ALK4:ActRIIB heteromultimers and uses thereof
In certain aspects, the disclosure provides soluble heteromeric polypeptide complexes comprising an extracellular domain of an ALK4 receptor and an extracellular domain of ActRIIB. In certain aspects, such soluble ALK4:ActRIIB complexes may be used to regulate (promote or inhibit) growth of tissues or cells including, for example, muscle, bone, cartilage, fat, neural tissue, tumors, and/or cancerous cells. In certain aspects, such ALK4:ActRIIB complexes are can be used to improve muscle formation, bone formation, metabolic parameters, and disorders associated with these tissues, cellular networks, kidney, and endocrine systems.
1. A soluble recombinant heteromultimer comprising an ALK4 polypeptide and an ActRIIB polypeptide, wherein the ALK4 polypeptide comprises an amino acid sequence that is at least 90% identical to amino acids 34-101 of SEQ ID NO: 9, and wherein the ActRIIB polypeptide comprises an amino acid sequence that is at least 90% identical to amino acids 29-109 of SEQ ID NO: 1, wherein the ActRIIB polypeptide does not comprise an aspartic acid (D) at the amino acid position corresponding to L79 of SEQ ID NO: 1; wherein the ALK4 and/or ActRIIB polypeptide is a fusion protein further comprising a heterologous domain, and wherein the heteromultimer binds to activin B.
2. The heteromultimer of claim 1 , wherein the ALK4 polypeptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that:
a) begins at any one of amino acids 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 of SEQ ID NO: 9, and
b) ends at any one of amino acids 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, or 126 of SEQ ID NO: 9.
3. The heteromultimer of claim 1 , wherein the ActRIIB polypeptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that:
a) begins at any one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of SEQ ID NO: 1, and
b) ends at any one of amino acids 109, 110, 111, 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: 1.
4. The heteromultimer of claim 1 , wherein the heterologous domain comprises an Fc immunoglobulin domain.
5. The heteromultimer of claim 4 , wherein the Fc immunoglobulin domain comprises one or more amino acid modifications that promote heterodimer formation.
6. The heteromultimer of claim 4 , wherein the immunoglobulin Fc domain comprises one or more amino acid modifications that inhibit homodimer formation.
7. The heteromultimer of claim 4 , wherein the heterologous domain comprises an Fc immunoglobulin domain of IgG1 immunoglobulin.
8. The heteromultimer of claim 1 , wherein the fusion protein further comprises a linker domain positioned between the ALK4 domain and the heterologous domain and/or a linker domain positioned between the ActRIIIB domain and the heterologous domain.
9. The heteromultimer of claim 1 , wherein the ALK4 polypeptide and/or ActRIIB polypeptide comprises one or more modified amino acid residues 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.
10. The heteromultimer of claim 1 , wherein the ALK4 polypeptide and/or ActRIIB polypeptide is glycosylated and has a glycosylation pattern obtainable from a Chinese hamster ovary cell line.
11. The heteromultimer of claim 1 , wherein the heteromultimer is an ALK4:ActRIIB heterodimer.
12. A pharmaceutical preparation comprising the heteromultimer of claim 1 and a pharmaceutically acceptable carrier.
13. The pharmaceutical preparation of claim 12 , wherein the pharmaceutical preparation is substantially pyrogen-free.
14. The pharmaceutical preparation of claim 12 , wherein the pharmaceutical preparation further comprises an additional active agent.
15. The pharmaceutical preparation of claim 12 , wherein the pharmaceutical preparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ALK4 homomultimers.
16. The pharmaceutical preparation of claim 12 , wherein the pharmaceutical preparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ActRIIB homomultimers.
17. The heteromultimer of claim 1 , wherein the ALK4 polypeptide comprises an amino acid sequence that is at least 95% identical to amino acids 34-101 of SEQ ID NO: 9, and wherein the ActRIIB polypeptide comprises an amino acid sequence that is at least 95% identical to amino acids 29-109 of SEQ ID NO: 1.
18. The heteromultimer of claim 1 , wherein the ALK4 polypeptide comprises the amino acid sequence corresponding to amino acids 34-101 of SEQ ID NO: 9, and wherein the ActRIIB polypeptide comprises the amino acid sequence corresponding to amino acids 29-109 of SEQ ID NO: 1.
19. The heteromultimer of claim 1 , wherein the ALK4 polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, and wherein the ActRIIB polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2.
20. The heteromultimer of claim 1 , wherein the ALK4 polypeptide comprises the amino acid sequence of SEQ ID NO: 10, and wherein the ActRIIB polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
21. The heteromultimer of claim 17 , wherein the heterologous domain comprises an Fc immunoglobulin domain, wherein the Fc immunoglobulin domain is an IgG1 immunoglobulin domain.
22. The heteromultimer of claim 18 , wherein the heterologous domain comprises an Fc immunoglobulin domain, wherein the Fc immunoglobulin domain is an IgG1 immunoglobulin domain.
23. The heteromultimer of claim 19 , wherein the heterologous domain comprises an Fc immunoglobulin domain, wherein the Fc immunoglobulin domain is an IgG1 immunoglobulin domain.
24. The heteromultimer of claim 20 , wherein the heterologous domain comprises an Fc immunoglobulin domain, wherein the Fc immunoglobulin domain is an IgG1 immunoglobulin domain.
25. The heteromultimer of claim 17 , wherein the fusion protein further comprises a linker domain positioned between the ALK4 domain and the heterologous domain and/or a linker domain positioned between the ActRIIB domain and the heterologous domain.
26. The heteromultimer of claim 18 , wherein the fusion protein further comprises a linker domain positioned between the ALK4 domain and the heterologous domain and/or a linker domain positioned between the ActRIIB domain and the heterologous domain.
27. The heteromultimer of claim 19 , wherein the fusion protein further comprises a linker domain positioned between the ALK4 domain and the heterologous domain and/or a linker domain positioned between the ActRIIB domain and the heterologous domain.
28. The heteromultimer of claim 20 , wherein the fusion protein further comprises a linker domain positioned between the ALK4 domain and the heterologous domain and/or a linker domain positioned between the ActRIIB domain and the heterologous domain.
29. The heteromultimer of claim 1 , wherein the ALK4 polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, and wherein the ActRIIB polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2.
30. The heteromultimer of claim 29 , wherein the heterologous domain comprises an Fc immunoglobulin domain, wherein the Fc immunoglobulin domain is an IgG1 immunoglobulin domain.
31. The heteromultimer of claim 29 , wherein the fusion protein further comprises a linker domain positioned between the ALK4 domain and the heterologous domain and/or a linker domain positioned between the ActRIIB domain and the heterologous domain.