Binders of TGFβ-superfamily ligands and uses thereof

1. A heteromultimer comprising an ActRIIB fusion protein and a TβRII fusion protein, wherein the ActRIIB fusion protein comprises an ActRIIB polypeptide comprising an amino acid sequence that is at least 98% identical to:

a. a sequence beginning at any one of positions 20 to 29 of SEQ ID NO: 50, and ending at any one of positions 109 to 134 of SEQ ID NO: 50;

b. a sequence beginning at position 20 of SEQ ID NO: 50, and ending at position 134 of SEQ ID NO: 50;

c. a sequence beginning at position 29 of SEQ ID NO: 50 and ending at position 109 of SEQ ID NO: 50;

d. a sequence beginning at position 25 of SEQ ID NO: 50 and ending at position 131 of SEQ ID NO: 50;

e. the sequence of SEQ ID NO: 51;

f. the sequence of SEQ ID NO: 52;

g. the sequence of SEQ ID NO: 54;

h. the sequence of SEQ ID NO: 55; or

i. the sequence of SEQ ID NO: 109;

wherein the TβRII fusion protein comprises a TβRII polypeptide comprising an amino acid sequence that is at least 98% identical to:

a. a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1;

b. a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2;

c. the sequence of SEQ ID NO: 18;

d. the sequence of SEQ ID NO: 27; or

e. the sequence of any one of SEQ ID NOs: 20, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38; and 39;

wherein the heteromultimer binds to and/or inhibits one or more of: GDF11, GDF8, activin A, activin B, BMP10, TGFβ1, and TGFβ3; and

wherein the ActRIIB fusion protein and the TβRII fusion protein further comprise a heterologous portion.

2. The heteromultimer of claim 1 , wherein the ActRIIB fusion protein further comprises a linker domain portion positioned between the ActRIIB portion and the heterologous portion, wherein the linker domain:

1) is between 10 and 25 amino acids in length, and comprises an amino acid sequence selected from:

a. (GGGGS)n, wherein n=≥2;

b. (GGGGS)n, wherein n=≥3;

c. (GGGGS)n, wherein n=≥4;

d. (GGGGS)n, wherein n≠>5; or

2) consists of the amino acid sequence of any one of SEQ ID Nos: 4-7, 19, 21, 25, 26, 40, and 63-67.

3. The heteromultimer of claim 1 , wherein the ActRIIB fusion protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 84 or 90.

4. The heteromultimer of claim 1 , wherein:

A. the ActRIIB polypeptide consists of:

a) an ActRIIB portion comprising the amino acid sequence of SEQ ID NO: 51 and no more than 2 or 1 additional amino acids;

b) a linker portion comprising an amino acid sequence that is at least 98°/, identical to the amino acid sequence of SEQ ID NO: 6;

c) a heterologous portion comprising an amino acid sequence that is at least 98°/A identical to an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, and 73; and

d) optionally a leader sequence; or

B. the ActRIIB polypeptide comprises:

a) an ActRIIB portion comprising the amino acid sequence of SEQ ID NO: 51;

b) a heterologous portion, wherein the heterologous portion comprises an amino acid sequence that is at least 98°/identical to an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, and 73; and

c) a linker portion connecting the ActRIIB polypeptide portion and the heterologous portion; wherein the linker comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 6.

5. The heteromultimer of claim 1 , wherein the ActRIIB fusion protein does not comprise an acidic amino acid at the residue corresponding to position 79 of SEQ ID NO: 50.

6. The heteromultimer of claim 1 , wherein the TβRII polypeptide comprises:

a. a sequence beginning at any one of positions 23 to 35 of SEQ ID NO: 1, and ending at any one of positions 153 to 159 of SEQ ID NO: 1;

b. a sequence beginning at any one of positions 23 to 60 of SEQ ID NO: 2, and ending at any one of positions 178 to 184 of SEQ ID NO: 2;

c. the sequence of SEQ ID NO: 18;

d. the sequence of SEQ ID NO: 27; or

e. the sequence of any one of SEQ ID NOs: 20, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38; and 39.

7. The heteromultimer of claim 1 , wherein the TβRII polypeptide comprises the amino acid sequence of SEQ ID NO: 18.

8. The heteromultimer of claim 1 , wherein the TβRII polypeptide is a TβRII fusion protein comprising:

a. a TβRII portion comprising an extracellular domain of TβRII; and

b. a heterologous portion.

9. The heteromultimer of claim 8 , wherein the TβRII fusion protein further comprises a linker domain portion positioned between the TβRII portion and the heterologous portion wherein the linker domain:

1) is between 10 and 25 amino acids in length, and comprises an amino acid sequence selected from:

a. (GGGGS)n, wherein n=≥2;

b. (GGGGS)n, wherein n=≥3;

c. (GGGGS)n, wherein n=≥4;

d. GGGGS)n, wherein n≠>5; or

2) consists of the amino acid sequence of any one of SEQ ID Nos: 4-7, 19, 21, 25, 26, 40, and 63 67.

10. The heteromultimer of claim 8 , wherein the TβRII fusion protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:87 or 93.

11. The heteromultimer of claim 8 , wherein:

A. the TβRII polypeptide consists of:

a) a TβRII polypeptide portion comprising the amino acid sequence of SEQ ID NO: 18;

b) a linker portion comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 6;

c) a heterologous portion comprising an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, and 73; and

d) optionally a leader sequence; or

B. the TβRII polypeptide comprises:

a) a TβRII polypeptide portion comprising the amino acid sequence of SEQ ID NO: 18;

b) a heterologous portion, wherein the heterologous portion comprises an amino acid sequence that is at least 9 identical to an amino acid sequence selected from SEQ ID NOs: 68, 69, 72, and 73; and

c) a linker portion connecting the TβRII polypeptide portion and the heterologous portion; wherein the linker comprises an amino acid sequence that is at least 98%, identical to the amino acid sequence of SEQ ID NO: 6.

12. The heteromultimer of claim 1 , wherein the heterologous portion comprises: 1) a first or second member of an interaction pair, and/or 2) one or more amino acid modifications that promotes heterodimer formation.

13. The heteromultimer of claim 1 , wherein the heterologous portion is an immunoglobulin Fc domain.

14. The heteromultimer of claim 13 , wherein the immunoglobulin Fc domain comprises an amino acid sequence that is at least 98% identical to:

a. the amino acid sequence of SEQ ID NO: 68, wherein the sequence comprises a lysine (K) at position 356 and a K at position 399 based on the amino acid positioning of the EU numbering scheme of Kabat;

b. the amino acid sequence of SEQ ID NO: 69, wherein the sequence comprises a aspartic acid (D) at position 392 and a D at position 409 based on the amino acid positioning of the EU numbering scheme of Kabat;

c. the amino acid sequence of SEQ ID NO: 72, wherein the sequence comprises a cysteine (C) at position 354 and a tryptophan (W) at position 366 based on the amino acid positioning of the EU numbering scheme of Kabat; or

d. the amino acid sequence of SEQ ID NO: 73, wherein the sequence comprises a C at position 349, a serine (S) at position 366, an alanine (A) at position 368, and a valine at position 407 based on the amino acid positioning of the EU numbering scheme of Kabat.