a metabolic disease or condition in a subject in need thereof, the method comprising administering the combination of any one of claims 1-67 to the subject.

70 . The method of claim 69 , wherein the subject is a human.

71 . The method of claim 69 or 70 , wherein the metabolic disorder is selected from the group consisting of obesity, diabetes, metabolic syndrome, anti-psychotic drug-associated obesity, glucocorticoid-induced obesity, hypothalamic obesity associated with craniopharyngioma, Prader-Willi syndrome, and a monogenetic disorder associated with obesity such as Bardet-Biedl syndrome.

72 . The method of claim 69 or 70 , wherein the metabolic disorder is selected from obesity, Type 2 diabetes, and pre-diabetes.

73 . The method of claim 71 , wherein the metabolic disorder is obesity.

74 . The method of any one of claims 69-73 , 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; cause a hematological complication in a subject; and/or does not cause a reduction in lean muscle mass in the subject.

75 . The method of any one of claims 69-74 , wherein the treatment increases lean mass in the subject.

76 . The method of any one of claims 69-75 , wherein the treatment reduces fat mass in the subject.