Growth differentiation factor 15 (GDF-15) constructs

Abstract

To provide constructs comprising GDF15 and mutants thereof.SOLUTION: A fusion protein comprising a GDF15 region and an Fc domain are provided. In various embodiments, constructs comprising GDF15 and mutants thereof can be of use in treating or ameliorating a metabolic disorder. In various embodiments, the metabolic disease or disorder is type 2 diabetes, obesity, dyslipidemia, elevated glucose levels, elevated insulin levels and diabetic nephropathy.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on July 29, 2014, is named A-1850-WO-PCT_SL.txt, and is 630,200 bytes in size.

[0002] The present disclosure relates to monomers and multimers that include polypeptides that include GDF15 regions. [Background technology]

[0003] Growth differentiation factor 15 (GDF15) is a divergent member of the TGFβ superfamily. It is also called macrophage inhibitory cytokine 1 (MIC1) (Bootcov, MR, 1997, Proc. Natl. Acad. Sci. 94:11514-9), placental bone morphogenetic factor (PLAB) (Hromas, R, 1997, Biochim. Biophys. Acta. 1354:40-4), placental transforming growth factor beta (PTGFB) (Lawton, LN, 1997, Gene. 203:17-26), prostate-derived factor (PDF) (Paralkar, VM, 1998, J. Biol. Chem. 273:13760-7) and nonsteroidal anti-inflammatory drug-activated gene (NAG-1) (Baek, SJ, 2001, J. Biol. Chem. 276:33384-92).

[0004] The human GDF15 gene is located on chromosome 19 p13.2-13.1, the rat GDF15 gene is located on chromosome 16, and the mouse GDF15 gene is located on chromosome 8. The open reading frame of GDF15 spans two exons (Bottner, M, 1999, Gene. 237:105-11 and NCBI). The mature GDF15 peptide shares low homology with other family members (Katoh, M, 2006, Int. J. Mol. Med. 17:951-5.).

[0005] GDF15 is synthesized as a large precursor protein that is cleaved at a dibasic cleavage site to release the mature peptide at the carboxy terminus. Both mouse and rat GDF15 prepropeptides contain 303 amino acids. The human full-length precursor contains 308 amino acids. The rodent mature peptide contains 115 amino acids after processing at the cleavage site RGRR (SEQ ID NO: 1). The human mature peptide contains 112 amino acids after processing at the cleavage site RGRRRAR (SEQ ID NO: 2). The human mature GDF15 peptide shares 66.1% and 68.1% sequence similarity with the rat and mouse mature GDF15 peptides (Bottner, M, 1999, Gene. 237:105-11; Bauskin, AR, 2000, EMBO J. 19:2212-20; NCBI). The mature GDF15 peptide has no glycosylation sites.

[0006] The mature GDF15 peptide contains the seven conserved cysteine ​​residues required for the formation of a cysteine ​​knot motif (with three intrachain disulfide bonds) and a single interchain disulfide bond that are typical of members of the TGFβ superfamily. The mature GDF15 peptide further contains two additional cysteine ​​residues that form a fourth intrachain disulfide bond. Biologically active GDF15 is a 25 KD homodimer of the mature peptide covalently linked by one interchain disulfide bond.

[0007] Circulating levels of GDF15 have been reported to be elevated in multiple pathological and physiological conditions, most notably in pregnancy (Moore, AG, 2000. J. Clin. Endocrinol. Metab. 85:4781-4788), β-thalassemia (Tanno, T, 2007, Nat. Med. 13:1096-101; Zimmermann, MB, 2008, Am. J. Clin. Nutr. 88:1026-31) and congenital dyserythrocytic anemia (Tamary, H, 2008, Blood. 112:5241-4). GDF15 has been associated with multiple biological activities in the literature. Studies of GDF15 knockout and transgenic mice have suggested that GDF15 may be protective against ischemia / reperfusion-induced or overload-induced cardiac injury (Kempf T, 2006, Circ. Res. 98: 351-60; Xu, J, 2006, Circ. Res. 98: 342-50), may be protective against age-related motor and sensory neuron loss (Strelau, J, 2009, J. Neurosci. 29: 13640-8), may be mildly protective against metabolic acidosis in the kidney, and may cause cachexia in cancer patients (Johnen, H, 2007, Nat. Med. 11: 1333-40). Numerous groups have also investigated the role of GDF15 in cell apoptosis and proliferation, reporting controversial results using different cell culture and xenograft models. Studies in transgenic mice have shown that GDF15 is protective against carcinogen- or Apc mutation-induced tumors in the intestine and lung (Baek, SJ, 2006, Gastroenterology. 131:1553-60; Cekanova, M, 2009, Cancer Prev. Res. 2:450-8). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Bootcov,MR,1997,Proc.Natl.Acad.Sci.94:11514-9

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[0009] Provided herein are fusion proteins comprising a GDF15 polypeptide or a GDF15 mutant polypeptide and an Fc domain.

[0010] In one embodiment, the Fc domain comprises a sequence selected from the group consisting of SEQ ID NOs: 16, 22, 28, 29, 33, 35, 38, 48, 85, 91, 106, 132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213, 220, 227, 233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 and 302. In another embodiment, the GDF15 polypeptide or GDF15 variant polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 25, 52 and 55. In another embodiment, the fusion protein further comprises a polypeptide linker. In one embodiment, the polypeptide linker comprises a sequence selected from the group consisting of SEQ ID NOs: 18, 30, 34, 40, 58, 61, 64, 69, 72, 75, 78, 113, 116, 119, 122, 125, 128. In one embodiment, the fusion protein comprises two or more Fc domains. In one embodiment, the fusion protein comprises two or more polypeptide linkers. In one embodiment, the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 46, 24, 27, 32, 37, 20, 42, 50, 54, 57, 60, 63, 66, 68, 71, 74, 77, 82, 84, 88, 93, 96, 98, 100, 102, 104, 108, 134, 137, 139, 143, 146, 150, 153, 269, 272, 276, 279, 157, 160, 164, 167, 171, 174, 178, 181, 185, 188, 194, 197, 201, 204, 208, 211, 215, 218, 222, 225, 229, 232, 233, 238, and 240.

[0011] Also provided is a dimer comprising (i) a first polypeptide chain comprising one of the aforementioned fusion proteins and (ii) a second polypeptide chain comprising an Fc domain. In yet further embodiments, the construct further comprises a sequence selected from the group consisting of SEQ ID NOs: 16, 22, 28, 29, 33, 35, 38, 48, 85, 91, 106, 132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213, 220, 227, 233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 and 302.

[0012] In one embodiment, the first and second polypeptide chains are non-covalently linked. In another embodiment, the first and second polypeptide chains are covalently linked. In one embodiment, the first and second polypeptide chains are covalently linked via a disulfide bond between their respective Fc domains. In another embodiment, the first and second polypeptide chains are linked by both covalent and non-covalent interactions.

[0013] In certain embodiments, a dimer is provided comprising: (a) two fusion proteins comprising the sequence of SEQ ID NO:46; (b) two fusion proteins comprising the sequence of SEQ ID NO:24; or (c) two fusion proteins comprising the sequence of SEQ ID NO:27.

[0014] In certain embodiments, a dimer is provided comprising: (a) two fusion proteins comprising the sequence of SEQ ID NO:32; or (b) two fusion proteins comprising the sequence of SEQ ID NO:37.

[0015] In certain embodiments, a dimer is provided that comprises a first polypeptide chain comprising the sequence of SEQ ID NO:20 and a second polypeptide chain comprising the sequence of SEQ ID NO:17.

[0016] In certain embodiments, the polypeptide chain includes (a) a first polypeptide chain comprising a sequence of SEQ ID NO: 42 and a second polypeptide chain comprising a sequence of SEQ ID NO: 39; (b) a first polypeptide chain comprising a sequence of SEQ ID NO: 50 and a second polypeptide chain comprising a sequence of SEQ ID NO: 47; (c) a first polypeptide chain comprising a sequence of SEQ ID NO: 54 and a second polypeptide chain comprising a sequence of SEQ ID NO: 47; (d) a first polypeptide chain comprising a sequence of SEQ ID NO: 57 and a second polypeptide chain comprising a sequence of SEQ ID NO: 47; (e) a first polypeptide chain comprising a sequence of SEQ ID NO: 60 and a second polypeptide chain comprising a sequence of SEQ ID NO: 47; (f) a first polypeptide chain comprising a sequence of SEQ ID NO: 63 and a second polypeptide chain comprising a sequence of SEQ ID NO: 47; (g) a first polypeptide chain comprising a sequence of SEQ ID NO: 66 and a second polypeptide chain comprising a sequence of SEQ ID NO: 47; (h) a first polypeptide chain comprising a sequence of SEQ ID NO: 66 and a second polypeptide chain comprising a sequence of SEQ ID NO: 47; (i) a first polypeptide chain comprising the sequence of SEQ ID NO: 68 and a second polypeptide chain comprising the sequence of SEQ ID NO: 47; (i) a first polypeptide chain comprising the sequence of SEQ ID NO: 71 and a second polypeptide chain comprising the sequence of SEQ ID NO: 47; (j) a first polypeptide chain comprising the sequence of SEQ ID NO: 74 and a second polypeptide chain comprising the sequence of SEQ ID NO: 47; (k) a first polypeptide chain comprising the sequence of SEQ ID NO: 77 and a second polypeptide chain comprising the sequence of SEQ ID NO: 47; (l) a first polypeptide chain comprising the sequence of SEQ ID NO: 80 and a second polypeptide chain comprising the sequence of SEQ ID NO: 47; (m) a first polypeptide chain comprising the sequence of SEQ ID NO: 82 and a second polypeptide chain comprising the sequence of SEQ ID NO: 47; or (n) a first polypeptide chain comprising the sequence of SEQ ID NO: 84 and a second polypeptide chain comprising the sequence of SEQ ID NO: 47.

[0017] In certain embodiments, a dimer is provided that comprises: (a) a first polypeptide chain comprising the sequence of SEQ ID NO:88 and a second polypeptide chain comprising the sequence of SEQ ID NO:86; (b) a first polypeptide chain comprising the sequence of SEQ ID NO:93 and a second polypeptide chain comprising the sequence of SEQ ID NO:90; (c) a first polypeptide chain comprising the sequence of SEQ ID NO:96 and a second polypeptide chain comprising the sequence of SEQ ID NO:90; (d) a first polypeptide chain comprising the sequence of SEQ ID NO:98 and a second polypeptide chain comprising the sequence of SEQ ID NO:90; (e) a first polypeptide chain comprising the sequence of SEQ ID NO:100 and a second polypeptide chain comprising the sequence of SEQ ID NO:90; (f) a first polypeptide chain comprising the sequence of SEQ ID NO:102 and a second polypeptide chain comprising the sequence of SEQ ID NO:90; (g) a first polypeptide chain comprising the sequence of SEQ ID NO:104 and a second polypeptide chain comprising the sequence of SEQ ID NO:90; or (h) a first polypeptide chain comprising the sequence of SEQ ID NO:108 and a second polypeptide chain comprising the sequence of SEQ ID NO:105.

[0018] In certain embodiments, a dimer is provided that comprises: (a) a first polypeptide chain comprising the sequence of SEQ ID NO:112 and a second polypeptide chain comprising the sequence of SEQ ID NO:12; (b) two polypeptide chains, each comprising the sequence of SEQ ID NO:112; (c) two polypeptide chains, each comprising the sequence of SEQ ID NO:115; (d) two polypeptide chains, each comprising the sequence of SEQ ID NO:118; (e) two polypeptide chains, each comprising the sequence of SEQ ID NO:121; (f) two polypeptide chains, each comprising the sequence of SEQ ID NO:124; (g) two polypeptide chains, each comprising the sequence of SEQ ID NO:127; (h) two polypeptide chains, each comprising the sequence of SEQ ID NO:130; or (i) two polypeptide chains, each comprising the sequence of SEQ ID NO:242.

[0019] In certain embodiments, the polypeptide chain comprises: (a) a first polypeptide chain comprising the sequence of SEQ ID NO: 134 and a second polypeptide chain comprising the sequence of SEQ ID NO: 131; (b) a first polypeptide chain comprising the sequence of SEQ ID NO: 137 and a second polypeptide chain comprising the sequence of SEQ ID NO: 131; (c) a first polypeptide chain comprising the sequence of SEQ ID NO: 139 and a second polypeptide chain comprising the sequence of SEQ ID NO: 131; (d) a first polypeptide chain comprising the sequence of SEQ ID NO: 143 and a second polypeptide chain comprising the sequence of SEQ ID NO: 140; (e) a first polypeptide chain comprising the sequence of SEQ ID NO: 146 and a second polypeptide chain comprising the sequence of SEQ ID NO: 140; (f) a first polypeptide chain comprising the sequence of SEQ ID NO: 150 and a second polypeptide chain comprising the sequence of SEQ ID NO: 150. (g) a first polypeptide chain comprising the sequence of SEQ ID NO:153 and a second polypeptide chain comprising the sequence of SEQ ID NO:147; (h) a first polypeptide chain comprising the sequence of SEQ ID NO:269 and a second polypeptide chain comprising the sequence of SEQ ID NO:267; (i) a first polypeptide chain comprising the sequence of SEQ ID NO:272 and a second polypeptide chain comprising the sequence of SEQ ID NO:267; (j) a first polypeptide chain comprising the sequence of SEQ ID NO:276 and a second polypeptide chain comprising the sequence of SEQ ID NO:274; or (k) a first polypeptide chain comprising the sequence of SEQ ID NO:279 and a second polypeptide chain comprising the sequence of SEQ ID NO:274.

[0020] In certain embodiments, dimers are provided that comprise: (a) a first polypeptide chain comprising the sequence of SEQ ID NO: 157 and a second polypeptide chain comprising the sequence of SEQ ID NO: 154; (b) a first polypeptide chain comprising the sequence of SEQ ID NO: 160 and a second polypeptide chain comprising the sequence of SEQ ID NO: 154; (c) a first polypeptide chain comprising the sequence of SEQ ID NO: 164 and a second polypeptide chain comprising the sequence of SEQ ID NO: 161; (d) a first polypeptide chain comprising the sequence of SEQ ID NO: 167 and a second polypeptide chain comprising the sequence of SEQ ID NO: 161.

[0021] In certain embodiments, a dimer is provided that comprises: (a) a first polypeptide chain comprising the sequence of SEQ ID NO:171 and a second polypeptide chain comprising the sequence of SEQ ID NO:168; or (b) a first polypeptide chain comprising the sequence of SEQ ID NO:174 and a second polypeptide chain comprising the sequence of SEQ ID NO:168.

[0022] In certain embodiments, a dimer is provided that comprises: (a) a first polypeptide chain comprising the sequence of SEQ ID NO:178 and a second polypeptide chain comprising the sequence of SEQ ID NO:175; (b) a first polypeptide chain comprising the sequence of SEQ ID NO:181 and a second polypeptide chain comprising the sequence of SEQ ID NO:175; (c) a first polypeptide chain comprising the sequence of SEQ ID NO:185 and a second polypeptide chain comprising the sequence of SEQ ID NO:182; (d) a first polypeptide chain comprising the sequence of SEQ ID NO:188 and a second polypeptide chain comprising the sequence of SEQ ID NO:182; (e) a first polypeptide chain comprising the sequence of SEQ ID NO:194 and a second polypeptide chain comprising the sequence of SEQ ID NO:191; (f) a first polypeptide chain comprising the sequence of SEQ ID NO:197 and a second polypeptide chain comprising the sequence of SEQ ID NO:191; (g) a first polypeptide chain comprising the sequence of SEQ ID NO:201 and a second polypeptide chain comprising the sequence of SEQ ID NO:198; or (h) a first polypeptide chain comprising the sequence of SEQ ID NO:204 and a second polypeptide chain comprising the sequence of SEQ ID NO:198.

[0023] In certain embodiments, a dimer is provided that comprises: (a) a first polypeptide chain comprising the sequence of SEQ ID NO:208 and a second polypeptide chain comprising the sequence of SEQ ID NO:205; (b) a first polypeptide chain comprising the sequence of SEQ ID NO:211 and a second polypeptide chain comprising the sequence of SEQ ID NO:205; (c) a first polypeptide chain comprising the sequence of SEQ ID NO:215 and a second polypeptide chain comprising the sequence of SEQ ID NO:212; or (d) a first polypeptide chain comprising the sequence of SEQ ID NO:218 and a second polypeptide chain comprising the sequence of SEQ ID NO:212.

[0024] In certain embodiments, a dimer is provided that comprises: (a) a first polypeptide chain comprising the sequence of SEQ ID NO:222 and a second polypeptide chain comprising the sequence of SEQ ID NO:219; (b) a first polypeptide chain comprising the sequence of SEQ ID NO:225 and a second polypeptide chain comprising the sequence of SEQ ID NO:219; (c) a first polypeptide chain comprising the sequence of SEQ ID NO:229 and a second polypeptide chain comprising the sequence of SEQ ID NO:226; or (d) a first polypeptide chain comprising the sequence of SEQ ID NO:232 and a second polypeptide chain comprising the sequence of SEQ ID NO:226.

[0025] In certain embodiments, a dimer is provided that comprises: (a) two polypeptide chains each comprising the sequence of SEQ ID NO:235; (b) two polypeptide chains each comprising the sequence of SEQ ID NO:238; or (c) two polypeptide chains each comprising the sequence of SEQ ID NO:240.

[0026] In certain embodiments, a tetramer is provided that includes (i) a first dimer that includes one of the aforementioned dimers, and (ii) a second dimer that includes one of the aforementioned dimers. In certain embodiments, the first polypeptide chain of the first dimer is linked to the first polypeptide chain of the second dimer via an interchain disulfide bond between their respective GDF15 domains.

[0027] In some embodiments, the dimer is DhCpmFc(-)-(G 4 S) 4 -GDF15:DhCpmFc(+), DhCpmFc(+)-(G 4 S) 4 -GDF15:DhCpmFc(-), DhCpmFc(-)-(G 4 S) 4 -GDF15(H6D):DhCpmFc(+), DhCpmFc(+)-(G 4 S) 4 -GDF15(H6D):DhCpmFc(-), DhCpmFc(+)-(G 4 S) 4 -GDF15(N3Q):DhCpmFc(-), DhCpmFc(+)-GDF15:DhCpmFc(-), DhCpmFc(+)-G 4-GDF15:DhCpmFc(-), DhCpmFc(+)-(G 4 S) 2 -GDF15:DhCpmFc(-), DhCpmFc(+)-(G 4 Q) 4 -GDF15:DhCpmFc(-), DhCpmFc(+)(L351C)-G 4 -GDF15:DhCpmFc(-)(L351C), DhCpmFc(+)(S354C)-G 4 -GDF15:DhCpmFc(-)(Y349C), CpmFc(-)-(G 4 S) 4 -GDF15: CpmFc(+), Fc-(G 4 S) 8 -Fc-GS(G 4 S) 4 -GDF15, Fc-(G 4 S) 3 -Fc-GS(G 4 S) 4 -GDF15 and Fc-(G 4 S) 5 -Fc-GS(G 4 S) 4 - Not selected from the group including GDF15.

[0028] In some embodiments, the dimer is DhCpmFc(-)-(G 4 S) 4 -GDF15:DhCpmFc(+), DhCpmFc(+)-(G 4 S) 4 -GDF15:DhCpmFc(-), DhCpmFc(-)-(G 4 S) 4 -GDF15(H6D):DhCpmFc(+), DhCpmFc(+)-(G 4 S) 4 -GDF15(H6D):DhCpmFc(-), DhCpmFc(+)-(G 4 S) 4 -GDF15(N3Q):DhCpmFc(-), DhCpmFc(+)-GDF15:DhCpmFc(-), DhCpmFc(+)-G 4 -GDF15:DhCpmFc(-), DhCpmFc(+)-(G4 S) 2 -GDF15:DhCpmFc(-), DhCpmFc(+)-(G 4 Q) 4 -GDF15:DhCpmFc(-), DhCpmFc(+)(L351C)-G 4 -GDF15: DhCpmFc(-)(L351C) and DhCpmFc(+)(S354C)-G 4 - GDF15:DhCpmFc(-)(Y349C).

[0029] In some embodiments, the fusion protein is an Fc-(G 4 S) 8 -Fc-GS(G 4 S) 4 -GDF15, Fc-(G 4 S) 3 -Fc-GS(G 4 S) 4 -GDF15 and Fc-(G 4 S) 5 -Fc-GS(G 4 S) 4 - Not selected from the group consisting of GDF15.

[0030] In some embodiments, the dimer is DhCpmFc(+)(L351C)-G 4 -GDF15: DhCpmFc(-)(L351C) and DhCpmFc(+)(S354C)-G 4 - GDF15:DhCpmFc(-)(Y349C).

[0031] In some embodiments, the dimer is DhCpmFc(-)-(G 4 S) 4 -GDF15:DhCpmFc(+), DhCpmFc(+)-(G 4 S) 4 -GDF15:DhCpmFc(-), DhCpmFc(-)-(G 4 S) 4 -GDF15(H6D):DhCpmFc(+), DhCpmFc(+)-(G 4 S) 4-GDF15(H6D):DhCpmFc(-), DhCpmFc(+)-(G 4 S) 4 -GDF15(N3Q):DhCpmFc(-), DhCpmFc(+)-GDF15:DhCpmFc(-), DhCpmFc(+)-G 4 -GDF15:DhCpmFc(-), DhCpmFc(+)-(G 4 S) 2 -GDF15:DhCpmFc(-), DhCpmFc(+)-(G 4 Q) 4 -GDF15:DhCpmFc(-), DhCpmFc(+)(L351C)-G 4 -GDF15:DhCpmFc(-)(L351C), DhCpmFc(+)(S354C)-G 4 -GDF15:DhCpmFc(-)(Y349C), CpmFc(-)-(G 4 S) 4 -GDF15: CpmFc(+), Fc-(G 4 S) 8 -Fc-GS(G 4 S) 4 -GDF15, Fc-(G 4 S) 3 -Fc-GS(G 4 S) 4 -GDF15 or Fc-(G 4 S) 5 -Fc-GS(G 4 S) 4 -Not GDF15.

[0032] In some embodiments, the dimer is DhCpmFc(-)-(G 4 S) 4 -GDF15:DhCpmFc(+), DhCpmFc(+)-(G 4 S) 4 -GDF15:DhCpmFc(-), DhCpmFc(-)-(G 4 S) 4 -GDF15(H6D):DhCpmFc(+), DhCpmFc(+)-(G 4 S) 4 -GDF15(H6D):DhCpmFc(-), DhCpmFc(+)-(G4 S) 4 -GDF15(N3Q):DhCpmFc(-), DhCpmFc(+)-GDF15:DhCpmFc(-), DhCpmFc(+)-G 4 -GDF15:DhCpmFc(-), DhCpmFc(+)-(G 4 S) 2 -GDF15:DhCpmFc(-), DhCpmFc(+)-(G 4 Q) 4 -GDF15:DhCpmFc(-), DhCpmFc(+)(L351C)-G 4 -GDF15: DhCpmFc(-)(L351C) or DhCpmFc(+)(S354C)-G 4 - Not GDF15:DhCpmFc(-)(Y349C).

[0033] In some embodiments, the dimer is an Fc-(G 4 S) 8 -Fc-GS(G 4 S) 4 -GDF15, Fc-(G 4 S) 3 -Fc-GS(G 4 S) 4 -GDF15 and Fc-(G 4 S) 5 -Fc-GS(G 4 S) 4 - Not selected from the group consisting of GDF15.

[0034] In some embodiments, the fusion protein is DhCpmFc(+)(L351C)-G 4 -GDF15: DhCpmFc(-)(L351C) and DhCpmFc(+)(S354C)-G 4 - GDF15:DhCpmFc(-)(Y349C).

[0035] In some embodiments, the dimer is DhCpmFc(-)-(G 4 S) 4 - GDF15:DhCpmFc(+). In some embodiments, the dimer is DhCpmFc(+)-(G 4 S)4 - GDF15:DhCpmFc(-). In some embodiments, the dimer is DhCpmFc(-)-(G 4 S) 4 - GDF15(H6D): is not DhCpmFc(+). In some embodiments, the dimer is DhCpmFc(+)-(G 4 S) 4 - GDF15(H6D): is not DhCpmFc(-). In some embodiments, the dimer is DhCpmFc(+)-(G 4 S) 4 In some embodiments, the dimer is not DhCpmFc(+)-GDF15:DhCpmFc(-). In some embodiments, the dimer is not DhCpmFc(+)-GDF15:DhCpmFc(-). 4 - GDF15:DhCpmFc(-). In some embodiments, the dimer is DhCpmFc(+)-(G 4 S) 2 - GDF15:DhCpmFc(-). In some embodiments, the dimer is DhCpmFc(+)-(G 4 Q) 4 - GDF15: is not DhCpmFc(-). In some embodiments, the dimer is DhCpmFc(+)(L351C)-G 4 - GDF15:DhCpmFc(-)(L351C). In some embodiments, the dimer is not DhCpmFc(+)(S354C)-G 4 - GDF15:DhCpmFc(-)(Y349C). In some embodiments, the dimer is not CpmFc(-)-(G 4 S) 4 - GDF15:CpmFc(+). In some embodiments, the dimer is Fc-(G 4 S) 8 -Fc-GS(G 4 S) 4 -GDF15. In some embodiments, the dimer is Fc-(G 4 S) 3 -Fc-GS(G 4 S) 4 -GDF15. In some embodiments, the dimer is Fc-(G 4 S) 5-Fc-GS(G 4 S) 4 -Not GDF15.

[0036] Also provided herein is a fusion protein comprising a GDF15 polypeptide or a GDF15 variant polypeptide and a human serum albumin (HSA) polypeptide. In a further embodiment, the HSA polypeptide is SEQ ID NO: 110. In another embodiment, the GDF15 polypeptide or the GDF15 variant polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 25, 52, and 55. In another embodiment, the fusion protein further comprises a polypeptide linker linking the GDF15 polypeptide or the GDF15 variant polypeptide to the HSA polypeptide. In one embodiment, the polypeptide linker has a sequence selected from the group consisting of SEQ ID NOs: 18, 30, 34, 40, 58, 61, 64, 69, 72, 75, 78, 113, 116, 119, 122, 125, 128. In one embodiment, the fusion protein comprises two or more HSA polypeptides. In one embodiment, the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 115, 118, 121, 124, 127, and 130.

[0037] Also provided herein is a dimer comprising (i) a first polypeptide chain comprising a fusion protein comprising a first GDF15 domain and a first HSA polypeptide, and (ii) a second polypeptide chain comprising a second HSA polypeptide. In some embodiments, the second polypeptide chain further comprises a second GDF15 domain. In some embodiments, the dimer is a heterodimer (i.e., the first and second polypeptide chains have different sequences). In some embodiments, the dimer is a homodimer (i.e., the first and second polypeptide chains have identical sequences). In some embodiments, the first polypeptide chain comprises a sequence selected from the group consisting of SEQ ID NOs: 115, 118, 121, 124, 127, and 130. In some embodiments, the second polypeptide chain comprises a sequence selected from the group consisting of SEQ ID NOs: 115, 118, 121, 124, 127, and 130. In one embodiment, the first and second polypeptide chains are non-covalently linked. In another embodiment, the first and second polypeptide chains are covalently linked. In one embodiment, the second polypeptide chain comprises a second GDF15 domain and the first and second polypeptide chains are covalently linked via a disulfide bond between their respective GDF15 domains, hi another embodiment, the first and second polypeptide chains are linked by both covalent and non-covalent interactions.

[0038] Also provided herein is a dimer comprising (i) a first polypeptide chain comprising a fusion protein comprising a first GDF15 domain and an HSA polypeptide, and (ii) a second polypeptide chain comprising a second GDF15 domain. In some embodiments, the first polypeptide chain comprises a sequence selected from the group consisting of SEQ ID NOs: 115, 118, 121, 124, 127, and 130. In some embodiments, the second polypeptide chain comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 25, 52, and 55. In one embodiment, the first and second polypeptide chains are non-covalently associated. In another embodiment, the first and second polypeptide chains are covalently associated. In another embodiment, the first and second polypeptide chains are associated by both covalent and non-covalent interactions. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 shows the knobs-holes construct, which contains a dimer of two DhknobFc-(G4S)4-GDF15:DhholeFc heterodimers. [Diagram 2] FIG. 1 shows a DhMonoFc construct comprising a dimer of two DhMonoFc-(G4S)4-GDF15 fusion proteins. [Diagram 3] FIG. 1 shows a HemiFc construct containing a dimer of two GGGFc-(G4S)4-Fc-S(G4S)4-GDF15 fusion proteins. [Figure 4] FIG. 1 shows a charge pair (delHinge) construct comprising a dimer of two DhCpmFc(-)-(G4S)2-GDF15:DhCpmFc(+) heterodimers. [Diagram 5] FIG. 1 shows a charge pair (delHinge) cysteine ​​clamp construct comprising a dimer of two DhCpmFc(-)(L351C)-(G4S)2-GDF15:DhCpmFc(-)(L351C) heterodimers. [Figure 6] FIG. 1 shows an HSA construct comprising a dimer of two HSA-(G4S)4-GDF15 fusion proteins. [Figure 7] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with dimers of DhknobFc-G4-GDF15:DholeFc heterodimer. [Figure 8] FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of the Fc-(G4S)4-GDF15 fusion protein. [Figure 9] FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of Fc-(G4S)4-GDF15(H6D) fusion protein. [Figure 10]FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of Fc-(G4S)4-Fc-(G4S)4-GDF15 fusion protein. [Figure 11] FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) using a dimer of DhFc-(G4S)5-DhFc-(G4S)4-GDF15 fusion protein. [Figure 12] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(+)-(1K)-GDF15:DhCpmFc(-) heterodimer. [Figure 13] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)-GDF15:DhCpmFc(+) heterodimers. [Figure 14] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) using dimers of DhCpmFc(-)-GDF15(N3D):DhCpmFc(+) heterodimers. [Figure 15] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with dimers of DhCpmFc(-)-GDF15(Ndel3):DhCpmFc(+) heterodimer. [Figure 16] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with dimers of DhCpmFc(-)-G4-GDF15(N3D):DhCpmFc(+) heterodimer. [Figure 17]FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with dimers of DhCpmFc(-)-G4S-GDF15:DhCpmFc(+) heterodimer. [Figure 18] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)-(G4S)2-GDF15:DhCpmFc(+) heterodimer. [Figure 19] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) using dimers of DhCpmFc(-)-(G4S)2-GDF15(N3D):DhCpmFc(+) heterodimer. [Figure 20] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) using dimers of DhCpmFc(-)-(G4Q)2-GDF15(N3D):DhCpmFc(+) heterodimer. [Figure 21] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)-G4P-GDF15:DhCpmFc(+) heterodimer. [Figure 22] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)-(G4P)2-GDF15:DhCpmFc(+) heterodimer. [Figure 23] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)-G4Q-GDF15:DhCpmFc(+) heterodimer. [Figure 24] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) using dimers of DhCpmFc(-)-(G4Q)2-GDF15(N3D):DhCpmFc(+) heterodimer. [Diagram 25] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) using dimers of DhCpmFc(-)-(G4Q)2-GDF15(Ndel3):DhCpmFc(+) heterodimer. [Figure 26] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(+)(Y349C)-GDF15(N3D):DhCpmFc(-)(S354C) heterodimer. [Figure 27] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(Y349C)-GDF15:DhCpmFc(+)(S354C) heterodimer. [Figure 28] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C) heterodimer. [Figure 29] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C) heterodimer. [Diagram 30]FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(Y349C)-G4-GDF15(N3D):DhCpmFc(+)(S354C) heterodimer. [Diagram 31] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(Y349C)-(G4S)2-GDF15(N3D):DhCpmFc(+)(S354C) heterodimer. [Diagram 32] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) using dimers of DhCpmFc(-)(Y349C)-(G4Q)2-GDF15(N3D):DhCpmFc(+)(S354C) heterodimer. [Diagram 33] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(L351C)-(G4S)2-GDF15:DhCpmFc(+)(L351C) heterodimer. [Diagram 34] FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of HSA-GSAAQAAQQGS-GDF15 fusion protein. [Diagram 35] Graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with a dimer of HSA-GSPAPAPGS-GDF15 fusion protein. [Diagram 36]FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of HSA-GS(AAQAAQQ)2GS-GDF15 fusion protein. [Figure 37] Graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of HSA-GS(PAPAP)2GS-GDF15 fusion protein. [Figure 38] FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of HSA-GDF15 fusion protein. [Figure 39] FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of HSA-GGNAEAAAKEAAAKEAAAKAGG-GDF15 fusion protein. [Diagram 40] FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of HSA-(G4S)6-GDF15 fusion protein. [Diagram 41] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) using dimers of DhCpmFc(-)(N297G)-GDF15(Ndel3):DhCpmFc(+)(N297G) heterodimer. [Diagram 42] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G) heterodimer. [Diagram 43]FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(N297G)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(S354C) heterodimer. [Diagram 44] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(N297G)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(S354C) heterodimer. [Diagram 45] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(N297G)(L351C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L351C) heterodimer. [Figure 46] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(N297G)(L351C)-GDF15(N3D):DhCpmFc(+)(N297G)(L351C) heterodimer. [Figure 47] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of DhCpmFc(-)(N297G)(A287C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C) heterodimer. [Figure 48] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) with dimers of CpmFc(-)(N297G)-GDF15(Ndel3):CpmFc(+)(N297G) heterodimer. [Figure 49] This is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) using dimers of Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(N297G) heterodimer. [Figure 50] This is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) using dimers of Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+)(N297G) heterodimer. [Figure 51] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log [g construct / kg body weight]) using dimers of Dh3CpmFc(-)(Y349C)-GDF15(Ndel3):Dh3CpmFc(+)(N297G)(S354C) heterodimer. [Figure 52] FIG. 13 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) using dimers of the Dh3CpmFc(-)(Y349C)-GDF15(N3D):Dh3CpmFc(-)(N297G)(S354C) heterodimer. [Diagram 53] FIG. 1 is a graph showing the effect on food intake (g food / g body weight) in ob / ob mice as a function of dose (log[g construct / kg body weight]) with a dimer of DhMonoFc(N297G)-GDF15 fusion protein. [Figure 54] FIG. 1 shows a graph of body weight (g) as a function of time (days after first injection) for vehicle, 0.1 nmol, 1 nmol and 10 nmol of Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, and 0.1 nmol, 1 nmol and 10 nmol of Dh3ComFc(-)-GDF15(Ndel3) heterodimer dimer. [Figure 55](a) vehicle, (b) 10 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (c) 1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (d) 0.1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (e) 10 mmolar Dh3CpmFc 13 is a bar graph of the area under the curve (AUC) of the glucose tolerance test at 2 weeks of treatment with (-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer dimer, (g) 1 mM Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer dimer, or (g) 0.1 mM Dh3CpmFc(-)-GDF15(Ndel):Dh3CpmFc(+) heterodimer dimer. [Figure 56] (a) vehicle, (b) 10 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (c) 1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (d) 0.1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (e) 10 mmolar Dh3CpmFc Bar graphs of insulin (ng / mL) (fed) at 3 weeks of treatment with (c) dimer of Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer, (g) 1 mmolar dimer of Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer, or (g) 0.1 mmolar dimer of Dh3CpmFc(-)-GDF15(Ndel):Dh3CpmFc(+) heterodimer. [Figure 57](a) vehicle, (b) 10 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (c) 1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (d) 0.1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (e) 10 mmolar Dh3CpmFc Bar graphs of triglycerides (mg / mL) (fed) at 3 weeks of treatment with (-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer dimer, (g) 1 mmolar Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer dimer, or (g) 0.1 mmolar Dh3CpmFc(-)-GDF15(Ndel):Dh3CpmFc(+) heterodimer dimer. [Figure 58] (a) vehicle, (b) 10 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (c) 1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (d) 0.1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (e) 10 mmolar Dh3CpmFc Bar graphs of cholesterol (mg / mL) (fed) at 3 weeks of treatment with (-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer dimer, (g) 1 mmolar Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer dimer, or (g) 0.1 mmolar Dh3CpmFc(-)-GDF15(Ndel):Dh3CpmFc(+) heterodimer dimer. [Figure 59](a) vehicle, (b) 10 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (c) 1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (d) 0.1 mmolar Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimer dimer, (e) 10 mmolar Dh3CpmFc 13 is a bar graph of the area under the curve (AUC) of the glucose tolerance test at 5 weeks of treatment with (-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer dimer, (g) 1 mM Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer dimer, or (g) 0.1 mM Dh3CpmFc(-)-GDF15(Ndel):Dh3CpmFc(+) heterodimer dimer. [Figure 60] (a) DhCpmFc(-)-GDF15(Ndel3):DhCpmFc(+) dimer, (b) DhCpmFc(-)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C) dimer; (c) Dh3CpmFc(-)-GDF15(Ndel3) dimer; (d) Dh3CpmFc(-)(Y349C)-GDF15(Ndel3)-Dh3CpmFc(-)(Y349C)-GDF15(Ndel3)-Dh3CpmFc(+)(S354C) dimer. 14 is a bar graph showing SPR binding (RU) for FcγRI, FcγRIIIA and FcγRIIA for (e) Dh3CpmFc(+)(S354C) dimer; (f) Dh3CpmFc(-)(Y349C)-GDF15(N3D):Dh3CpmFc(+)(S354C) dimer (left to right for each receptor). [Figure 61](a) (for the pair of bars named "N3D"), the dimers DhCpmFc(-)-GDF15(N3D):DhCpmFc(+) and Dh3CpmFc(-)-GDF15(N3D):DhCpmFc(+); (b) (for the pair of bars named "Ndel3"), the dimers DhCpmFc(-)-GDF15(Ndel3):DhCpmFc(+) and Dh3CpmFc(-)-GDF15(Ndel3):DhCpmFc(+); (c) 1 is a bar graph showing the DSC first Tm (° C.) for (a) (for the pair of bars labeled "N3D+CC"), the dimers DhCpmFc(-)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C) and the dimers Dh3CpmFc(-)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C); and (b) (for the pair of bars labeled "Ndel3+CC"), the dimers DhCpmFc(-)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C) and the dimers Dh3CpmFc(-)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The present disclosure provides fusion proteins comprising GDF15 polypeptides or GDF15 mutant polypeptides and constructs comprising such fusion proteins. Also provided are the production and use of the disclosed molecules in treating metabolic disorders, such as, for example, type 2 diabetes, high glucose levels, high insulin levels, dyslipidemia, or obesity. GDF15 polypeptides, GDF15 mutant polypeptides, and certain polypeptide constructs comprising GDF15 polypeptides and GDF15 mutant polypeptides are described in co-owned PCT / US2012 / 032415, filed April 5, 2012, and PCT / 2013 / 023465, filed January 28, 2013, both of which are expressly incorporated herein by reference for any purpose.

[0041] Recombinant polypeptide and nucleic acid methods used herein, including those in the Examples, are generally those referenced in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) or Current Protocols in Molecular Biology (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons, 1994).

[0042] I. General Definitions Following convention, as used herein, "a" and "an" mean "one or more" unless otherwise indicated.

[0043] As used herein, the terms "amino acid" and "residue" are interchangeable and, when used in the context of a peptide or polypeptide, refer to both naturally occurring and synthetic amino acids, as well as amino acid analogs, amino acid mimetics, and non-naturally occurring amino acids that are chemically similar to naturally occurring amino acids.

[0044] The terms "naturally occurring amino acid" and "naturally encoded amino acid" are used interchangeably and refer to those amino acids encoded by the genetic code, as well as those amino acids encoded by the genetic code that are modified after synthesis, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

[0045] An "amino acid analog" is a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but will retain the same basic chemical structure as a naturally occurring amino acid.

[0046] An "amino acid mimetic" is a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid. Examples include methacryloyl or acryloyl derivatives of amides, β-, γ-, δ-imino acids (e.g., piperidine-4-carboxylic acid), and the like.

[0047] The terms "non-naturally occurring amino acid" and "non-naturally encoded amino acid" are used interchangeably and refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, but that are not incorporated into a growing polypeptide chain by the translation complex. "Non-naturally occurring amino acids" also include, but are not limited to, amino acids that arise by modification (e.g., post-translational modification) of a naturally encoded amino acid (including, but not limited to, the 20 common amino acids), but that are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. A non-limiting list of examples of non-naturally occurring amino acids that can be inserted into or substituted for wild-type residues in a polypeptide sequence include β-amino acids, homoamino acids, cyclic amino acids, and amino acids with derivatized side chains. Examples include (in L- or D-form; abbreviated in parentheses): citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit), Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn), Nα-methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Nα-methylaminoglutamine (hMethylaminoglutamine) (HMG), homoglycoside (HGA ... N-methyl-arginine (NMeR), N-methyl-leucine (Nα-MeL or NMeL), N-methyl-homolysine (NMeHoK), N-methyl-glutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-Tetrahydroisoquinoline (Tic), 2-indanylglycine (IgI), para-indophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or 4-amino-Phe), 4-guanidinophenylalanine (Guf), glycyrrhizin (abbreviated as "K(Nε-glycyl)" or "K(glycyl)" or "K(gly)"), nitrophenylalanine (nitroPhe), aminophenylalanine (aminop he or amino-Phe), benzylphenylalanine (benzylphe), gamma-carboxyglutamic acid (gamma-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), alpha-aminoadipic acid (Aad), N-α-methylvaline (NMeVal), N-α-methylleucine (NMeLeu), N-α-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Cpg), Cylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine; 4Bip), α-amino-isobutyric acid (A ib), β-alanine, β-aminopropionic acid, piperidinic acid, aminocaprionic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), g-carboxyglutamic acid, eN,N,Included are N-trimethyllysine, eN-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-amino-O-phthalic acid (4APA), N-acetylglucosaminyl-L-serine, N-acetylglucosylaminyl-L-threonine, O-phosphotyrosine and other similar amino acids, and derivatized forms of any of those specifically listed.

[0048] Also included in the definition of "naturally occurring amino acid" are: structure [ka] where the R group is any substituent other than those used in the 20 amino acids.

[0049] In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group. structure [ka] wherein n is 0 to 10; 1 is alkyl, aryl, substituted alkyl, or substituted aryl; R 2 is H, alkyl, aryl, substituted alkyl, and substituted aryl; and R 3 is H, an amino acid, a polypeptide, or an amino-terminus modification group, and R 4 is H, an amino acid, a polypeptide, or a carboxy-terminal modification group.

[0050] In some embodiments, the non-naturally encoded amino acid comprises an aminooxy group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazide group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazine group. In some embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group.

[0051] In some embodiments, the non-naturally encoded amino acid residue comprises an azide group. structure [ka] wherein n is 0 to 10; 1 is alkyl, aryl, substituted alkyl, substituted aryl, or absent; X is O, N, S, or absent; m is 0 to 10; R 2 is H, an amino acid, a polypeptide, or an amino-terminus modification group, and R 3 is H, an amino acid, a polypeptide, or a carboxy-terminal modification group.

[0052] In some embodiments, the non-naturally encoded amino acid comprises an alkyne group. structure [ka] wherein n is 1 to 10; R 1 is alkyl, aryl, substituted alkyl, or substituted aryl; X is O, N, S, or absent; m is 0-10, and R 2 is H, an amino acid, a polypeptide, or an amino-terminus modification group, and R 3 is H, an amino acid, a polypeptide, or a carboxy-terminal modification group.

[0053] The term "substituted" means that a hydrogen atom in a molecule or group is replaced by a group or atom called a substituent. Typical substituents include halogen, C 1-8 Alkyl, Hydroxyl, C 1-8 Alkoxy, -NR x R x , nitro, cyano, halo or perhalo C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8Alkynyl, -SR x , -S(=O) 2 R x , -C(=O)OR x , -C(=O)R x In this case, each R x are independently hydrogen or C 1 -C 8 It is an alkyl group. The substituent is -NR x R x If R x It is noted that the groups may be linked together with the nitrogen atom to form a ring.

[0054] The term "alkyl" means a straight or branched chain hydrocarbon. Representative examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, and hexyl. Typical alkyl groups are those where the group is generally C 1-8 It is an alkyl group having 1 to 8 carbon atoms, denoted as alkyl.

[0055] The term "alkoxy" refers to an alkyl group attached to an oxygen atom. Representative examples of alkoxy groups include methoxy, ethoxy, tert-butoxy, propoxy, and isobutoxy. Common alkoxy groups include C 1-8 It is an alkoxy group.

[0056] The term "halogen" or "halo" means chlorine, fluorine, bromine or iodine.

[0057] The term "alkenyl" means a branched or straight chain hydrocarbon having one or more carbon / carbon double bonds. Representative examples of alkenyl groups include ethenyl, propenyl, allyl, butenyl, and 4-methylbutenyl. Common alkenyl groups include C 2-8 It is an alkenyl group.

[0058] The term "alkynyl" means a branched or straight chain hydrocarbon having one or more carbon / carbon triple bonds. Representative examples of alkynyl groups include ethynyl, propynyl (propargyl) and butynyl. Common alkynyl groups include C 2-8 It is an alkynyl group.

[0059] The term "cycloalkyl" means a cyclic non-aromatic hydrocarbon. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Cycloalkyl groups can contain one or more double bonds. Examples of cycloalkyl groups containing double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl, and cyclobutadienyl. Common cycloalkyl groups are C 3-8 It is a cycloalkyl group.

[0060] The term "perfluoroalkyl" refers to an alkyl group in which all of the hydrogen atoms have been replaced with fluorine atoms. Common perfluoroalkyl groups are C 1-8 A common example of a perfluoroalkyl group is -CF 3 It is.

[0061] The term "acyl" refers to a group derived from an organic acid by removal of the hydroxy group (-OH). For example, the acyl group CH 3 C(=O)- is CH 3 It is formed by removal of the hydroxy group from C(=O)OH.

[0062] The term "aryl" means a cyclic aromatic hydrocarbon. Examples of aryl groups include phenyl and naphthyl. Common aryl groups are 6-13 membered rings.

[0063] The term "heteroatom" as used herein means an oxygen, nitrogen, or sulfur atom.

[0064] The term "heteroaryl" refers to a cyclic aromatic hydrocarbon in which one or more carbon atoms of an aryl group are replaced by a heteroatom. If a heteroaryl group contains more than one heteroatom, the heteroatoms may be the same or different. Examples of heteroaryl include pyridyl, pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl, indolyl, triazolyl, pyridazinyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl, isothiazolyl, and benzo[b]thienyl. Common heteroaryl groups are 5- to 13-membered rings containing 1-4 heteroatoms. Heteroaryl groups that are 5- and 6-membered rings containing 1-3 heteroatoms are particularly common.

[0065] The term "heterocycloalkyl" refers to a cycloalkyl group in which one or more of the carbon atoms are replaced by a heteroatom. If the heterocycloalkyl group contains more than one heteroatom, the heteroatoms may be the same or different. Examples of heterocycloalkyl groups include tetrahydrofuryl, morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl. It is possible for a heterocycloalkyl group to have one or more double bonds, but it is not aromatic. Examples of heterocycloalkyl groups containing double bonds include dihydrofuran. Common heterocycloalkyl groups are 3- to 10-membered rings containing 1-4 heteroatoms. Heterocycloalkyl groups that are 5- and 6-membered rings containing 1-2 heteroatoms are particularly common.

[0066] It is also noted that cyclic ring groups, i.e., aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, can contain more than one ring. For example, a naphthyl group is a fused bicyclic ring system. It is also intended that the present invention encompasses ring groups having bridging atoms or ring groups having a spiro orientation.

[0067] Representative examples of 5-6 membered aromatic rings optionally having one or two heteroatoms are phenyl, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyridinyl, pyridiazinyl, pyrimidinyl and pyrazinyl.

[0068] Representative examples of partially saturated, fully saturated or fully unsaturated 5-8 membered rings, optionally having 1-3 heteroatoms, are cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and phenyl. Further examples of 5 membered rings are furyl, thienyl, pyrrolyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 1,3-dioxolanyl, oxazolyl, thiazolyl, imidazolyl, 2H-imidazolyl, 2-imidazolinyl, imidazolidinyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2-dithiolyl, 1,3-dithiolyl, 3H-1,2-oxathiolyl, 1,2, 3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4 oxadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-thiadiazolyl, 3H-1,2,3-dioxazolyl, 1,2,4-dioxazolyl, 1,3,2-dioxazolyl, 1,3,4-dioxazolyl, 5H-1,2,5-oxathiazolyl and 1,3-oxthiolyl.

[0069] Further examples of 6-membered rings are 2H-pyranyl, 4H-pyranyl, pyridinyl, piperidinyl, 1,2-dioxinyl, 1,3-dioxinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, pindazinyl, pyrimidinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl, 1,3,5-trithianyl, 4H-1,2- oxazinyl, 2H-1,3-oxazinyl, 6H-1,3-oxazinyl, 6H-1,2-oxazinyl, 1,4-oxazinyl, 2H-1,2-oxazinyl, 4H-1,4-oxazinyl, 1,2,5-oxathiazinyl, 1,4-oxazinyl, o-isoxazinyl, p-isoxazinyl, 1,2,5-oxathiazinyl, 1,2,6-(3oxathiazinyl, and 1,4,2-oxadiazinyl.

[0070] Further exemplary seven-membered rings are azepinyl, oxepinyl, thiepinyl and 1,2,4-triazepinyl.

[0071] Further exemplary eight-membered rings are cyclooctyl, cyclooctenyl, and cyclooctadienyl.

[0072] Exemplary bicyclic rings consisting of two fused partially saturated, fully saturated or fully unsaturated 5- and / or 6-membered rings, optionally having 1-4 heteroatoms, are indolizinyl, indolyl, isoindolyl, indolinyl, cyclopenta(b)pyridinyl, pyrano(3,4-b)pyrrolyl, benzofuryl, isobenzofuryl, benzo(b)thienyl, benzo(c)thienyl, 1H-indazolyl, indoxazinyl, benzoxazolyl, anthranilyl, benzimidazolyl, benzthiazolyl, purinyl, quinolinyl, isoquinolinyl, cinnolinyl, and cyclopenta(b)pyridinyl. These include, for example, pyrido(3,4-b)pyridinyl, pyrido(3,2-b)pyridinyl, pyrido(4,3-b)-pyridinyl, 2H-1,3-benzoxazinyl, 2H-1,4-benzoxazinyl, 1H-2,3-benzoxazinyl, 4H-3,1-benzoxazinyl, 2H-1,2-benzoxazinyl and 4H-1,4-benzoxazinyl.

[0073] A cyclic ring group may be bonded to another group in more than one way. If no particular bonding arrangement is specified, all possible arrangements are intended. For example, the term "pyridyl" includes 2-, 3-, or 4-pyridyl, and the term "thienyl" includes 2- or 3-thienyl.

[0074] The term "isolated nucleic acid molecule" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end (e.g., the nucleic acid sequences of GDF15 provided herein), or analogs thereof that are separated from at least about 50% of the polypeptides, peptides, lipids, carbohydrates, polynucleotides or other materials with which the nucleic acid is naturally found when the entire nucleic acid is isolated from a source cell. Preferably, an isolated nucleic acid molecule is substantially free of any other contaminating nucleic acid molecules or other molecules found in the nucleic acid's natural environment that would interfere with its use in the production of polypeptides or its therapeutic, diagnostic, prophylactic or research uses.

[0075] The term "isolated polypeptide" refers to a polypeptide (e.g., a GDF15 polypeptide or a GDF15 variant polypeptide provided herein) that has been separated from at least about 50% of the polypeptides, peptides, lipids, carbohydrates, polynucleotides or other materials with which the polypeptide is naturally found when isolated from a source cell. Preferably, an isolated polypeptide is substantially free of any other contaminating polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research uses.

[0076] The term "encoding" refers to a polynucleotide sequence that encodes one or more amino acids. The term does not require a start codon and a stop codon. The amino acid sequence can be encoded in any one of the various reading frames provided by the polynucleotide sequence.

[0077] The terms "identical" and percent "identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are identical. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps (if any) in sequence comparisons can be addressed by certain mathematical models or computer programs (i.e., "algorithms"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed.), (1988), New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., (1987), Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., (1988), SIAM J. Applied Math. 48:1073.

[0078] In calculating percent identity, the sequences to be compared are aligned to obtain the maximum match between the sequences. The computer program used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., (1984), Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for the best match of each amino acid or nucleotide (the "matched distance" determined by the algorithm). A gap opening penalty (calculated as 3 times the average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix used; the "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (usually 1 / 10 times the gap opening penalty) and a comparison matrix such as, for example, PAM250 or BLOSUM62 are used in conjunction with the algorithm. In certain embodiments, standard comparison matrices (for the PAM250 comparison matrix, see Dayhoff et al., (1978), Atlas of Protein Sequence and Structure, 5:345-352; for the BLOSUM62 comparison matrix, see Henikoff et al., (1992), Proc. Natl. Acad. Sci. USA 89:10915-10919) are also used by the algorithm.

[0079] Recommended parameters for determining percent identity for polypeptide or nucleotide sequences using the GAP program are as follows: Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-13; Comparison matrix: Henikoff et al., 1992, BLOSUM 62, supra; Gap penalty: 12 (but no penalty for end gaps) Gap Length Penalty: 4 Similarity threshold: 0

[0080] Certain sequence comparison schemes for aligning two amino acid sequences can produce matches of only short regions of the two sequences, and these small aligned regions can have very high sequence identity even if there is no significant relationship between the two full-length sequences. Thus, the selected sequence comparison method (e.g., the GAP program) can be adjusted to produce a sequence comparison over at least 50 contiguous amino acids of the target polypeptide, if so desired.

[0081] The term "GDF15 polypeptide" refers to naturally occurring or "wild type" GDF15 expressed in a mammal, including, but not limited to, a human, rabbit, monkey (e.g., cynomolgus monkey), dog, rat, mouse, or pig. In one embodiment, GDF15 polypeptide refers to any full-length GDF15 consisting of 308 amino acid residues and encoded by the nucleotide sequence SEQ ID NO:3, e.g., SEQ ID NO:4; any form of the polypeptide consisting of 279 amino acid residues and encoded by the nucleotide sequence SEQ ID NO:7, with the 29 amino acid residues at the amino terminus of full-length GDF15 (constituting the signal peptide) removed, e.g., SEQ ID NO:8; and any form of GDF15 consisting of an active domain and a prodomain with the prodomain and signal sequence removed, consisting of 112 amino acid residues and encoded by SEQ ID NO:11, with the signal sequence and prodomain removed, e.g., SEQ ID NO:12. GDF15 polypeptides may or may not include an amino-terminal methionine, which may be introduced by engineering or as a result of a bacterial expression process.

[0082] The term "GDF15 mutant polypeptide" refers to a modified GDF15 polypeptide (e.g., SEQ ID NO: 4, 8, or 12). Such modifications include, but are not limited to, one or more substitutions, additions, or deletions, including substitutions with non-naturally occurring amino acids, non-naturally occurring amino acid analogs, and amino acid mimetics. In one embodiment, the term "GDF15 mutant polypeptide" refers to a GDF15 polypeptide in which at least one residue normally found at a given position in a naturally occurring GDF15 polypeptide is deleted or replaced by a residue not normally found at that position in a naturally occurring GDF15 polypeptide. In some cases, it will be desirable to replace a single residue normally found at a given position in the sequence of a naturally occurring GDF15 polypeptide with more than one residue not normally found at that position; in still other cases, it may be desirable to maintain the sequence of a naturally occurring GDF15 polypeptide and insert one or more residues at a given position in the protein; in still other cases, it may be desirable to delete a given residue entirely; all of these constructs are encompassed by the term "GDF15 mutant polypeptide".

[0083] In various embodiments, a GDF15 mutant polypeptide comprises an amino acid sequence that is at least about 85 percent identical to a naturally occurring GDF15 polypeptide (e.g., SEQ ID NO: 4, 8, or 12). In other embodiments, a GDF15 mutant polypeptide comprises an amino acid sequence that is at least about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to a naturally occurring GDF15 polypeptide (e.g., SEQ ID NO: 4, 8, or 12). Such GDF15 mutant polypeptides preferably, but may not, have at least one activity of a naturally occurring GDF15 polypeptide, such as, for example, the ability to reduce blood glucose, insulin, triglyceride, or cholesterol levels; the ability to reduce body weight; the ability to improve glucose tolerance, lipid tolerance, or insulin sensitivity; or the ability to reduce urinary glucose and protein excretion.

[0084] The term "GDF15 domain" encompasses GDF15 polypeptides and GDF15 mutant polypeptides and sequences as defined above.

[0085] The GDF15 polypeptide or GDF15 variant polypeptide is preferably biologically active. The GDF15 polypeptide or GDF15 variant polypeptide used to treat or ameliorate a metabolic disorder in a subject, optionally from or derived from a species different from the subject. The GDF15 polypeptide or GDF15 variant polypeptide used to treat or ameliorate a metabolic disorder in a subject, optionally from or derived from the same species as the subject.

[0086] The term "native Fc" refers to a molecule or sequence that includes the sequence of a non-antigen-binding fragment resulting from digestion of a whole antibody, whether in monomeric or multimeric form. The original source of immunoglobulin for native Fc is preferably of human origin and may be any immunoglobulin, although IgG1 and IgG2 are preferred. Native Fc is composed of monomeric polypeptides that may be linked by covalent (i.e., disulfide bonds) and non-covalent bonds into a dimeric or multimeric form. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of IgG (Ellison et al. (1982), Nucleic Acids Res. 10:4071-9). The term "native Fc" as used herein is inclusive to monomeric, dimeric and multimeric forms.

[0087] The term "Fc variant" refers to a molecule or sequence that is modified from a native Fc. Such modifications include, but are not limited to, substitution, deletion, or addition of one or more amino acids, including substitution with non-naturally occurring amino acids, non-naturally occurring amino acid analogs, and amino acid mimetics. Thus, the term "Fc variant" includes molecules or sequences that are humanized from a non-human native Fc. Additionally, a native Fc contains sites that may be removed because these sites provide structural features or biological activities that are not required for the fusion molecules of the present invention. Thus, the term "Fc variant" includes molecules or sequences that lack one or more sites or residues of the native Fc that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in more detail below. In various embodiments, the Fc variants comprise an amino acid sequence that is at least about 85 percent identical to a native Fc, hi other embodiments, the Fc variants comprise an amino acid sequence that is at least about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to a native Fc.

[0088] The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined above. As well as Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form.

[0089] The term "multimer," as applied to an Fc domain or a molecule containing an Fc domain, refers to a molecule having two or more polypeptide chains that are covalently linked, non-covalently linked, or linked by both covalent and non-covalent interactions. IgG molecules normally form dimers, IgM forms pentamers, IgD forms dimers, and IgA forms monomers, dimers, trimers, or tetramers. Multimers may be formed by utilizing the sequence and resulting activity of a native Ig source of Fc or by derivatizing such a native Fc.

[0090] The term "dimer," as applied to an Fc domain or a molecule containing an Fc domain, refers to a molecule having two polypeptide chains that are covalently linked, non-covalently linked, or linked by both covalent and non-covalent interactions.

[0091] The term "hinge" or "hinge region" as used herein includes the flexible polypeptide that comprises the amino acids between the first and second constant domains of an antibody. The "hinge region" referred to herein is a sequence region of 6-62 amino acids in length that is present only in IgA, IgD and IgG and encompasses the cysteine ​​residues that bridge the two heavy chains.

[0092] The term "polypeptide linker" refers to a short polypeptide, generally 1-30 amino acid residues in length, that covalently links two polypeptides together, usually via a peptide bond.

[0093] As used herein, the term HSA polypeptide includes naturally occurring "wild-type" human serum albumin. The term also includes various biologically active fragments and variants, fusion proteins and modified forms of wild-type HSA protein. Such biologically active fragments or variants, fusion proteins and modified forms of wild-type HSA protein have at least a portion of the amino acid sequence of substantial sequence identity to wild-type HSA protein. In certain embodiments, such biologically active fragments or variants, fusion proteins and modified forms of wild-type HSA protein have an amino acid sequence that is at least about 85 percent identical to wild-type HSA. In other embodiments, such biologically active fragments or variants, fusion proteins and modified forms of wild-type HSA protein have an amino acid sequence that is at least about 90 percent, or about 95, 96, 97, 98 or 99 percent identical to wild-type HSA.

[0094] II. Fc fusions comprising GDF15 polypeptides or GDF15 mutant polypeptides, and polynucleotides Various Fc fusion proteins comprising GDF15 polypeptides or GDF15 mutant polypeptides are provided herein. In some embodiments, the fusion protein comprises a native Fc. In some embodiments, the fusion protein comprises an engineered Fc domain.

[0095] In some embodiments, an Fc fusion protein containing a GDF15 polypeptide (or a GDF15 mutant polypeptide) associates with another polypeptide chain consisting of or including an Fc domain to form a heterodimer. In some embodiments, two such heterodimers associate to form a heterotetramer. Some of these polypeptide constructs (i.e., Fc fusion proteins containing a GDF15 polypeptide (or a GDF15 mutant polypeptide) and multimers comprising Fc fusion proteins containing one or more such GDF15 polypeptides (or GDF15 mutant polypeptides)) have been experimentally investigated, as described in the Examples presented herein below.

[0096] Antibodies belong to the immunoglobulin class of proteins, which includes IgG, IgA, IgE, IgM, and IgD. The most abundant immunoglobulin class in human serum is IgG (Deisenhofer, J. 1981, Biochem. 20:2361-2370; Huber, R. 1984 Behring Inst Mitt 76:1-14; Roux KH 1999 Int. Arch. Allergy Immunol. 120:85-99). The structure of IgG has four chains, two light chains and two heavy chains, with each light chain having two domains and each heavy chain having four domains. The antigen-binding site is located in the Fab domain (fragment antigen-binding), which contains the light chain variable (VL) domain and the heavy chain variable (VH) domain, as well as the light chain constant (LC) domain and the heavy chain constant (CH1) domain. The regions of the CH2 and CH3 domains of the heavy chains are called Fc (fragment crystallizable). An IgG molecule can be considered as a heterotetramer with two heavy chains and two light chains held together by disulfide bonds (-SS-) at the hinge region. The number of disulfide bonds in the hinge varies among immunoglobulin subclasses (Papadea, C. 1989, Crit. Rev. Clin. Lab. Sci. 27:27-58). The FcRn binding site is located within the Fc domain of the antibody (Martin, WL. 2001, Mol. Cell, 7:867-877), so the extended serum half-life property of the antibody is retained in the Fc fragment. The Fc domain alone can be considered as a homodimer of the heavy chain containing the CH2 and CH3 domains.

[0097] In certain preferred embodiments, the fusion proteins described herein comprise an IgGFc domain derived from the Fc domain of wild-type human IgG. By "wild-type human" IgG Fc is meant a sequence of amino acids that occur naturally in the human population. Of course, just as Fc sequences may vary slightly between individuals, one or more changes may be made in the wild-type sequence and still be within the scope of the present invention. For example, the Fc domain may contain additional changes that are not relevant to the present invention, such as mutations at glycosylation sites or the inclusion of non-natural amino acids. In certain embodiments, the polypeptide containing the CH3 region is an IgG molecule, further comprising a CH1 domain and a CH2 domain. Exemplary human IgG sequences include the constant regions of IgG1, IgG2, IgG3 and IgG4. The Fc domain may also be comprised within the constant regions of the heavy chains of IgA, IgD, IgE and IgM.

[0098] Fc fusion proteins containing a GDF15 polypeptide or a GDF15 mutant polypeptide, and portions of multimers that contain such Fc fusion proteins, include those described below.

[0099] II.A. DhMonoFc Construct In this disclosure, the designation "Mono-" or "MonoFc domain" refers to an Fc domain that has been engineered to reduce or prevent homodimer formation. In one embodiment, a MonoFc domain is provided by introducing a tyrosine to threonine mutation (Y349T) and two lysine to aspartic acid mutations (K392D and K409D) in a native Fc or an Fc mutant.

[0100] The C-terminal lysine (K447) may optionally be deleted in MonoFc, which may be advantageous, for example, when a peptide is fused at the C-terminus to reduce proteolysis of the fusion protein.

[0101] The fusion protein includes MonoFc domain and GDF15 domain.Usually, the N-terminus of GDF15 domain is linked to the C-terminus of MonoFc domain directly or via a polypeptide linker.However, in some embodiments, the N-terminus of MonoFc domain is linked to the C-terminus of GDF15 domain directly or via a polypeptide linker.

[0102] In certain embodiments, a dimer is provided that comprises two such fusion proteins, linked via interchain disulfide bonds between their respective GDF15 domains.In some embodiments, the dimer is a homodimer.In other embodiments, the dimer is a heterodimer.

[0103] The designation "DhMono-" or "DhMonoFc domain" in this disclosure refers to an Fc domain that has had all or part of its hinge region removed and has been engineered to reduce or prevent homodimer formation. In one embodiment, a DhMonoFc domain is provided that introduces a tyrosine to threonine mutation (Y349T) and two lysine to aspartic acid mutations (K392D and K409D) in a native Fc or an Fc mutant that has had all or part of its hinge region removed.

[0104] The C-terminal lysine (K447) in DhMonoFc may optionally be deleted, which may be advantageous, for example, when a peptide is fused at the C-terminus to reduce proteolysis of the fusion protein.

[0105] Provided is a fusion protein comprising DhMonoFc domain and GDF15 domain.Usually, the N-terminus of GDF15 domain is linked directly or via a polypeptide linker to the C-terminus of DhMonoFc domain.However, in some embodiments, the N-terminus of DhMonoFc domain is linked directly or via a polypeptide linker to the C-terminus of GDF15 domain.

[0106] In certain embodiments, a dimer is provided that comprises two such fusion proteins, linked via interchain disulfide bonds between their respective GDF15 domains.In some embodiments, the dimer is a homodimer.In other embodiments, the dimer is a heterodimer.

[0107] II.A.1. DhMonoFc-GDF15 The designation "MonoFc-GDF15" in this disclosure refers to a fusion protein comprising a GDF15 polypeptide, the N-terminus of which is directly linked to the C-terminus of a MonoFc domain.

[0108] In certain embodiments, homodimers are provided that comprise two such fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains.

[0109] More particularly, in specific embodiments, the homodimer comprises (a) Sequence: Two MonoFc domains (one for each monomer) containing APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:22); (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12.

[0110] In a preferred embodiment, the fusion protein has the amino acid sequence: 46, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.

[0111] As discussed above, in a specific embodiment, a homodimer is provided that comprises two monomers having the sequence of SEQ ID NO:46.

[0112] II.A.2. DhMonoFc-(G 4 S) 4 -GDF15 In this disclosure, the name "DhMonofc-(G 4 S) 4 "-GDF15" refers to a fusion protein comprising a GDF15 polypeptide linked to a DhMonoFc domain via a polypeptide linker comprising the sequence of SEQ ID NO:18, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the DhMonoFc domain.

[0113] In certain embodiments, homodimers are provided that comprise two such fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains.

[0114] More particularly, in specific embodiments, the homodimer comprises (a) two DhMonoFc domains (one for each monomer) comprising the sequence of SEQ ID NO: 22; (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer), each containing the sequence: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18), linking the N-terminus of the GDF15 polypeptide to the C-terminus of the DhMonoFc domain.

[0115] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0116] As discussed above, in a specific embodiment, a homodimer is provided that comprises two monomers having the sequence of SEQ ID NO:24.

[0117] II.A.3. DhMonoFc-(G 4 S) 4 -GDF15(H6D) In this disclosure, the name "DhMonofc-(G 4 S) 4 "-GDF15(H6D)" refers to a fusion protein comprising a GDF15 polypeptide linked to a DhMonoFc domain via a polypeptide linker comprising the sequence of SEQ ID NO: 18, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the DhMonoFc domain. The GDF15(H6D) variant is a naturally occurring human GDF15 variant.

[0118] In certain embodiments, homodimers are provided that comprise two such fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains.

[0119] More particularly, in specific embodiments, the homodimer comprises (a) two DhMonoFc domains (one for each monomer) comprising the sequence of SEQ ID NO: 22; (b) Sequence: two GDF15 (H6D) polypeptides (one for each monomer) comprising the sequence: ARNGDDCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 25); (c) two polypeptide linkers (one for each monomer) each comprising the sequence of SEQ ID NO:18 linking the N-terminus of the GDF15 polypeptide to the C-terminus of the DhMonoFc domain.

[0120] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0121] As discussed above, in a specific embodiment, a homodimer is provided that comprises two monomers having the sequence of SEQ ID NO:27.

[0122] II.B. HemiFc The designation "Hemi-" or "HemiFc domain" in this disclosure refers to a polypeptide chain comprising a first Fc domain linked to a second Fc domain, either directly or via a polypeptide linker. In one embodiment, the first Fc domain and the second Fc domain have identical sequences. In another embodiment, the first Fc domain and the second Fc domain have different sequences. Typically, the first and second Fc domains are covalently linked via a disulfide bond between their respective hinge regions.

[0123] The C-terminal lysine (K447) may optionally be deleted in the first Fc domain, the second Fc domain, or both, which may be advantageous, for example, when a peptide is fused at the C-terminus to reduce proteolysis of the fusion protein.

[0124] Provided is a fusion protein comprising HemiFc domain and GDF15 region.Usually, the N-terminus of GDF15 region is linked to the C-terminus of HemiFc domain directly or via a polypeptide linker.However, in some embodiments, the N-terminus of HemiFc domain is linked to the C-terminus of GDF15 region directly or via a polypeptide linker.

[0125] In certain embodiments, a dimer is provided that comprises two such fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains. In some embodiments, the dimer is a homodimer. In other embodiments, the dimer is a heterodimer. 4 S) 4 -Fc-S(G 4 S) 4-See FIG. 3 for a diagrammatic depiction of a homodimeric embodiment of the GDF15 fusion protein.

[0126] GGGFc-(G 4 S) 4 -Fc-S(G 4 S) 4 -GDF15 The name "GGGFc-(G 4 S) 4 -Fc-S(G 4 S) 4 "-GDF15" refers to a fusion protein comprising a GDF15 polypeptide linked to a HemiFc domain via a first polypeptide linker comprising the sequence of SEQ ID NO: 30, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HemiFc domain. The HemiFc domain comprises a first Fc domain linked to a second Fc domain via a polypeptide linker comprising the sequence of SEQ ID NO: 18, which connects the N-terminus of the first Fc domain to the C-terminus of the second Fc domain (having three glycines added to its N-terminus).

[0127] In certain embodiments, homodimers are provided that comprise two such fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains.

[0128] More particularly, in specific embodiments, the homodimer comprises (a) Sequence (part of the hinge region is in parentheses): two second Fc domains (one in each monomer) comprising: GGG(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 28); (b) Sequence (part of the hinge region is in parentheses): (ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 29); (c) two second polypeptide linkers (one for each monomer) comprising the sequence of SEQ ID NO: 18 linking the N-terminus of the first Fc domain to the C-terminus of the second Fc domain; (d) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO: 12; (e) two first polypeptide linkers (one for each monomer) comprising the sequence: SGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 30) linking the N-terminus of the GDF15 polypeptide to the C-terminus of the first Fc domain.

[0129] In a preferred embodiment, the fusion protein has the amino acid sequence (part of the hinge region in brackets; double underlined linker sequence): [ka] which comprises the nucleic acid sequence:

[0130] As discussed above, in a specific embodiment, a homodimer is provided that comprises two monomers having the sequence of SEQ ID NO:32.

[0131] II.C. DhHemiFc The designation "DhHemi-" or "DhHemiFc domain" in this disclosure refers to a polypeptide chain comprising a first Fc domain with all or part of its hinge region removed ("DhFc" domain), linked directly or via a polypeptide linker to a second DhFc domain. In a particular embodiment, the N-terminal 12 amino acids in the hinge region are removed, e.g., ERKSSVECPPCP (SEQ ID NO:15). In one embodiment, the first DhFc domain and the second DhFc domain have identical sequences. In another embodiment, the first DhFc domain and the second DhFc domain have different sequences.

[0132] Provided is a fusion protein comprising DhHemiFc domain and GDF15 domain.Usually, the N-terminus of GDF15 domain is linked directly or via a polypeptide linker to the C-terminus of DhHemiFc domain.However, in some embodiments, the N-terminus of DhHemiFc domain is linked directly or via a polypeptide linker to the C-terminus of GDF15 domain.

[0133] In certain embodiments, a dimer is provided that comprises two such fusion proteins, linked via interchain disulfide bonds between their respective GDF15 domains.In some embodiments, the dimer is a homodimer.In other embodiments, the dimer is a heterodimer.

[0134] GGGDhFc-(G 4 S) 5 -DhFc-S(G 4 S) 4 -GDF15 In this disclosure, the name “GGGDhFc-(G 4 S) 5-DhFc-S(G 4 S) 4 "-GDF15" refers to a fusion protein comprising a GDF15 polypeptide linked to a DhHemiFc domain via a first polypeptide linker comprising the sequence of SEQ ID NO: 30, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HemiFc domain. The HemiFc domain comprises a first DhFc domain linked to a second DhFc domain via a polypeptide linker comprising the sequence of SEQ ID NO: 34, which connects the N-terminus of the first DhFc domain to the C-terminus of the second DhFc domain (having three glycine residues added to its N-terminus).

[0135] In certain embodiments, homodimers are provided that comprise two such fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains.

[0136] More particularly, in specific embodiments, the homodimer comprises (a) Sequence: two second DhFc domains (one in each monomer) comprising: GGGAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 33); (b) Sequence: and two first DhFc domains (one in each monomer) comprising: APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35); (c) two second polypeptide linkers (one for each monomer) comprising the sequence: GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 34) linking the N-terminus of the first DhFc domain to the C-terminus of the second DhFc domain; (d) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (e) two first polypeptide linkers (one for each monomer) comprising the sequence of SEQ ID NO:30 linking the N-terminus of the GDF15 polypeptide to the C-terminus of the first DhFc domain.

[0137] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0138] As discussed above, in a specific embodiment, a homodimer is provided that comprises two monomers having the sequence of SEQ ID NO:37.

[0139] II.D. Knob / Hole The designation "knob-" or "knob Fc domain" in this disclosure refers to an Fc domain that comprises a "knob" mutation. The designation "hole-" or "hole Fc domain" in this disclosure refers to a native Fc or an Fc variant that comprises a "hole" mutation.

[0140] A "knob" can be created by replacing a small amino acid side chain with a larger one, and a "hole" can be created by replacing a large amino acid side chain with a small one. See, e.g., Ridgway, JBB. 1996, Protein Eng. 9:617-621; Merchant, AM, 1998, Nature Biotech. 16:677-681.

[0141] In one embodiment, a "knobFc" domain is provided by introducing a threonine to tryptophan mutation (T366W) in the sequence of the Fc domain. In one embodiment, a "holeFc" domain is provided by introducing a threonine to serine mutation (T366S), a leucine to alanine mutation (L368A), and a tyrosine to valine mutation (Y407V) in the sequence of the Fc domain.

[0142] The C-terminal lysine (K447) can optionally be deleted in the knobFc domain, the holeFc domain, or both, which can be advantageous, for example, when a peptide is fused at the C-terminus to reduce proteolysis of the fusion protein.

[0143] In one embodiment, a "DhknobFc" domain is provided by introducing a threonine to tryptophan mutation (T366W) into the sequence of an Fc domain from which all or part of its hinge region has been deleted. In one embodiment, a "DhholeFc" domain is provided by introducing a threonine to serine mutation (T366S), a leucine to alanine mutation (L368A) and a tyrosine to valine mutation (Y407V) into the sequence of an Fc domain from which all or part of its hinge region has been deleted.

[0144] The C-terminal lysine (K447) may be optionally deleted in DhknobFc, DhholeFc, or both, which may be advantageous, for example, when a peptide is fused at the C-terminus to reduce proteolysis of the fusion protein.

[0145] In some embodiments, heterodimers are provided that include (i) a first polypeptide chain that includes a GDF15 region linked directly or via a polypeptide linker to a holeFc domain, and (ii) a second polypeptide chain that includes a knobFc domain. In other embodiments, heterodimers are provided that include (i) a first polypeptide chain that includes a GDF15 region linked directly or via a polypeptide linker to a knobFc domain, and (ii) a second polypeptide chain that includes a holeFc domain.

[0146] In some embodiments, heterodimers are provided that include (i) a first polypeptide chain that includes a GDF15 region linked directly or via a polypeptide linker to a DhholeFc domain, and (ii) a second polypeptide chain that includes a DhknobFc domain. In other embodiments, heterodimers are provided that include (i) a first polypeptide chain that includes a GDF15 region linked directly or via a polypeptide linker to a DhknobFc domain, and (ii) a second polypeptide chain that includes a DhholeFc domain.

[0147] In some embodiments, a tetramer is provided that comprises two such heterodimers linked via an interchain disulfide bond between the GDF15 domains of each first polypeptide chain of the heterodimers. See Figure 1 for a diagrammatic representation of an embodiment of a heterotetramer comprising two heterodimers, each heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhknobFc domain via a polypeptide linker, and (ii) a second polypeptide chain comprising a DhholeFc domain.

[0148] DhknobFc-(G 4 S) 4 -GDF15:DhholeFc In this disclosure, the name "DhknobFc-(G 4 S) 4 "-GDF15:DhholeFc" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhknobFc domain via a linker comprising the sequence of SEQ ID NO:18 connecting the N-terminus of the GDF15 polypeptide to the C-terminus of the DhknobFc domain, and (ii) a polypeptide chain comprising a DhholeFc domain.

[0149] In certain embodiments, the first polypeptide chain of each heterodimer comprises two DhknobFc-(GDF15 domains) linked via an interchain disulfide bond between their respective GDF15 domains. 4 S) 4 - A tetramer comprising a dimer of the GDF15:DholeFc heterodimer is provided.

[0150] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two DhknobFc domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16); (b) Sequence: Two DhholeFc domains (one for each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 281); (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO: 12; (d) two polypeptide linkers (one for each heterodimer) each comprising the sequence of SEQ ID NO: 18, which link the N-terminus of the GDF15 polypeptide to the C-terminus of the DhknobFc domain via a peptide bond.

[0151] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0152] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17), which comprises the nucleic acid sequence: (SEQ ID NO:21).

[0153] As discussed above, in a specific embodiment, a tetramer is provided that comprises two monomers having the sequence of SEQ ID NO:20 and two monomers having the sequence of SEQ ID NO:17.

[0154] II.E. Charge Pair (delHinge) The designation "CpmFc(+) domain" in this disclosure refers to an Fc domain that comprises a "positive" charge pair mutation. The designation "CpmFc(-) domain" in this disclosure refers to an Fc domain that comprises a "negative" charge pair mutation. Note that the use of the terms "positive" and "negative" is for ease of reference (i.e., to describe the charge pair nature of the Fc domain) and does not indicate that the entire sequence or construct is necessarily positively or negatively charged.

[0155] In one embodiment, "positive" electron pair mutations are provided by introducing a glutamic acid to lysine mutation (E356K) and an aspartic acid to lysine mutation (D399K) in the sequence of the Fc domain, while in one embodiment, "negative" electron pair mutations are provided by introducing two lysine to aspartic acid mutations (K392D, K409D) in the sequence of the Fc domain.

[0156] When incubated together, the aspartic acid residues associate with the lysine residues through electrostatic forces, promoting the formation of heterodimers between the CpmFc(+) and CpmFc(-) domains and reducing or preventing the formation of homodimers between the CpmFc(+) or CpmFc(-) sequences.

[0157] The C-terminal lysine (K447) may optionally be deleted in the CpmFc(+) domain, the CpmFc(-) domain, or both, which may be advantageous, for example, when a peptide is fused at the C-terminus to reduce proteolysis of the fusion protein.

[0158] In one embodiment, a "DhCpmFc(+)" domain is provided by introducing a glutamic acid to lysine mutation (E356K) and an aspartic acid to lysine mutation (D399K) in the sequence of an Fc domain from which all or part of its hinge region has been deleted. In one embodiment, a "DhCpmFc(-)" domain is provided by introducing two lysine to aspartic acid mutations (K392D, K409D) in the sequence of an Fc domain from which all or part of its hinge region has been deleted.

[0159] When incubated together, the aspartic acid residues associate with the lysine residues through electrostatic forces, promoting the formation of heterodimers between the DhCpmFc(+) and DhCpmFc(-) domains and reducing or preventing the formation of homodimers between the DhCpmFc(+) or DhCpmFc(-) sequences.

[0160] The C-terminal lysine (K447) may optionally be deleted in DhCpmFc(+), DhCpmFc(-), or both, which may be advantageous, for example, when a peptide is fused at the C-terminus to reduce proteolysis of the fusion protein.

[0161] In some embodiments, a heterodimer is provided that includes (i) a first polypeptide chain that includes a GDF15 region linked to a CpmFc(+) domain, and (ii) a second polypeptide chain that includes a CpmFc(-) domain. In some embodiments, the N-terminus of the GDF15 region is linked to the C-terminus of the CpmFc(+) domain, either directly or via a polypeptide linker. In other embodiments, the N-terminus of the CpmFc(+) domain is linked to the C-terminus of the GDF15 region, either directly or via a polypeptide linker.

[0162] In other embodiments, a heterodimer is provided that includes (i) a first polypeptide chain that includes a GDF15 region linked to a CpmFc(-) domain, and (ii) a second polypeptide chain that includes a CpmFc(+) domain. In some embodiments, the N-terminus of the GDF15 region is linked to the C-terminus of the CpmFc(-) domain, either directly or via a polypeptide linker. In other embodiments, the N-terminus of the CpmFc(-) domain is linked to the C-terminus of the GDF15 region, either directly or via a polypeptide linker.

[0163] In some embodiments, a heterodimer is provided that includes (i) a first polypeptide chain that includes a GDF15 region linked to a DhCpmFc(+) domain, and (ii) a second polypeptide chain that includes a DhCpmFc(-) domain. In some embodiments, the N-terminus of the GDF15 region is linked to the C-terminus of the DhCpmFc(+) domain, either directly or via a polypeptide linker. In other embodiments, the N-terminus of the DhCpmFc(+) domain is linked to the C-terminus of the GDF15 region, either directly or via a polypeptide linker.

[0164] In other embodiments, heterodimers are provided that include (i) a first polypeptide chain that includes a GDF15 region linked to a DhCpmFc(-) domain, and (ii) a second polypeptide chain that includes a DhCpmFc(+) domain. In some embodiments, the N-terminus of the GDF15 region is linked to the C-terminus of the DhCpmFc(-) domain, either directly or via a polypeptide linker. In other embodiments, the N-terminus of the DhCpmFc(-) domain is linked to the C-terminus of the GDF15 region, either directly or via a polypeptide linker.

[0165] In some embodiments, a tetramer is provided that comprises a dimer of two such heterodimers, where the two first polypeptide chains of the heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains. See Figure 4 for a diagrammatic representation of an embodiment of a tetramer that comprises two heterodimers, each heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhCpmFc(-) domain via a polypeptide linker, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0166] II.E.1. DhCpmFc(+)-(1K)-GDF15:DhCpmFc(-) In this disclosure, the name "DhCpmFc(+)-(1K)-GDF15:DhCpmFc(-)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhCpmFc(+) domain via a linker comprising the sequence of SEQ ID NO:40 connecting the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(+) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(-) domain.

[0167] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(+)-(1K)-GDF15:DhCpmFc(-) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0168] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two DhCpmFc(+) domains (one for each heterodimer) containing APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 38); (b) Sequence: Two DhCpmFc(-) domains (one for each heterodimer) containing APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 282); (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO: 12; (d) two polypeptide linkers (one for each heterodimer) each containing the sequence: GSGSATGGSGSVASSGSGSATHL (SEQ ID NO:40), which link the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(+) domain via a peptide bond.

[0169] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0170] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:39), which has the nucleic acid sequence: (SEQ ID NO:43).

[0171] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:42 and two polypeptide chains comprising the sequence of SEQ ID NO:39.

[0172] II.E.2. DhCpmFc(-)-GDF15:DhCpmFc(+) In this disclosure, the name "DhCpmFc(-)-GDF15:DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide whose N-terminus is directly linked to the C-terminus of a DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0173] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)-GDF15:DhCpmFc(+) heterodimers in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0174] More particularly, in specific embodiments, the tetramer comprises: (a) Amino acid sequence: Two DhCpmFc(+) domains (one for each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 283); (b) Amino acid sequence: Two DhCpmFc(-) domains (one in each heterodimer) containing APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 48); (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:12.

[0175] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 50, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.

[0176] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47), which comprises the nucleic acid sequence: (SEQ ID NO:51).

[0177] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:50 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0178] II.E.3. DhCpmFc(-)-GDF15(N3D):DhCpmFc(+) The designation "DhCpmFc(-)-GDF15(N3D):DhCpmFc(+)" in this disclosure refers to a heterodimer comprising (i) a first polypeptide chain comprising a naturally occurring variant of GDF15 having an asparagine to aspartic acid mutation (N3D) directly linked at its N-terminus to the C-terminus of the DhCpmFc(-) domain ("GDF15(N3D)"), and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain. N3D may reduce deamidation-induced heterogeneity.

[0179] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)-GDF15(N3D):DhCpmFc(+) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0180] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) Sequence: and two GDF15(N3D) polypeptides (one for each heterodimer) comprising: ARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:52).

[0181] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 54, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.

[0182] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0183] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:54 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0184] II.E.4. DhCpmFc(-)-GDF15(Ndel3):DhCpmFc(+) In this disclosure, the designation "DhCpmFc(-)-GDF15(Ndel3):DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a mutant of GDF15 lacking the first three amino acids ("GDF15(Ndel3)"), whose N-terminus is directly linked to the C-terminus of the DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising the DhCpmFc(+) domain. The Ndel3 mutant may reduce heterogeneity induced by deamidation of N3 and subsequent isomerization of D3.

[0185] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)-GDF15(Ndel3):DhCpmFc(+) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0186] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) Sequence: and two GDF15 (Ndel3) polypeptides (one for each heterodimer) having the following sequence: GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:55).

[0187] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 57, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI. It is encoded by [[SEQ ID NO:56]]: gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0188] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0189] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:57 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0190] II.E.5. DhCpmFc(-)-G 4 -GDF15(N3D):DhCpmFc(+) In this disclosure, the name "DhCpmFc(-)-G 4 "-GDF15(N3D):DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(-) domain via a linker comprising the sequence of SEQ ID NO:58, which connects the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0191] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-Gp2H2O2 linked via an interchain disulfide bond between their respective GDF15 domains. 4 - A tetramer comprising a dimer of the GDF15(N3D):DhCpmFc(+) heterodimer is provided.

[0192] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) having the sequence of SEQ ID NO: 283; (b) two DhCpmFc(+) domains (one for each heterodimer) having the sequence of SEQ ID NO: 48; (c) two GDF15(N3D) polypeptides (one for each heterodimer) having the sequence of SEQ ID NO:52; (d) two polypeptide linkers (one for each heterodimer) each comprising the sequence: GGGG (SEQ ID NO:58), which link the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0193] In a preferred embodiment, the first polypeptide has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0194] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0195] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:60 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0196] II.E.6. DhCpmFc(-)-G 4 S-GDF15:DhCpmFc(+) In this disclosure, the name "DhCpmFc(-)-G 4 "S-GDF15:DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhCpmFc(-) domain via a polypeptide linker comprising the sequence of SEQ ID NO:61, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0197] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-Gp2H2O2 linked via an interchain disulfide between their respective GDF15 domains. 4 Tetramers comprising dimers of S-GDF15:DhCpmFc(+) heterodimers are provided.

[0198] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO: 12; (d) two polypeptide linkers (one for each heterodimer) containing the sequence GGGGS (SEQ ID NO: 61), each linking the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0199] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0200] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0201] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:63 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0202] II.E.7. DhCpmFc(-)-(G 4 S) 2 -GDF15:DhCpmFc(+) In this disclosure, the name “DhCpmFc(-)-(G 4 S) 2 "-GDF15:DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhCpmFc(-) domain via a polypeptide linker comprising the sequence of SEQ ID NO:64 connecting the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0203] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-(G 4 S) 2 - A tetramer comprising a dimer of the GDF15:DhCpmFc(+) heterodimer is provided.

[0204] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO: 12; (d) two polypeptide linkers (one for each heterodimer) comprising the sequence GGGGSGGGGS (SEQ ID NO:64), each linking the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0205] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0206] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0207] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:66 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0208] II.E.8. DhCpmFc(-)-(G 4 S) 2 -GDF15(N3D):DhCpmFc(+) In this disclosure, the name “DhCpmFc(-)-(G 4 S) 2 "-GDF15(N3D):DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(-) domain via a linker comprising the sequence of SEQ ID NO:64 connecting the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0209] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-(G 4 S) 2 - A tetramer comprising a dimer of the GDF15(N3D):DhCpmFc(+) heterodimer is provided.

[0210] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52; (d) two polypeptide linkers (one for each heterodimer) comprising SEQ ID NO:64, each linking the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0211] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0212] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0213] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:68 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0214] II.E.9. DhCpmFc(-)-G 4 P-GDF15:DhCpmFc(+) In this disclosure, the name "DhCpmFc(-)-G 4 "P-GDF15:DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhCpmFc(-) domain via a linker comprising the sequence of SEQ ID NO:69 connecting the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0215] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-Gp2H2O2 linked via an interchain disulfide bond between their respective GDF15 domains. 4 Tetramers comprising dimers of P-GDF15:DhCpmFc(+) heterodimers are provided.

[0216] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO: 12; (d) two polypeptide linkers (one for each heterodimer) each containing the sequence GGGGP (SEQ ID NO:69), which link the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0217] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0218] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0219] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:71 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0220] II.E.10. DhCpmFc(-)-(G 4 P) 2 -GDF15:DhCpmFc(+) In this disclosure, the name “DhCpmFc(-)-(G 4 P) 2 "-GDF15:DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhCpmFc(-) domain via a linker comprising the sequence of SEQ ID NO:72 connecting the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain, and (ii) a second v comprising a DhCpmFc(+) domain.

[0221] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-(G 4 P) 2 - A tetramer comprising a dimer of the GDF15:DhCpmFc(+) heterodimer is provided.

[0222] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO: 12; (d) two polypeptide linkers (one for each heterodimer) each containing the sequence GGGGPGGGGP (SEQ ID NO:72), which links the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0223] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0224] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0225] As discussed above, a tetramer is provided that comprises two polypeptide chains having the sequence of SEQ ID NO:74 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0226] II.E.11. DhCpmFc(-)-G 4 Q-GDF15:DhCpmFc(+) In this disclosure, the name "DhCpmFc(-)-G 4 "Q-GDF15:DhCpmFc(+)" refers to a heterodimer comprising (ii) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhCpmFc(-) domain via a linker comprising the sequence of SEQ ID NO:75 connecting the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain, and (iii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0227] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-Gp2H2O2 linked via an interchain disulfide bond between their respective GDF15 domains. 4 A tetramer comprising a dimer of the Q-GDF15:DhCpmFc(+) heterodimer is provided.

[0228] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) chains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) chains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO: 12; (d) two polypeptide linkers (one for each heterodimer) each containing the sequence GGGGQ (SEQ ID NO: 75), which link the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0229] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0230] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0231] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:77 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0232] II.E.12. DhCpmFc(-)-(G 4 Q) 2 -GDF15:DhCpmFc(+) In this disclosure, the name “DhCpmFc(-)-(G 4 Q) 2 "-GDF15:DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhCpmFc(-) domain via a linker comprising the sequence of SEQ ID NO:78 connecting the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0233] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-(G 4 Q) 2 - A tetramer comprising a dimer of the GDF15:DhCpmFc(+) heterodimer is provided.

[0234] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO: 12; (d) two polypeptide linkers (one for each heterodimer) each comprising the sequence GGGGQGGGGQ (SEQ ID NO:78), which link the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0235] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0236] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0237] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:80 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0238] II.E.13. DhCpmFc(-)-(G 4 Q) 2 -GDF15(N3D):DhCpmFc(+) In this disclosure, the name “DhCpmFc(-)-(G 4 Q) 2 "-GDF15(N3D):DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(-) domain via a linker comprising the sequence of SEQ ID NO:78, which connects the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0239] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-(G 4 Q) 2 - A tetramer comprising a dimer of the GDF15(N3D):DhCpmFc(+) heterodimer is provided.

[0240] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52; (d) two polypeptide linkers (one for each heterodimer) comprising SEQ ID NO:78, each linking the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0241] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0242] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0243] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:82 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0244] II.E.14. DhCpmFc(-)-(G 4 Q) 2 -GDF15(Ndel3):DhCpmFc(+) In this disclosure, the name “DhCpmFc(-)-(G 4 Q) 2 "-GDF15(Ndel3):DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide linked to a DhCpmFc(-) domain via a linker comprising the sequence of SEQ ID NO:78, which connects the N-terminus of the GDF15(Ndel3) polypeptide to the C-terminus of the DhCpmFc(-) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

[0245] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)-(G 4 Q) 2 - A tetramer comprising a dimer of the GDF15(Ndel3):DhCpmFc(+) heterodimer is provided.

[0246] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 283; (b) two DhCpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 48; (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55; (d) two polypeptide linkers (one for each heterodimer) comprising SEQ ID NO:78, each linking the N-terminus of the GDF15(Ndel3) polypeptide to the C-terminus of the DhCpmFc(-) domain via a peptide bond.

[0247] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0248] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, encoded by the nucleic acid sequence of SEQ ID NO:51.

[0249] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:84 and two polypeptide chains having the sequence of SEQ ID NO:47.

[0250] II.F. Charge-paired (delHinge) cysteine ​​clamp For example, a "cysteine ​​clamp" mutation may be introduced into an Fc domain, such as a CpmFc(+) domain, a CpmFc(-) domain, a DhCpmFc(+) domain, or a DhCpmFc(-) domain. A "cysteine ​​clamp" mutation usually involves introducing a cysteine ​​into the CH3 domain of an Fc domain at a specific position via mutation, so that when incubated with another Fc domain having a cysteine ​​introduced into the CH3 domain at a specific position via mutation, a disulfide bond (cysteine ​​clamp) may form between the two Fc domains (e.g., between a CpmFc(+) domain having a "cysteine ​​clamp" mutation and a CpmFc(-) domain having a "cysteine ​​clamp" mutation, or between a DhCpmFc(+) domain having a "cysteine ​​clamp" mutation and a DhCpmFc(-) domain having a "cysteine ​​clamp" mutation). An Fc domain may contain one or more such cysteine ​​clamp mutations.

[0251] In one embodiment, the cysteine ​​clamp is provided by introducing a serine to cysteine ​​mutation (S354C) into the first Fc domain and a tyrosine to cysteine ​​mutation (Y349C) into the second Fc domain.

[0252] The designation "DhCpmFc(-)(S354C)" domain in this disclosure refers to a DhCpmFc(-) domain containing a serine to cysteine ​​mutation (S354C). The designation "DhCpmFc(+)(S354C)" domain in this disclosure refers to a DhCpmFc(+) domain containing a serine to cysteine ​​mutation (S354C). The designation "DhCpmFc(-)(Y349C)" domain in this disclosure refers to a DhCpmFc(-) domain containing a serine to cysteine ​​mutation (Y349C). The designation "DhCpmFc(+)(Y349C)" domain in this disclosure refers to a DhCpmFc(+) domain containing a serine to cysteine ​​mutation (Y349C).

[0253] In this disclosure, the designation "CpmFc(-)(S354C)" domain refers to a CpmFc(-) domain containing a serine to cysteine ​​mutation (S354C). In this disclosure, the designation "CpmFc(+)(S354C)" domain refers to a CpmFc(+) domain containing a serine to cysteine ​​mutation (S354C). In this disclosure, the designation "CpmFc(-)(Y349C)" domain refers to a CpmFc(-) domain containing a serine to cysteine ​​mutation (Y349C). In this disclosure, the designation "CpmFc(+)(Y349C)" domain refers to a CpmFc(+) domain containing a serine to cysteine ​​mutation (Y349C).

[0254] In another embodiment, a cysteine ​​clamp is provided by introducing a leucine to cysteine ​​mutation (L351C) in both the first domain and the Fc domain.

[0255] In this disclosure, the designation "DhCpmFc(-)(L351C)" domain refers to a DhCpmFc(-) domain containing a serine to cysteine ​​mutation (L351C). In this disclosure, the designation "DhCpmFc(+)(L351C)" domain refers to a DhCpmFc(+) domain containing a serine to cysteine ​​mutation (L351C).

[0256] In this disclosure, the designation "CpmFc(-)(L351C)" domain refers to a CpmFc(-) domain containing a serine to cysteine ​​mutation (L351C). In this disclosure, the designation "CpmFc(+)(L351C)" domain refers to a CpmFc(+) domain containing a serine to cysteine ​​mutation (L351C).

[0257] The C-terminal lysine (K447) may optionally be deleted in the CpmFc(+) domain, the CpmFc(-) domain, or both, which may be advantageous, for example, when a peptide is fused at the C-terminus to reduce proteolysis of the fusion protein.

[0258] In some embodiments, the heterodimers provided include (i) a first polypeptide chain comprising a GDF15 region linked to a CpmFc(+) domain comprising a cysteine ​​clamp mutation, and (ii) a second polypeptide chain comprising a CpmFc(-) domain comprising a cysteine ​​clamp mutation. In some embodiments, the N-terminus of the GDF15 region is linked, either directly or via a polypeptide linker, to the C-terminus of the CpmFc(+) domain comprising a cysteine ​​clamp mutation. In other embodiments, the N-terminus of the CpmFc(+) domain comprising a cysteine ​​clamp mutation is linked, either directly or via a polypeptide linker, to the C-terminus of the GDF15 region.

[0259] In some embodiments, the heterodimers provided include (i) a first polypeptide chain comprising a GDF15 region linked to a CpmFc(-) domain comprising a cysteine ​​clamp mutation, and (ii) a second polypeptide chain comprising a CpmFc(+) domain comprising a cysteine ​​clamp mutation. In some embodiments, the N-terminus of the GDF15 region is linked, either directly or via a polypeptide linker, to the C-terminus of the CpmFc(-) domain comprising a cysteine ​​clamp mutation. In other embodiments, the N-terminus of the CpmFc(-) domain comprising a cysteine ​​clamp mutation is linked, either directly or via a polypeptide linker, to the C-terminus of the GDF15 region.

[0260] In some embodiments, the heterodimers provided include (i) a first polypeptide chain including a GDF15 region linked to a DhCpmFc(+) domain including a cysteine ​​clamp mutation, and (ii) a second polypeptide chain including a DhCpmFc(-) domain including a cysteine ​​clamp mutation. In some embodiments, the N-terminus of the GDF15 region is linked, either directly or via a polypeptide linker, to the C-terminus of the DhCpmFc(+) domain including a cysteine ​​clamp mutation. In other embodiments, the N-terminus of the DhCpmFc(+) domain including a cysteine ​​clamp mutation is linked, either directly or via a polypeptide linker, to the C-terminus of the GDF15 region.

[0261] In some embodiments, the heterodimers provided include (i) a first polypeptide chain including a GDF15 region linked to a DhCpmFc(-) domain including a cysteine ​​clamp mutation, and (ii) a second polypeptide chain including a DhCpmFc(+) domain including a cysteine ​​clamp mutation. In some embodiments, the N-terminus of the GDF15 region is linked, either directly or via a polypeptide linker, to the C-terminus of the DhCpmFc(-) domain including a cysteine ​​clamp mutation. In other embodiments, the N-terminus of the DhCpmFc(-) domain including a cysteine ​​clamp mutation is linked, either directly or via a polypeptide linker, to the C-terminus of the GDF15 region.

[0262] In some embodiments, a tetramer is provided that includes two such heterodimer dimers, where the two first polypeptide chains of the heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.See FIG. 5 for a diagrammatic representation of an embodiment of a tetramer that includes two heterodimers, each heterodimer including (i) a first polypeptide chain that includes a GDF15 polypeptide linked to a DhCpmFc(+)(L351C) domain via a polypeptide linker, and (ii) a second polypeptide chain that includes a DhCpmFc(-)(L351C) domain.See FIG. 5 for a diagrammatic representation of an embodiment of a tetramer that includes two heterodimers, each heterodimer including a first polypeptide chain that includes a GDF15 polypeptide linked to a DhCpmFc(+)(L351C) domain via a polypeptide linker.

[0263] II.F.1. DhCpmFc(+)(S354C)-GDF15(N3D):DhCpmFc(-)(Y349C) In this disclosure, the designation "DhCpmFc(+)(S354C)-GDF15(N3D):DhCpmFc(-)(Y349C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a DhCpmFc(+)(S354C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(-)(Y349C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C354 of the first polypeptide chain and C349 of the second polypeptide chain.

[0264] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(+)(S354C)-GDF15(N3D):DhCpmFc(-)(Y349C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0265] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two DhCpmFc(+)(S354C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 85); (b) Sequence: Two DhCpmFc(-)(Y349C) (one in each heterodimer) containing APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 284); (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0266] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 88, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.

[0267] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 86), which comprises the nucleic acid sequence: (SEQ ID NO:89).

[0268] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:88 and two polypeptide chains having the sequence of SEQ ID NO:86.

[0269] II.F.2. DhCpmFc(-)(Y349C)-GDF15:DhCpmFc(+)(S354C) In this disclosure, the designation "DhCpmFc(-)(Y349C)-GDF15:DhCpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide whose N-terminus is directly linked to the C-terminus of a DhCpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0270] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(Y349C)-GDF15:DhCpmFc(+)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0271] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two DhCpmFc(+)(S354C) domains (one in each heterodimer) containing APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 285); (b) Sequence: Two DhCpmFc(-)(Y349C) domains (one in each heterodimer) containing APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 91); (c) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:12.

[0272] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 93, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.

[0273] In a preferred embodiment, the second polypeptide chain has the sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 90), which comprises the nucleic acid sequence: (SEQ ID NO:94).

[0274] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:93 and two polypeptide chains having the sequence of SEQ ID NO:90.

[0275] II.F.3. DhCpmFc(-)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C) In this disclosure, the designation "DhCpmFc(-)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a DhCpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide and C354 of the second polypeptide.

[0276] In certain embodiments, a tetramer is provided that includes a dimer of two DhCpmFc(-)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains. The N3D mutation in the GDF15 sequence may be introduced to, for example, eliminate heterogeneity caused by N-deamidation.

[0277] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 285; (b) two DhCpmFc(-)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 91; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0278] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 96, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.

[0279] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90, which is encoded by the nucleic acid sequence of SEQ ID NO:94.

[0280] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:96 and two polypeptide chains having the sequence of SEQ ID NO:90.

[0281] II.F.4. DhCpmFc(-)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C) In this disclosure, the designation "DhCpmFc(-)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a DhCpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0282] In certain embodiments, a tetramer is provided that includes a dimer of two DhCpmFc(-)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains. An N-terminal three amino acid deletion (Ndel3) in the GDF15 sequence may be introduced to eliminate heterogeneity caused, for example, by deamidation of asparagine or isomerization of aspartic acid.

[0283] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 285; (b) two DhCpmFc(-)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 91; (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0284] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 98, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI. It is encoded by gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata (SEQ ID NO: 97).

[0285] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90, which is encoded by the nucleic acid sequence of SEQ ID NO:94.

[0286] As discussed above, a tetramer is provided that includes two polypeptide chains having the sequence of SEQ ID NO:98 and two polypeptide chains having the sequence of SEQ ID NO:90.

[0287] II.F.5. DhCpmFc(-)(Y349C)-G 4 -GDF15(N3D):DhCpmFc(+)(S354C) The name in this disclosure is "DhCpmFc(-)(Y349C)-G 4 "-GDF15(N3D):DhCpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(-)(Y349C) domain via a linker comprising the sequence of SEQ ID NO:58, which connects the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0288] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)(Y349C)-G linked via an interchain disulfide bond between their respective GDF15 domains. 4 - A tetramer comprising a dimer of the GDF15(N3D):DhCpmFc(+)(S354C) heterodimer is provided.

[0289] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 285; (b) two DhCpmFc(-)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 91; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52; (d) two polypeptide linkers (one for each heterodimer) each comprising the sequence of SEQ ID NO:58, which link the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-)(Y349C) domain via a peptide bond.

[0290] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0291] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90, which is encoded by the nucleic acid sequence of SEQ ID NO:94.

[0292] As discussed above, in a specific embodiment, a tetramer is provided that comprises two monomers having the sequence of SEQ ID NO:100 and two monomers having the sequence of SEQ ID NO:90.

[0293] II.F.6. DhCpmFc(-)(Y349C)-(G 4 S) 2 -GDF15(N3D):DhCpmFc(+)(S354C) In this disclosure, the name “DhCpmFc(-)(Y349C)-(G 4 S) 2 "-GDF15(N3D):DhCpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(-)(Y349C) domain via a linker comprising the sequence of SEQ ID NO:64, which connects the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0294] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)(Y349C)-(GDF15) heterodimers linked via interchain disulfide bonds between their respective GDF15 domains. 4 S) 2 - A tetramer comprising a dimer of the GDF15(N3D):DhCpmFc(+)(S354C) heterodimer is provided.

[0295] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 285; (b) two DhCpmFc(-)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 91; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52; (d) Two polypeptide linkers (one for each heterodimer) each comprising the sequence of SEQ ID NO:64 linking the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-)(Y349C) domain via a peptide bond.

[0296] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0297] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90, which is encoded by the nucleic acid sequence of SEQ ID NO:94.

[0298] As discussed above, in a specific embodiment, a tetramer is provided that comprises two monomers having the sequence of SEQ ID NO:102 and two monomers having the sequence of SEQ ID NO:90.

[0299] II.F.7. DhCpmFc(-)(Y349C)-(G 4 Q) 2 -GDF15(N3D):DhCpmFc(+)(S354C) In this disclosure, the name “DhCpmFc(-)(Y349C)-(G 4 Q) 2 "-GDF15(N3D):DhCpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(-)(Y349C) domain via a linker comprising the sequence of SEQ ID NO:78, which connects the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0300] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)(Y349C)-(GDF15) heterodimers linked via interchain disulfide bonds between their respective GDF15 domains. 4 Q) 2 - A tetramer comprising a dimer of the GDF15(N3D):DhCpmFc(+)(S354C) heterodimer is provided.

[0301] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 285; (b) two DhCpmFc(-)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 91; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52; (d) two polypeptide linkers (one for each heterodimer) each comprising the sequence of SEQ ID NO:78, which link the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-)(Y349C) domain via a peptide bond.

[0302] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0303] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90, which is encoded by the nucleic acid sequence of SEQ ID NO:94.

[0304] As discussed above, in a specific embodiment, a tetramer is provided that comprises two monomers having the sequence of SEQ ID NO:104 and two monomers having the sequence of SEQ ID NO:90.

[0305] II.F.8. DhCpmFc(-)(L351C)-(G 4 S) 2 -GDF15:DhCpmFc(+)(L351C) In this disclosure, the name “DhCpmFc(-)(L351C)-(G 4 S) 2 "-GDF15:DhCpmFc(+)(L351C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to a DhCpmFc(-)(L351C) domain via a linker comprising the sequence of SEQ ID NO:64, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-)(L351C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(L351C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C351 of the first polypeptide chain and C351 of the second polypeptide chain.

[0306] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)(L351C)-(GDF15)-GDF15 domains linked via interchain disulfide bonds between the two first polypeptide chains of each heterodimer. 4 S) 2 - A tetramer comprising a dimer of the GDF15:DhCpmFc(+)(L351C) heterodimer is provided.

[0307] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two DhCpmFc(+)(L351C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 286); (b) Sequence: Two DhCpmFc(-)(L351C) domains (one in each heterodimer) containing APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 106); (c) two GDF15 polypeptide chains (one for each heterodimer) comprising the sequence of SEQ ID NO: 12; (d) two polypeptide linkers (one for each heterodimer) each comprising the sequence of SEQ ID NO:64, which link the N-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(-)(L351C) domain via a peptide bond.

[0308] In a preferred embodiment, the first polypeptide chain has the amino acid sequence (double underlined is the linker sequence): [ka] which comprises the nucleic acid sequence:

[0309] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 105), which comprises the nucleic acid sequence: (SEQ ID NO: 109).

[0310] As discussed above, in a specific embodiment, a heterodimer is provided comprising two monomers having the sequence of SEQ ID NO:108 and two monomers having the sequence of SEQ ID NO:105.

[0311] II.G. HSA The designation "HSA" or "human serum albumin" in this disclosure refers to a fusion protein that includes a GDF15 region linked, either directly or via a polypeptide linker, to a human serum albumin (HSA) polypeptide. In some embodiments, the fusion protein includes two or more HSA polypeptides.

[0312] Typically, the N-terminus of the GDF15 domain is linked to the C-terminus of the HSA polypeptide, either directly or via a polypeptide linker. However, in some embodiments, the N-terminus of the HSA polypeptide is linked to the C-terminus of the GDF15 domain, either directly or via a polypeptide linker.

[0313] In certain embodiments, homodimers are provided that comprise two such fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains. See Figure 6 for a diagrammatic depiction of such a homodimer embodiment. Alternatively, heterodimers are provided that comprise one such fusion protein and a GDF15 or GDF mutant polypeptide linked via an interchain disulfide bond between the GDF15 domain of the fusion protein and the GDF15 or mutant polypeptide.

[0314] II.G.1. HSA-(G 4 S) 4 -GDF15:GDF15 heterodimer The name "HSA-(G 4 S) 4 "-GDF15:GDF15" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15 polypeptide linked to an HSA polypeptide via a linker comprising the sequence of SEQ ID NO:18, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide, and (ii) a second polypeptide chain comprising a GDF15 polypeptide.

[0315] Typically, the first and second polypeptide chains are linked via an interchain disulfide bond between their respective GDF15 domains.

[0316] More particularly, in specific embodiments, the heterodimer comprises (a) Sequence: (SEQ ID NO: 110), and (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) one polypeptide linker (first monomer) comprising the sequence of SEQ ID NO: 18, which links the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide via a peptide bond.

[0317] In a preferred embodiment, the first polypeptide has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0318] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:12, which is encoded by the nucleic acid sequence of SEQ ID NO:11.

[0319] As discussed above, a heterodimer is provided that comprises a first polypeptide chain having the sequence of SEQ ID NO:112 and a second polypeptide chain having the sequence of SEQ ID NO:12.

[0320] II.G.2. HSA-(G 4 S) 4 -GDF15 The name "HSA-(G 4 S) 4 "-GDF15" refers to a fusion protein comprising a GDF15 polypeptide linked to an HSA polypeptide via a linker comprising the sequence of SEQ ID NO:18, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide.

[0321] In certain embodiments, two HSA-(G 4 S) 4 -GDF15 fusion protein comprising two HSA-(G 4 S) 4 -GDF15 fusion protein-containing homodimers are provided.

[0322] More particularly, in specific embodiments, the homodimer comprises (a) two HSA polypeptides (one for each monomer) comprising the sequence of SEQ ID NO: 110; (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer) each comprising the sequence of SEQ ID NO:18, which link the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide via a peptide bond.

[0323] In a preferred embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:112, encoded by the nucleic acid sequence of SEQ ID NO:111.

[0324] As discussed above, in a specific embodiment, a homodimer is provided that comprises two fusion proteins having the sequence of SEQ ID NO:112.

[0325] II.G.3. HSA-GSPAPAPGS-GDF15 The name "HSA-(GSPAPAPGS)-GDF15" in this disclosure refers to a fusion protein comprising a GDF15 polypeptide linked to an HSA polypeptide via a linker comprising the sequence of SEQ ID NO:113, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide.

[0326] In certain embodiments, a homodimer is provided comprising two HSA-(GSPAPAPGS)-GDF15 fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains.

[0327] More particularly, in specific embodiments, the homodimer comprises (a) two HSA polypeptides (one for each monomer) comprising the sequence of SEQ ID NO: 110; (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer) each comprising the sequence: GSPAPAGS (SEQ ID NO: 113), which link the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide via a peptide bond.

[0328] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0329] As discussed above, in a specific embodiment, a homodimer is provided that comprises two fusion proteins having the sequence of SEQ ID NO:115.

[0330] II.G.4. HSA-GS (PAPAP) 2 GS-GDF15 The name in this disclosure is "HSA-GS (PAPAP) 2 "GS-GDF15" refers to a fusion protein comprising a GDF15 polypeptide linked to an HSA polypeptide via a linker comprising the sequence of SEQ ID NO:116, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide.

[0331] In certain embodiments, two HSA-GS (PAPAP) 2 A GS-GDF15 fusion protein, comprising two HSA-GS (PAPAP) proteins linked via interchain disulfide bonds between their respective GDF15 domains. 2 Homodimers comprising GS-GDF15 fusion proteins are provided.

[0332] More particularly, in specific embodiments, the homodimer comprises (a) two HSA polypeptides (one for each monomer) comprising the sequence of SEQ ID NO: 110; (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer) each comprising the sequence: GSPAPAPAPAGS (SEQ ID NO: 116), which link the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide via a peptide bond.

[0333] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0334] As discussed above, in a specific embodiment, a homodimer is provided that comprises two fusion proteins having the sequence of SEQ ID NO:118.

[0335] II.G.5. HSA-GSAAQAAQQGS-GDF15 The name "HSA-GSAAQAAQQGS-GDF15" in this disclosure refers to a fusion protein comprising a GDF15 polypeptide linked to an HSA polypeptide via a linker comprising the sequence of SEQ ID NO:119, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide.

[0336] In certain embodiments, a homodimer is provided comprising two HSA-GSAAQAAQQGS-GDF15 fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains.

[0337] More particularly, in specific embodiments, the homodimer comprises (a) two HSA polypeptides (one for each monomer) comprising the sequence of SEQ ID NO: 110; (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer), each containing the sequence: GSAAQAAQQGS (SEQ ID NO: 119), linking the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide via a peptide bond.

[0338] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0339] As discussed above, in a specific embodiment, a homodimer is provided comprising two fusion proteins having the sequence of SEQ ID NO:121.

[0340] II.G.6. HSA-GS(AAQAAQQ) 2 GS-GDF15 The name in this disclosure is "HSA-GS (AAQAAQQ) 2 "GS-GDF15" refers to a fusion protein comprising a GDF15 polypeptide linked to an HSA polypeptide via a linker comprising the sequence of SEQ ID NO:122, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide.

[0341] In certain embodiments, two HSA-GS (AAQAAQQ) 2 A GS-GDF15 fusion protein, comprising two HSA-GS (AAQAAQQ) linked via an interchain disulfide bond between their respective GDF15 domains. 2 Homodimers comprising GS-GDF15 fusion proteins are provided.

[0342] More particularly, in specific embodiments, the homodimer comprises (a) two HSA polypeptides (one for each monomer) comprising the sequence of SEQ ID NO: 110; (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer), each containing the sequence: GSAAQAAQQAAQAAQQGS (SEQ ID NO: 122), linking the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide via a peptide bond.

[0343] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0344] As discussed above, in a specific embodiment, a homodimer is provided comprising two fusion proteins having the sequence of SEQ ID NO:124.

[0345] II.G.7. HSA-GGNAEAAAKEAAAKAAAKAGG-GDF15 The name "HSA-GGNAEAAAKEAAAKEAAAKAGG-GDF15" in this disclosure refers to a fusion protein comprising a GDF15 polypeptide linked to an HSA polypeptide via a linker comprising the sequence of SEQ ID NO: 125, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide.

[0346] In certain embodiments, a homodimer is provided comprising two HSA-GGNAEAAAKEAAAKEAAAKAGG-GDF15 fusion proteins linked via an interchain disulfide bond between their respective GDF15 domains.

[0347] More particularly, in specific embodiments, the homodimer comprises (a) two HSA polypeptides (one for each monomer) comprising the sequence of SEQ ID NO: 110; (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer), each containing the sequence: GGNAEAAAAKEAAAKEAAAKAGG (SEQ ID NO: 125), which link the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide via a peptide bond.

[0348] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0349] As discussed above, in a specific embodiment, a homodimer is provided that comprises two fusion proteins having the sequence of SEQ ID NO:127.

[0350] II.G.8. HSA-(G 4 S) 6 -GDF15 The name "HSA-(G 4 S) 6 "-GDF15" refers to a fusion protein comprising a GDF15 polypeptide linked to an HSA polypeptide via a linker comprising the sequence of SEQ ID NO:128, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide.

[0351] In certain embodiments, two HSA-(G 4 S) 6 -GDF15 fusion protein comprising two HSA-(G 4 S) 6 -GDF15 fusion protein-containing homodimers are provided.

[0352] More particularly, in specific embodiments, the homodimer comprises (a) two HSA polypeptides (one for each monomer) comprising the sequence of SEQ ID NO: 110; (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer), each comprising the sequence: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 128), linking the N-terminus of the GDF15 polypeptide to the C-terminus of the HSA polypeptide via a peptide bond.

[0353] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0354] As discussed above, in a specific embodiment, a homodimer is provided comprising two fusion proteins having the sequence of SEQ ID NO:130.

[0355] II.G.9. HSA-GS(AAQAAQQ) 2 GS-GDF15(N3D) The name in this disclosure is "HSA-GS (AAQAAQQ) 2 "GS-GDF15(N3D)" refers to a fusion protein comprising a GDF15 polypeptide (N3D) linked to an HSA polypeptide via a linker comprising the sequence of SEQ ID NO: 122, which connects the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the HSA polypeptide.

[0356] In certain embodiments, two HSA-GS (AAQAAQQ) 2 GS-GDF15(N3D) fusion protein, which is composed of two HSA-GS(AAQAAQQ) linked via an interchain disulfide bond between their respective GDF15 domains. 2 Homodimers comprising a GS-GDF15(N3D) fusion protein are provided.

[0357] More particularly, in specific embodiments, the homodimer comprises (a) two HSA polypeptides (one for each monomer) comprising the sequence of SEQ ID NO: 110; (b) two GDF15(N3D) polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:52; (c) two polypeptide linkers (one for each monomer) each comprising SEQ ID NO:122, which link the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the HSA polypeptide via a peptide bond.

[0358] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence: (SEQ ID NO:241).

[0359] As discussed above, in a specific embodiment, a homodimer is provided comprising two fusion proteins having the sequence of SEQ ID NO:242.

[0360] II.H. Constructs with Mutations Addressing Affinity for FcγR Binding Certain delHinge multimers have been found to exhibit Fcγ receptor (FcγR) binding, particularly binding to FcγRI and FcγRIII. See, e.g., Example 7. In some cases, the FcγR binding affinity is comparable to that seen with multimers containing the hinge region. This was unexpected, since Fcγ receptors interact with the hinge region, and these multimers were delHinge, lacking all or part of the hinge region, as described above. Mutational analysis of Fc residues involved in binding to FcγR suggests that the major interaction sites are located in the hinge region and CH2 domain (Tamm, A, 1997, Int. Rev. Immunol. 16:57-85). See also Radaev, S., et al., J. Biol. Chem. 276:16469-16477; Sondermann, P., et al., 2000, Nature, 406:267-273.

[0361] Antibody-dependent cellular cytotoxicity (ADCC), an immune response primarily mediated by natural killer (NK) cells in humans, depends on the interaction of FcγRs, particularly FcγRIIIA, with the Fc domain of antibodies or Fc-containing proteins in humans. In ADCC, binding of Fc to FcγRIII on the surface of NK cells activates the NK cells, which release perforin and granzymes.

[0362] Thus, constructs are provided that have additional modifications that modulate the construct's interaction with FcγR. In one series of embodiments, an asparagine to glycine mutation (N297G) is introduced into a native Fc or an Fc variant comprising the various Fc domains described above. The N297G mutation removes a conserved N-glycosylation site in the CH2 domain.

[0363] In another series of embodiments, additional N-terminal amino acid residues are deleted from the Fc domain from which all or part of the hinge region has been removed. For example, the amino acid sequence: GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 303) is provided by deletion of amino acid residues from the N-terminus through G236 of the Fc domain of wild type IgG1. In some embodiments, the C-terminal lysine (K447) may optionally be deleted from this Fc variant. The amino acid sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 304) is provided by deletion of amino acid residues from the N-terminus through G237 of the Fc domain of wild type IgG1. In some embodiments, the C-terminal lysine (K447) may optionally be deleted from this Fc variant. Amino acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 305) is provided by deletion of amino acid residues from the N-terminus through P238 of the Fc domain of wild type IgG1. In some embodiments, the C-terminal lysine (K447) may optionally be deleted from this Fc variant.

[0364] In one embodiment, a DhCpmFc(-) domain is provided in which an asparagine to glycine mutation (N297G) has been introduced (referred to herein as the "DhCpmFc(-)(N297G)" domain). In another embodiment, a DhCpmFc(+) domain is provided in which an asparagine to glycine mutation (N297G) has been introduced (referred to herein as the "DhCpmFc(+)(N297G)" domain).

[0365] In another embodiment, a DhCpmFc(-)(Y349C) domain is provided in which an asparagine to glycine mutation (N297G) has been introduced (referred to herein as the "DhCpmFc(-)(N297G)(Y349C)" domain). In another embodiment, a DhCpmFc(+)(S354C) domain is provided in which an asparagine to glycine mutation (N297G) has been introduced (referred to herein as the "DhCpmFc(+)(N297G)(S354C)" domain).

[0366] In another embodiment, a DhCpmFc(+)(Y349C) domain is provided in which an asparagine to glycine mutation (N297G) has been introduced (referred to herein as the "DhCpmFc(+)(N297G)(Y349C)" domain). In another embodiment, a DhCpmFc(-)(S354C) domain is provided in which an asparagine to glycine mutation (N297G) has been introduced (referred to herein as the "DhCpmFc(-)(N297G)(S354C)" domain).

[0367] In another embodiment, there is provided a DhCpmFc(+)(L351C) domain having an asparagine to glycine mutation (N297G) introduced therein (referred to herein as the "DhCpmFc(+)(N297G)(L351C)" domain). In another embodiment, there is provided a DhCpmFc(-)(L351C) domain having an asparagine to glycine mutation (N297G) introduced therein (referred to herein as the "DhCpmFc(-)(N297G)(L351C)" domain).

[0368] In another embodiment, a DhCpmFc(-) domain is provided having an alanine to cysteine ​​mutation (A287C) introduced therein (referred to herein as the "DhCpmFc(-)(A287C)" domain). In another embodiment, a DhCpmFc(+) domain is provided having an alanine to cysteine ​​mutation (A287C) introduced therein (referred to herein as the "DhCpmFc(+)(A287C)" domain).

[0369] In another embodiment, there is provided a DhCpmFc(-) domain having a leucine to cysteine ​​mutation (L306C) introduced therein (referred to herein as the "DhCpmFc(-)(L306C)" domain). In another embodiment, there is provided a DhCpmFc(+) domain having a leucine to cysteine ​​mutation (L306C) introduced therein (referred to herein as the "DhCpmFc(+)(L306C)" domain).

[0370] In another embodiment, there is provided a DhCpmFc(-)(A287C) domain having a tyrosine to cysteine ​​mutation (Y349C) introduced therein (referred to herein as the "DhCpmFc(-)(A287C)(Y349C)" domain). In another embodiment, there is provided a DhCpmFc(+)(A287C) domain having a tyrosine to cysteine ​​mutation (Y349C) introduced therein (referred to herein as the "DhCpmFc(+)(A287C)(Y349C)" domain).

[0371] In another embodiment, a DhCpmFc(-)(A287C) domain is provided having a serine to cysteine ​​mutation (S354C) introduced therein (referred to herein as the "DhCpmFc(-)(A287C)(S354C)" domain). In another embodiment, a DhCpmFc(+)(A287C) domain is provided having a serine to cysteine ​​mutation (S354C) introduced therein (referred to herein as the "DhCpmFc(+)(A287C)(S354C)" domain).

[0372] In another embodiment, there is provided a DhCpmFc(-)(L306C) domain having a tyrosine to cysteine ​​mutation (Y349C) introduced therein (referred to herein as the "DhCpmFc(-)(L306C)(Y349C)" domain). In another embodiment, there is provided a DhCpmFc(+)(L306C) domain having a tyrosine to cysteine ​​mutation (Y349C) introduced therein (referred to herein as the "DhCpmFc(+)(L306C)(Y349C)" domain).

[0373] In another embodiment, a DhCpmFc(-)(L306C) domain is provided having a serine to cysteine ​​mutation (S354C) introduced therein (referred to herein as the "DhCpmFc(-)(L306C)(S354C)" domain). In another embodiment, a DhCpmFc(+)(L306C) domain is provided having a serine to cysteine ​​mutation (S354C) introduced therein (referred to herein as the "DhCpmFc(+)(A287C)(L306C)" domain).

[0374] In another embodiment, a DhCpmFc(-) domain is provided in which the N-terminal seven amino acids have been removed (referred to herein as the "Dh2CpmFc(-)" domain). In another embodiment, a DhCpmFc(+) domain is provided in which the N-terminal seven amino acids have been removed (referred to herein as the "Dh2CpmFc(+)" domain).

[0375] In another embodiment, a DhCpmFc(-)(Y349C) domain is provided in which the N-terminal seven amino acids have been removed (referred to herein as the "Dh2CpmFc(-)(Y349C)" domain). In another embodiment, a DhCpmFc(+)(S354C) domain is provided in which the N-terminal seven amino acids have been removed (referred to herein as the "Dh2CpmFc(+)(S354C)" domain).

[0376] In another embodiment, a DhCpmFc(+)(Y349C) domain is provided in which the N-terminal seven amino acids have been removed (referred to herein as the "Dh2CpmFc(+)(Y349C)" domain). In another embodiment, a DhCpmFc(-)(S354C) domain is provided in which the N-terminal seven amino acids have been removed (referred to herein as the "Dh2CpmFc(-)(S354C)" domain).

[0377] In another embodiment, a CpmFc(+) domain is provided in which an asparagine to glycine mutation (N297G) has been introduced (referred to herein as the "CpmFc(+)(N297G)" domain). In another embodiment, a CpmFc(-) domain is provided in which an asparagine to glycine mutation (N297G) has been introduced (referred to herein as the "CpmFc(-)(N297G)" domain).

[0378] In another embodiment, a Dh2CpmFc(+) domain is provided having an asparagine to glycine mutation (N297G) introduced therein (referred to herein as the "Dh2CpmFc(+)(N297G)" domain). In another embodiment, a Dh2CpmFc(-) domain is provided having an asparagine to glycine mutation (N297G) introduced therein (referred to herein as the "Dh2CpmFc(-)(N297G)" domain).

[0379] In another embodiment, a Dh2CpmFc(-)(N297G) domain is provided in which an alanine to cysteine ​​mutation (A287C) has been introduced (referred to herein as the "Dh2CpmFc(-)(N297G)(A287C)" domain). In another embodiment, a Dh2CpmFc(+)(N297G) domain is provided in which a leucine to cysteine ​​mutation (L306C) has been introduced (referred to herein as the "Dh2CpmFc(+)(N297G)(L306C)" domain).

[0380] In another embodiment, there is provided a Dh2CpmFc(-)(N297G)(A287C) domain having a tyrosine to cysteine ​​mutation (Y349C) introduced therein (referred to herein as the "Dh2CpmFc(-)(N297G)(A287C)(Y349C)" domain). In another embodiment, there is provided a Dh2CpmFc(+)(N297G)(L306C) domain having a serine to cysteine ​​mutation (S354C) introduced therein (referred to herein as the "Dh2CpmFc(+)(N297G)(L306C)(S354C)" domain).

[0381] In another embodiment, a Dh2CpmFc(-) domain is provided with two glycines added to the N-terminus (referred to herein as the "GG-Dh2CpmFc(-)(N297G)" domain). In another embodiment, a Dh2CpmFc(+) domain is provided with two glycines added to the N-terminus (referred to herein as the "GG-Dh2CpmFc(+)(N297G)" domain).

[0382] In another embodiment, a GG-Dh2CpmFc(-) domain is provided in which a tyrosine to cysteine ​​mutation (Y349C) has been introduced (referred to herein as the "GG-Dh2CpmFc(-)(Y349C)" domain). In another embodiment, a GG-Dh2CpmFc(+) domain is provided in which a serine to cysteine ​​mutation (S354C) has been introduced (referred to herein as the "GG-Dh2CpmFc(+)(S354C)" domain).

[0383] In another embodiment, a GG-Dh2CpmFc(+) domain is provided in which a tyrosine to cysteine ​​mutation (Y349C) has been introduced (referred to herein as the "GG-Dh2CpmFc(+)(Y349C)" domain). In another embodiment, a GG-Dh2CpmFc(-) domain is provided in which a serine to cysteine ​​mutation (S354C) has been introduced (referred to herein as the "GG-Dh2CpmFc(-)(S354C)" domain).

[0384] In another embodiment, a Dh2CpmFc(-) domain is provided with a glycine added to the N-terminus (referred to herein as the "Dh3CpmFc(-)(N297G)" domain). In another embodiment, a Dh2CpmFc(+) domain is provided with a glycine added to the N-terminus (referred to herein as the "Dh3CpmFc(+)" domain).

[0385] In another embodiment, a Dh3CpmFc(-) domain is provided having a tyrosine to cysteine ​​mutation (Y349C) introduced therein (referred to herein as the "Dh3CpmFc(-)(Y349C) domain"). In another embodiment, a Dh3CpmFc(+) domain is provided having a serine to cysteine ​​mutation (S354C) introduced therein (referred to herein as the "Dh3CpmFc(+)(S354C) domain).

[0386] In another embodiment, an asparagine to glycine mutation (N297G) is introduced into the DhMonoFc domain (referred to herein as "DhMonoFc(N297G)").

[0387] II.H.1. DhCpmFc(-)(N297G)-GDF15(Ndel3):DhCpmFc(+)(N297G) In this disclosure, the name "DhCpmFc(-)(N297G)-GDF15(Ndel3):DhCpmFc(+)(N297G)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a DhCpmFc(-)(N297G) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G) domain.

[0388] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)-GDF15(Ndel3):DhCpmFc(+)(N297G) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0389] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two DhCpmFc(+)(N297G) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 287); (b) Sequence: Two DhCpmFc(-)(N297G) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 132); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0390] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 134, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 134). It is encoded by [[SEQ ID NO: 133]]: gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0391] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 131), which comprises the nucleic acid sequence: (SEQ ID NO: 135).

[0392] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:134 and two polypeptide chains comprising the sequence of SEQ ID NO:131.

[0393] II.H.2. DhCpmFc(-)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G) In this disclosure, the name "DhCpmFc(-)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G)" refers to a heterodimer comprising (i) a first polypeptide chain comprising the GDF15(N3D) sequence whose N-terminus is directly linked to the C-terminus of the DhCpmFc(-)(N297G) domain, and (ii) a second polypeptide chain comprising the DhCpmFc(+)(N297G) domain.

[0394] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0395] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(N297G) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 287; (b) two DhCpmFc(-)(N297G) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 132; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0396] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 137, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.

[0397] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:131, encoded by the nucleic acid sequence of SEQ ID NO:135.

[0398] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:137 and two polypeptide chains comprising the sequence of SEQ ID NO:131.

[0399] II.H.3. DhCpmFc(-)(N297G)-G 4 -GDF15(N3D):DhCpmFc(+)(N297G) In this disclosure, the name "DhCpmFc(-)(N297G)-G 4 "-GDF15(N3D):DhCpmFc(+)(N297G)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(-)(N297G) domain via a linker comprising the sequence of SEQ ID NO:58 connecting the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-)(N297G) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G) domain.

[0400] In certain embodiments, the two first polypeptide chains of each heterodimer are two DhCpmFc(-)(N297G)-G linked via an interchain disulfide bond between their respective GDF15 domains. 4 - A tetramer comprising a dimer of the GDF15(N3D):DhCpmFc(+)(N297G) heterodimer is provided.

[0401] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(N297G) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 287; (b) two DhCpmFc(-)(N297G) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 132; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52; (d) two polypeptide linkers (one for each heterodimer) each comprising the sequence of SEQ ID NO:58, which link the N-terminus of the GDF15(N3D) polypeptide to the C-terminus of the DhCpmFc(-)(N297G) domain via a peptide bond.

[0402] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: [ka] which comprises the nucleic acid sequence:

[0403] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:131, encoded by the nucleic acid sequence of SEQ ID NO:135.

[0404] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:139 and two polypeptide chains comprising the sequence of SEQ ID NO:131.

[0405] II.H.4. DhCpmFc(-)(N297G)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(S354C) In this disclosure, the designation "DhCpmFc(-)(N297G)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide linked at its N-terminus directly to the C-terminus of a DhCpmFc(-)(N297G)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0406] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0407] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two DhCpmFc(+)(N297G)(S354C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 288); (b) Sequence: Two DhCpmFc(-)(N297G)(S349C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 141); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0408] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 143, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 143), It is encoded by [[SEQ ID NO: 142]]: gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0409] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: 140, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 140). (SEQ ID NO: 144).

[0410] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:143 and two polypeptide chains comprising the sequence of SEQ ID NO:140.

[0411] II.H.5. DhCpmFc(-)(N297G)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(S354C) In this disclosure, the designation "DhCpmFc(-)(N297G)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide linked at its N-terminus directly to the C-terminus of a DhCpmFc(-)(N297G)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G)(S345C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0412] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0413] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(N297G)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 288; (b) two DhCpmFc(-)(N297G)(S349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 141; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0414] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 146, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.

[0415] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:140, which is encoded by the nucleic acid sequence of SEQ ID NO:144.

[0416] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:146 and two polypeptide chains comprising the sequence of SEQ ID NO:140.

[0417] II.H.6. DhCpmFc(-)(N297G)(L351C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L351C) In this disclosure, the designation "DhCpmFc(-)(N297G)(L351C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L351C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide linked at its N-terminus directly to the C-terminus of a DhCpmFc(-)(N297G)(L351C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G)(L351C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C351 of the first polypeptide chain and C351 of the second polypeptide chain.

[0418] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)(L351C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L351C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0419] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two DhCpmFc(+)(N297G)(L351C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 289); (b) Sequence: Two DhCpmFc(-)(N297G)(L351C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 148); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0420] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 150, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 150),

[0421] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHELHNHYTQKSLSLSPGK (SEQ ID NO: 147), which comprises the nucleic acid sequence: (SEQ ID NO: 151).

[0422] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:150 and two polypeptide chains comprising the sequence of SEQ ID NO:147.

[0423] II.H.7. DhCpmFc(-)(N297G)(L351C)-GDF15(N3D):DhCpmFc(+)(N297G)(L351C) In this disclosure, the designation "DhCpmFc(-)(N297G)(L351C)-GDF15(N3D):DhCpmFc(+)(N297G)(L351C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide linked at its N-terminus directly to the C-terminus of a DhCpmFc(-)(N297G)(L351C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G)(L351C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C351 of the first polypeptide chain and C351 of the second polypeptide chain.

[0424] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)(L351C)-GDF15(N3D):DhCpmFc(+)(N297G)(L351C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0425] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(N297G)(L351C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 289; (b) two DhCpmFc(-)(N297G)(L351C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 148; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0426] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 153, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 153),

[0427] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:147, encoded by the nucleic acid sequence of SEQ ID NO:151.

[0428] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:153 and two polypeptide chains comprising the sequence of SEQ ID NO:147.

[0429] II.H.8. DhCpmFc(-)(N297G)(A287C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C) In this disclosure, the name "DhCpmFc(-)(N297G)(A287C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a DhCpmFc(-)(N297G)(A287C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G)(L306C) domain.

[0430] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)(A287C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0431] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence Two DhCpmFc(+)(N297G)(L306C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 290); (b) Sequence: Two DhCpmFc(-)(N297G)(A287C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 268); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0432] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 269, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 269), It is encoded by [[SEQ.ID.NO:270]]: gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0433] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: 267, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 267). (SEQ ID NO:271).

[0434] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:269 and two polypeptide chains comprising the sequence of SEQ ID NO:267.

[0435] II.H.9. DhCpmFc(-)(N297G)(A287C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C) In this disclosure, the name "DhCpmFc(-)(N297G)(A287C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a DhCpmFc(-)(N297G)(A287C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G)(L306C) domain.

[0436] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)(A287C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0437] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(N297G)(L306C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 290; (b) two DhCpmFc(-)(N297G)(A287C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 268; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0438] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 272, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 272),

[0439] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:267, encoded by the nucleic acid sequence of SEQ ID NO:271.

[0440] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:272 and two polypeptide chains comprising the sequence of SEQ ID NO:267.

[0441] II.H.10. DhCpmFc(-)(N297G)(A287C)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)(S354C) In this disclosure, the name "DhCpmFc(-)(N297G)(A287C)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide directly linked at its N-terminus to the C-terminus of a DhCpmFc(-)(N297G)(A287C)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G)(L306C)(S354C) domain.

[0442] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)(A287C)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0443] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence and two DhCpmFc(+)(N297G)(L306C)(Y349C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 291); (b) Sequence: Two DhCpmFc(-)(N297G)(A287C)(S354C) domains (one in each heterodimer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 275); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0444] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 276, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 276), It is encoded by [[SEQ ID NO: 277]] gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0445] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: 274, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 274). (SEQ ID NO:278).

[0446] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:276 and two polypeptide chains comprising the sequence of SEQ ID NO:274.

[0447] II.H.11. DhCpmFc(-)(N297G)(A287C)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)(S354C) In this disclosure, the designation "DhCpmFc(-)(N297G)(A287C)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a DhCpmFc(-)(N297G)(A287C)(Y349C) domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)(N297G)(L306C)(S354C) domain.

[0448] In certain embodiments, a tetramer is provided that comprises a dimer of two DhCpmFc(-)(N297G)(A287C)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0449] More particularly, in specific embodiments, the tetramer comprises: (a) two DhCpmFc(+)(N297G)(L306C)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 291; (b) two DhCpmFc(-)(N297G)(A287C)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 275; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0450] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 279, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 279),

[0451] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:274, encoded by the nucleic acid sequence of SEQ ID NO:278.

[0452] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:279 and two polypeptide chains comprising the sequence of SEQ ID NO:274.

[0453] II.H.12. Dh2CpmFc(-)-GDF15(Ndel3):Dh2CpmFc(+) In this disclosure, the name "Dh2CpmFc(-)-GDF15(Ndel3):Dh2CpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+) domain.

[0454] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)-GDF15(Ndel3):Dh2CpmFc(+) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0455] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two Dh2CpmFc(+) domains (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 292); (b) Sequence: Two Dh2CpmFc(-) domains (one in each heterodimer) containing PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 155); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0456] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 157, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 157), It is encoded by ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata

[0457] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: 154, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 154). (SEQ ID NO: 158).

[0458] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:157 and two polypeptide chains comprising the sequence of SEQ ID NO:154.

[0459] II.H.13. Dh2CpmFc(-)-GDF15(N3D):Dh2CpmFc(+) In this disclosure, the name "Dh2CpmFc(-)-GDF15(N3D):Dh2CpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked by a peptide bond to the C-terminus of the Dh2CpmFc(-) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+) domain.

[0460] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)-GDF15(N3D):Dh2CpmFc(+) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0461] More particularly, in specific embodiments, the tetramer comprises: (a) two Dh2CpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 292; (b) two Dh2CpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 155; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0462] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 160, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI. It is encoded by [[SEQ.ID.NO:159]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0463] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:154, encoded by the nucleic acid sequence of SEQ ID NO:158.

[0464] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:160 and two polypeptide chains comprising the sequence of SEQ ID NO:154.

[0465] II.H.14. Dh2CpmFc(-)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(S354C) In this disclosure, the designation "Dh2CpmFc(-)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0466] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0467] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two Dh2CpmFc(+)(S354C) domains (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 293); (b) Sequence: Two Dh2CpmFc(-)(Y349C) domains (one in each heterodimer) containing PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 162); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0468] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 164, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 164). It is encoded by ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata

[0469] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: 161, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 165).

[0470] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:164 and two polypeptide chains comprising the sequence of SEQ ID NO:161.

[0471] II.H.15. Dh2CpmFc(-)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(S354C) In this disclosure, the designation "Dh2CpmFc(-)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0472] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0473] More particularly, in specific embodiments, the tetramer comprises: (a) two Dh2CpmFc(+)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 293; (b) two Dh2CpmFc(-)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 162; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0474] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 167, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 167), It is encoded by [[SEQ.ID.NO:166]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0475] In a preferred embodiment, the second monomer comprises the amino acid sequence of SEQ ID NO:161, encoded by the nucleic acid sequence of SEQ ID NO:165.

[0476] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:167 and two polypeptide chains comprising the sequence of SEQ ID NO:161.

[0477] II.H.16. CpmFc(-)(N297G)-GDF15(Ndel3):CpmFc(+)(N297G) In this disclosure, the name "CpmFc(-)(N297G)-GDF15(Ndel3):CpmFc(+)(N297G)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide directly linked at its N-terminus to the C-terminus of a CpmFc(-)(N297G) domain, and (ii) a second polypeptide chain comprising a CpmFc(+)(N297G) domain.

[0478] In certain embodiments, a tetramer is provided that comprises a dimer of two CpmFc(-)(N297G)-GDF15(Ndel3):CpmFc(+)(N297G) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0479] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence (part of the hinge region is in parentheses): (DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 294), (b) Sequence (part of the hinge region is in parentheses): (DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 169), and (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0480] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 171, which comprises the nucleic acid sequence:

[0481] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: (DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 168), which comprises the nucleic acid sequence: (SEQ ID NO: 172).

[0482] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:171 and two polypeptide chains comprising the sequence of SEQ ID NO:168.

[0483] II.H.17. CpmFc(-)(N297G)-GDF15(N3D):CpmFc(+)(N297G) In this disclosure, the designation "CpmFc(-)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G) refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide directly linked at its N-terminus to the C-terminus of a CpmFc(-)(N297G) domain, and (ii) a second polypeptide chain comprising a CpmFc(+)(N297G) domain.

[0484] In certain embodiments, a tetramer is provided that comprises a dimer of two CpmFc(-)(N297G)-GDF15(N3D):CpmFc(+)(N297G) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0485] More particularly, in specific embodiments, the tetramer comprises: (a) two CpmFc(+)(N297G) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 294; (b) two CpmFc(-)(N297G) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 169; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0486] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 174, which comprises the nucleic acid sequence:

[0487] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:168, which is encoded by the nucleic acid sequence of SEQ ID NO:172.

[0488] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:174 and two polypeptide chains comprising the sequence of SEQ ID NO:168.

[0489] II.H.18. Dh2CpmFc(-)(N297G)-GDF15(Ndel3):Dh2CpmFc(+)(N297G) In this disclosure, the name "Dh2CpmFc(-)(N297G)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-)(N297G) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(N297G) domain.

[0490] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(N297G)-GDF15(Ndel3):Dh2CpmFc(+)(N297G) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0491] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two Dh2CpmFc(+)(N297G) domains (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 295); (b) Sequence: Two Dh2CpmFc(-)(N297G) domains (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 176); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0492] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 178, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 178), It is encoded by [[SEQ ID NO:177]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0493] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: 175, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 175). (SEQ ID NO: 179).

[0494] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:178 and two polypeptide chains comprising the sequence of SEQ ID NO:175.

[0495] II.H.19. Dh2CpmFc(-)(N297G)-GDF15(N3D):Dh2CpmFc(+)(N297G) In this disclosure, the name "Dh2CpmFc(-)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-)(N297G) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(N297G) domain.

[0496] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(N297G)-GDF15(N3D):Dh2CpmFc(+)(N297G) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0497] More particularly, in specific embodiments, the tetramer comprises: (a) two Dh2CpmFc(+)(N297G) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 295; (b) two Dh2CpmFc(-)(N297G) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 176; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0498] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 181, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 181), It is encoded by [[SEQ ID NO: 180]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0499] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:175, encoded by the nucleic acid sequence of SEQ ID NO:179.

[0500] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:181 and two polypeptide chains comprising the sequence of SEQ ID NO:175.

[0501] II.H.20. Dh2CpmFc(-)(N297G)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(S354C) The designation "Dh2CpmFc(-)(N297G)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(S354C)" in this disclosure refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide linked at its N-terminus directly to the C-terminus of a Dh2CpmFc(-)(N297G)(Y349C) polypeptide, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(N297G)(S345C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0502] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(N297G)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0503] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two Dh2CpmFc(+)(N297G)(S354C) domains (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 296); (b) Sequence: Two Dh2CpmFc(-)(N297G)(Y349C) domains (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 183); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0504] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 185, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 185), It is encoded by [[SEQ.ID.NO:184]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0505] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: 182, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 186).

[0506] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:185 and two polypeptide chains comprising the sequence of SEQ ID NO:182.

[0507] II.H.21. Dh2CpmFc(-)(N297G)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(S354C) The designation "Dh2CpmFc(-)(N297G)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(S354C)" in this disclosure refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-)(N297G)(Y349C) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(N297G)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide and C354 of the second polypeptide.

[0508] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(N297G)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0509] More particularly, in specific embodiments, the tetramer comprises: (a) two Dh2CpmFc(+)(N297G)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 296; (b) two Dh2CpmFc(-)(N297G)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 183; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0510] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 188, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 188), It is encoded by [[SEQ ID NO:187]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0511] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:182, encoded by the nucleic acid sequence of SEQ ID NO:186.

[0512] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:188 and two polypeptide chains comprising the sequence of SEQ ID NO:182.

[0513] II.H.22. Dh2CpmFc(-)(N297G)(A287C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C) The designation "Dh2CpmFc(-)(N297G)(A287C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)" in this disclosure refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-)(N297G)(A287C) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(N297G)(L306C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C287 of the first monomer and C306 of the second monomer.

[0514] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(N297G)(A287C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0515] More specifically, in a specific embodiment, (a) Sequence: Two Dh2CpmFc(+)(N297G)(L306C) domains (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 297); (b) Sequence: Two Dh2CpmFc(-)(N297G)(A287C) (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 192); (c) a tetramer comprising two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0516] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 194, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 194). It is encoded by [[SEQ ID NO:193]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0517] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: 191, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 195).

[0518] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:194 and two polypeptide chains comprising the sequence of SEQ ID NO:191.

[0519] II.H.23. Dh2CpmFc(-)(N297G)(A287C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C) The designation "Dh2CpmFc(-)(N297G)(A287C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)" in this disclosure refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-)(N297G)(A287C) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(N297G)(L306C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C287 of the first polypeptide chain and C306 of the second polypeptide chain.

[0520] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(N297G)(A287C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0521] More specifically, in a specific embodiment, (a) two Dh2CpmFc(+)(N297G)(L306C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 297; (b) two Dh2CpmFc(-)(N297G)(A287C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 192; (c) a tetramer comprising two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0522] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 197, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI. It is encoded by [[SEQ ID NO:196]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0523] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:191, encoded by the nucleic acid sequence of SEQ ID NO:195.

[0524] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:197 and two polypeptide chains comprising the sequence of SEQ ID NO:191.

[0525] II.H.24. Dh2CpmFc(-)(N297G)(A287C)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)(S354C) In this disclosure, the name "Dh2CpmFc(-)(N297G)(A287C)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-)(N297G)(A287C)(Y349C) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(N297G)(L306C)(S354C) domain. The cysteine ​​clamp mutations allow the first and second polypeptide chains to be linked via interchain disulfide bonds between C287 of the first polypeptide chain and C306 of the second polypeptide chain, and between C349 of the first polypeptide chain and C349 of the second polypeptide chain.

[0526] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(N297G)(A287C)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0527] More specifically, in a specific embodiment, (a) Sequence: and two Dh2CpmFc(+)(N297G)(L306C)(S354C) domains (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 298); (b) Sequence: Two Dh2CpmFc(-)(N297G)(A287C)(Y349C) domains (one in each heterodimer) comprising PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 199); (c) a tetramer comprising two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0528] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 201, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 201), It is encoded by [[SEQ.ID.NO:200]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0529] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: 198, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 202).

[0530] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:201 and two polypeptide chains comprising the sequence of SEQ ID NO:198.

[0531] II.H.25. Dh2CpmFc(-)(N297G)(A287C)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)(S354C) In this disclosure, the name "Dh2CpmFc(-)(N297G)(A287C)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)(S354C)" refers to a heterodimer comprising (i) a first monomer comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh2CpmFc(-)(N297G)(A287C)(Y349C) domain, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)(N297G)(L306C)(S354C) domain. The cysteine ​​clamp mutations allow the first and second polypeptide chains to be linked via interchain disulfide bonds between C287 of the first polypeptide chain and C306 of the second polypeptide chain, and between C349 of the first polypeptide chain and C349 of the second polypeptide chain.

[0532] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh2CpmFc(-)(N297G)(A287C)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0533] More specifically, in a specific embodiment, (a) two Dh2CpmFc(+)(N297G)(L306C)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 298; (b) two Dh2CpmFc(-)(N297G)(A287C)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 199; (c) a tetramer comprising two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0534] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 204, which comprises the nucleic acid sequence: PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 204). It is encoded by [[SEQ.ID.NO:203]]: ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0535] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:198, encoded by the nucleic acid sequence of SEQ ID NO:202.

[0536] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:204 and two polypeptide chains comprising the sequence of SEQ ID NO:198.

[0537] II.H.26. GG-Dh2CpmFc(-)-GDF15(Ndel3):GG-Dh2CpmFc(+) In this disclosure, the name "GG-Dh2CpmFc(-)-GDF15(Ndel3):GG-Dh2CpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a GG-Dh2CpmFc(-) domain, and (ii) a second polypeptide chain comprising a GG-Dh2CpmFc(+) domain.

[0538] In certain embodiments, a tetramer is provided that comprises a dimer of two GG-Dh2CpmFc(-)-GDF15(Ndel3):GG-Dh2CpmFc(+) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0539] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: Two GG-Dh2CpmFc(+) domains (one for each heterodimer) comprising GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 299); (b) Sequence: GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 206), and (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0540] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 208, which comprises the nucleic acid sequence: It is encoded by [[SEQ ID NO:207]]: ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0541] In embodiments employing a VH21 signal sequence, in a preferred embodiment, the first polypeptide chain has the amino acid sequence (single underline is the signal sequence): MEWSWVFLFFLSVTTGVHS GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 243), which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0542] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 205), which comprises the nucleic acid sequence: (SEQ ID NO: 209).

[0543] In embodiments employing a VH21 signal sequence, in a preferred embodiment, the second polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0544] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:208 and two polypeptide chains comprising the sequence of SEQ ID NO:205.

[0545] II.H.27. GG-Dh2CpmFc(-)-GDF15(N3D):GG-Dh2CpmFc(+) In this disclosure, the name "GG-Dh2CpmFc(-)-GDF15(N3D):GG-DhCpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a GG-Dh2CpmFc(-) domain, and (ii) a second polypeptide chain comprising a GG-DhCpmFc(+) domain.

[0546] In certain embodiments, a tetramer is provided that comprises a dimer of two GG-Dh2CpmFc(-)-GDF15(N3D):GG-Dh2CpmFc(+) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0547] More particularly, in specific embodiments, the tetramer comprises: (a) two GG-Dh2CpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 299; (b) two GG-Dh2CpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 206; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0548] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 211, which comprises the nucleic acid sequence: GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 211), It is encoded by [[SEQ.ID.NO:210]]: ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0549] In embodiments employing a VH21 signal sequence, in a preferred embodiment, the first polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0550] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:205, encoded by the nucleic acid sequence of SEQ ID NO:209.

[0551] In embodiments employing a VH21 signal sequence, in a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:245, which is encoded by the nucleic acid sequence of SEQ ID NO:246.

[0552] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:211 and two polypeptide chains comprising the sequence of SEQ ID NO:205.

[0553] II.H.28. GG-Dh2CpmFc(-)(Y349C)-GDF15(Ndel3):GG-Dh2CpmFc(+)(S354C) In this disclosure, the designation "GG-Dh2CpmFc(-)(Y349C)-GDF15(Ndel3):GG-Dh2CpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a GG-Dh2CpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a GG-Dh2CpmFc(+)(S345C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0554] In certain embodiments, a tetramer is provided that comprises a dimer of two GG-Dh2CpmFc(-)(Y349C)-GDF15(Ndel3):GG-Dh2CpmFc(+)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0555] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 300), and (b) Sequence: and two GG-Dh2CpmFc(-)(Y349C) chains (one for each heterodimer) comprising GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 213); (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0556] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 215, which comprises the nucleic acid sequence: It is encoded by [[SEQ.ID.NO:214]]: ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0557] In embodiments employing a VH21 signal sequence, in a preferred embodiment, the first polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0558] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 212), which comprises the nucleic acid sequence: (SEQ ID NO:216).

[0559] In embodiments employing a VH21 signal sequence, in a preferred embodiment, the second polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0560] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:215 and two polypeptide chains comprising the sequence of SEQ ID NO:212.

[0561] II.H.29. GG-Dh2CpmFc(-)(Y349C)-GDF15(N3D):GG-Dh2CpmFc(+)(S354C) The designation "GG-Dh2CpmFc(-)(Y349C)-GDF15(N3D):GG-Dh2CpmFc(+)(S354C)" in this disclosure refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a GG-Dh2CpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a GG-Dh2CpmFc(+)(S345C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0562] In certain embodiments, a tetramer is provided that comprises a dimer of two GG-Dh2CpmFc(-)(Y349C)-GDF15(N3D):GG-Dh2CpmFc(+)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via an interchain disulfide bond between their respective GDF15 domains.

[0563] More particularly, in specific embodiments, the tetramer comprises: (a) two GG-Dh2CpmFc(+)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 300; (b) two GG-Dh2CpmFc(-)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 213; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0564] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: 218, which comprises the nucleic acid sequence: It is encoded by ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata (SEQ ID NO: 217).

[0565] In embodiments employing a VH21 signal sequence, in a preferred embodiment, the first polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0566] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:212, encoded by the nucleic acid sequence of SEQ ID NO:218.

[0567] In embodiments employing a VH21 signal sequence, in a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:251, which is encoded by the nucleic acid sequence of SEQ ID NO:252.

[0568] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:218 and two polypeptide chains comprising the sequence of SEQ ID NO:212.

[0569] II.H.30. Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(+) In this disclosure, the name "Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh3CpmFc(-) domain, and (ii) a second polypeptide chain comprising a Dh3CpmFc(+) domain.

[0570] In certain embodiments, a tetramer is provided comprising a dimer of two Dh3CpmFc(-)-GDF15(Ndel3):Dh3CpmFc(+) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0571] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 301), and (b) Sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 220), and (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0572] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 222), which comprises the nucleic acid sequence: It is encoded by ggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata (SEQ ID NO: 221).

[0573] In embodiments employing the VK1 signal sequence, in preferred embodiments, the first polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0574] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 219), which comprises the nucleic acid sequence: ggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctg aatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgta caccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaa tgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa (SEQ ID NO: 223).

[0575] In embodiments employing a VK1 signal sequence, in a preferred embodiment, the second polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0576] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:222 and two polypeptide chains comprising the sequence of SEQ ID NO:219.

[0577] II.H.31. Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) In this disclosure, the name "Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh3CpmFc(-) domain, and (ii) a second polypeptide chain comprising a Dh3CpmFc(+) domain.

[0578] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh3CpmFc(-)-GDF15(N3D):Dh3CpmFc(+) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0579] More particularly, in specific embodiments, the tetramer comprises: (a) two Dh3CpmFc(+) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 301; (b) two Dh3CpmFc(-) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 220; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0580] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 225), which comprises the nucleic acid sequence:

[0581] In embodiments employing the VK1 signal sequence, in preferred embodiments, the first polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0582] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:219, encoded by the nucleic acid sequence of SEQ ID NO:223.

[0583] In embodiments employing a VK1 signal sequence, in a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:257, which is encoded by the nucleic acid sequence of SEQ ID NO:258.

[0584] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:225 and two polypeptide chains comprising the sequence of SEQ ID NO:219.

[0585] II.H.32. Dh3CpmFc(-)(Y349C)-GDF15(Ndel3):Dh3CpmFc(+)(S354C) In this disclosure, the designation "Dh3CpmFc(-)(Y349C)-GDF15(Ndel3):Dh3CpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(Ndel3) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh3CpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a Dh3CpmFc(+)(S345C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide and C354 of the second polypeptide.

[0586] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh3CpmFc(-)(Y349C)-GDF15(Ndel3):Dh3CpmFc(+)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0587] More particularly, in specific embodiments, the tetramer comprises: (a) Sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 302), and (b) Sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 227), and (c) two GDF15(Ndel3) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:55.

[0588] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 229), which comprises the nucleic acid sequence:

[0589] In embodiments employing the VK1 signal sequence, in preferred embodiments, the first polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0590] In a preferred embodiment, the second polypeptide chain has the amino acid sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 226), which comprises the nucleic acid sequence: (SEQ ID NO: 230).

[0591] In embodiments employing a VK1 signal sequence, in a preferred embodiment, the second polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0592] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:229 and two polypeptide chains comprising the sequence of SEQ ID NO:226.

[0593] II.H.33. Dh3CpmFc(-)(Y349C)-GDF15(N3D):Dh3CpmFc(+)(S354C) In this disclosure, the designation "Dh3CpmFc(-)(Y349C)-GDF15(N3D):Dh3CpmFc(+)(S354C)" refers to a heterodimer comprising (i) a first polypeptide chain comprising a GDF15(N3D) polypeptide whose N-terminus is directly linked to the C-terminus of a Dh3CpmFc(-)(Y349C) domain, and (ii) a second polypeptide chain comprising a Dh3CpmFc(+)(S354C) domain. The cysteine ​​clamp mutation allows the first and second polypeptide chains to be linked via an interchain disulfide bond between C349 of the first polypeptide chain and C354 of the second polypeptide chain.

[0594] In certain embodiments, a tetramer is provided that comprises a dimer of two Dh3CpmFc(-)(Y349C)-GDF15(N3D):Dh3CpmFc(+)(S354C) heterodimers, in which the two first polypeptide chains of each heterodimer are linked via interchain disulfide bonds between their respective GDF15 domains.

[0595] More particularly, in specific embodiments, the tetramer comprises: (a) two Dh3CpmFc(+)(S354C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 302; (b) two Dh3CpmFc(-)(Y349C) domains (one for each heterodimer) comprising the sequence of SEQ ID NO: 227; (c) two GDF15(N3D) polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:52.

[0596] In a preferred embodiment, the first polypeptide chain has the amino acid sequence: GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 232), which comprises the nucleic acid sequence: It is encoded by [[SEQ.ID.NO:231]]: ggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

[0597] In embodiments employing a VK1 signal sequence, in preferred embodiments, the first polypeptide chain has the amino acid sequence (single underline is the signal sequence): [ka] which comprises the nucleic acid sequence (underlined is the signal sequence): [ka] is coded by

[0598] In a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:226, encoded by the nucleic acid sequence of SEQ ID NO:230.

[0599] In embodiments employing a VK1 signal sequence, in a preferred embodiment, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:263, which is encoded by the nucleic acid sequence of SEQ ID NO:264.

[0600] As discussed above, a tetramer is provided that comprises two polypeptide chains comprising the sequence of SEQ ID NO:232 and two polypeptide chains comprising the sequence of SEQ ID NO:226.

[0601] II.H.34. DhMonoFc(N297G)-GDF15 The designation "DhMonoFc(N297G)-GDF15" in this disclosure refers to a fusion protein comprising a GDF15 polypeptide linked at its N-terminus directly to the C-terminus of DhMonoFc(N297G) by a peptide bond.

[0602] In certain embodiments, homodimers are provided that comprise two such fusion proteins, linked via an interchain disulfide between their respective GDF15 polypeptides.

[0603] More particularly, in specific embodiments, the homodimer comprises (a) Sequence: Two DhMonoFc (N297G) domains (one in each monomer) containing APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 233); (b) two GDF15 polypeptides (one for each heterodimer) comprising the sequence of SEQ ID NO:12.

[0604] In a preferred embodiment, the fusion protein has the amino acid sequence: 235, which comprises the nucleic acid sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 235),

[0605] As discussed above, in a specific embodiment, a homodimer is provided that comprises two monomers comprising the sequence of SEQ ID NO:235.

[0606] II.H.35. DhMonoFc(N297G)-(G 4 S) 4 -GDF15 In this disclosure, the name “DhMonoFc(N297G)-(G 4 S) 4 "-GDF15" refers to a fusion protein comprising a GDF15 polypeptide linked to a DhMonoFc(N297G) domain via a linker comprising the sequence of SEQ ID NO: 18, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the DhMonoFc(N297G) domain by a peptide bond.

[0607] In certain embodiments, homodimers are provided that comprise two such fusion proteins, linked via an interchain disulfide bond between their respective GDF15 polypeptides.

[0608] More particularly, in specific embodiments, the homodimer comprises (a) Sequence: Two DhMonoFc (N297G) domains (one in each monomer) comprising APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 236); (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer) each comprising the sequence of SEQ ID NO:18, which link the N-terminus of the GDF15 polypeptide to the C-terminus of the DhMonoFc(N297G) domain via a peptide bond.

[0609] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0610] As discussed above, in a specific embodiment, a homodimer is provided that comprises two monomers having the sequence of SEQ ID NO:238.

[0611] II.H.36. DhMonoFc(N297G)-G 4 -GDF15 The name in this disclosure is “DhMonoFc(N297G)-G 4 "-GDF15" refers to a fusion protein comprising a GDF15 polypeptide linked to a DhMonoFc(N297G) domain via a linker comprising the sequence of SEQ ID NO:58, which connects the N-terminus of the GDF15 polypeptide to the C-terminus of the DhMonoFc(N297G) domain by a peptide bond.

[0612] In certain embodiments, homodimers are provided that comprise two such fusion proteins, linked via an interchain disulfide bond between their respective GDF15 polypeptides.

[0613] More particularly, in specific embodiments, the homodimer comprises (a) two DhMonoFc(N297G) domains (one for each monomer) comprising the sequence of SEQ ID NO: 236; (b) two GDF15 polypeptides (one for each monomer) comprising the sequence of SEQ ID NO:12; (c) two polypeptide linkers (one for each monomer) each comprising the sequence of SEQ ID NO:58, which link the N-terminus of the GDF15 polypeptide to the C-terminus of the DhMonoFc(N297G) domain via a peptide bond.

[0614] In a preferred embodiment, the fusion protein has the amino acid sequence (double underlined is the linker): [ka] which comprises the nucleic acid sequence:

[0615] As discussed above, in a specific embodiment, a homodimer is provided that comprises two monomers having the sequence of SEQ ID NO:240.

[0616] III. GDF15-Containing Constructs, Including GDF15 Polypeptides and Mutant Forms Thereof As disclosed herein, the GDF15 polypeptides described in this disclosure (including the full-length and mature forms of human GDF15) and constructs comprising GDF15 can be manipulated and / or engineered using standard molecular biology methods to form mutant forms and constructs of the GDF15 polypeptides provided herein. In various examples, nucleic acid sequences encoding mutant forms and constructs of the GDF15 polypeptides provided herein, which may include all or part of SEQ ID NO: 4, 8 or 12, can be isolated and / or amplified from genomic DNA or cDNA using appropriate oligonucleotide primers. Primers can be designed based on the nucleic acid and amino acid sequences provided herein according to standard (RT)-PCR amplification methods. The amplified GDF15 mutant polypeptide nucleic acid can then be cloned into a suitable vector and characterized by DNA sequence analysis.

[0617] Oligonucleotides for use as probes to isolate or amplify all or part of the mutant forms and constructs of GDF15 polypeptides provided herein can be designed and produced using standard synthetic techniques, e.g., an automated DNA synthesizer, or can be isolated from longer sequences of DNA.

[0618] III.A. GDF15 Polypeptide and Polynucleotide Sequences In vivo, GDF15 is expressed as a contiguous amino acid sequence that includes a signal sequence, a prodomain and an activation domain.

[0619] The sequence of the 308 amino acid sequence of the full-length human GDF15 is: The DNA sequence: MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 4). It is encoded by atgcccgggcaagaactcaggacggtgaatggctctcagatgctcctggtgttgctggtgctctcgtggctgccgcatgggggcgccctgtctctggccgaggcgagccgcgcaagtttcccgggaccctcagagttgcactccgaagactccagattccgagagttgcggaaacgctacgaggacctgctaaccaggctgcgggccaaccagagctgggaagattcgaacaccgacctcgtcccggcccctgcagtccggatactcacgccagaagtgcggctgggatccggcggccacctgcacctgcgtatctctcgggccgcccttcccgaggggctccccgaggcctcccgccttcaccgggctctgttccggctgtccccgacggcgtcaaggtcgtgggacgtgacacgaccgctgcggcgtcagctcagccttgcaagaccccaggcgcccgcgctgcacctgcgactgtcgccgccgccgtcgcagtcggaccaactgctggcagaatcttcgtccgcacggccccagctggagttgcacttgcggccgcaagccgccagggggcgccgcagagcgcgtgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga (SEQ ID NO: 3).

[0620] The sequence of the 303 amino acid sequence of the full-length mouse GDF15 is MAPPALQAQPPGGSQLRFLLFLLLLLLLLSWPSQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLHANQSREDSNSEPSPPDPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTARPWDITRPLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGVSLQTYDDLVARGCHCA (SEQ ID NO: 6), atggccccgcccgcgctccaggcccagcctccaggcggctctcaactgaggttcctgctgttcctgctgctgttgctgctgctgctgtcatggccatcgcagggggacgccctggcaatgcctgaacagcgaccctccggccctgagtcccaactcaacgccgacgagctacggggtcgcttccaggacctgctgagccggctgcatgccaaccagagccgagaggactcgaactcagaaccaagtcctgacccagctgtccggatactcagtccagaggtgagattggggtcccacggccagctgctactccgcgtcaaccgggcgtcgctgagtcagggtctccccgaagcctaccgcgtgcaccgagcgctgctcctgctgacgccgacggcccgcccctgggacatcactaggcccctgaagcgtgcgctcagcctccggggaccccgtgctcccgcattacgcctgcgcctgacgccgcctccggacctggctatgctgccctctggcggcacgcagctggaactgcgcttacgggtagccgccggcagggggcgccgaagcgcgcatgcgcacccaagagactcgtgcccactgggtccggggcgctgctgtcacttggagactgtgcaggcaactcttgaagacttgggctggagcgactgggtgctgtccccgcgccagctgcagctgagcatgtgcgtgggcgagtgtccccacctgtatcgctccgcgaacacgcatgcgcagatcaaagcacgcctgcatggcctgcagcctgacaaggtgcctgccccgtgctgtgtcccctccagctacaccccggtggttcttatgcacaggacagacagtggtgtgtcactgcagacttatgatgacctggtggcccggggctgccactgcgcttga (SEQ ID NO: 5).

[0621] The amino acid sequence of human GDF15 after cleavage of the 29-residue signal sequence is The DNA sequence: LSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 8). It is encoded by [[SEQ ID NO:7]]: ctgtctctggccgaggcgagccgcgcaagtttcccgggaccctcagagttgcactccgaagactccagattccgagagttgcggaaacgctacgaggacctgctaaccaggctgcgggccaaccagagctgggaagattcgaacaccgacctcgtcccggcccctgcagtccggatactcacgccagaagtgcggctgggatccggcggccacctgcacctgcgtatctctcgggccgcccttcccgaggggctccccgaggcctcccgccttcaccgggctctgttccggctgtccccgacggcgtcaaggtcgtgggacgtgacacgaccgctgcggcgtcagctcagccttgcaagaccccaggcgcccgcgctgcacctgcgactgtcgccgccgccgtcgcagtcggaccaactgctggcagaatcttcgtccgcacggccccagctggagttgcacttgcggccgcaagccgccagggggcgccgcagagcgcgtgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

[0622] The amino acid sequence of mouse GDF15 after cleavage of the 32-residue signal sequence is SQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLHANQSREDSNSEPSPPDPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTARPWDITRPLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGVSLQTYDDLVARGCHCA (SEQ ID NO: 10), (SEQ ID NO:9).

[0623] Biologically active forms of GDF15 include homodimers comprising two mature GDF15 monomers, each comprising SEQ ID NO: 12. The monomers that homodimerize to form human native mature GDF15 have the nucleic acid sequence: and the amino acid sequence: gcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga (SEQ ID NO: 11), Contains ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (sequence number 12).

[0624] Thus, the "human native mature GDF15 dimer" comprises two covalently linked monomers comprising SEQ ID NO:12.

[0625] The amino acid sequence of the recombinant act...

Claims

1. A fusion protein comprising a GDF15 region and an Fc domain.

2. 2. The fusion protein of claim 1, wherein the Fc domain comprises a sequence selected from the group consisting of SEQ ID NOs: 16, 22, 28, 29, 33, 35, 38, 48, 85, 91, 106, 132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213, 220, 227, 233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 and 302.

3. The fusion protein of claim 2, wherein the GDF15 region comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 25, 52 and 55.

4. The fusion protein of claim 2 , wherein the GDF15 region and the Fc domain are linked by a polypeptide linker.

5. The fusion protein of claim 4, wherein the polypeptide linker is selected from the group consisting of SEQ ID NOs: 18, 30, 34, 40, 58, 61, 64, 69, 72, 75, 78, 113, 116, 119, 122, 125, and 128.

6. The fusion protein is selected from the group consisting of SEQ ID NOs: 46, 24, 27, 32, 37, 20, 42, 50, 54, 57, 60, 63, 66, 68, 71, 74, 77, 82, 84, 88, 93, 96, 98, 100, 102, 104, 108, 134, 137, 139, 143, 146, 150, 153, 269, 272, 276 279, 157, 160, 164, 167, 171, 174, 178, 181, 185, 188, 194, 197, 201, 204, 208, 211, 215, 218, 222, 225, 229, 232, 233, 238 and 240.

7. The fusion protein of claim 1 , wherein the fusion protein comprises two or more Fc domains.

8. A dimer comprising (i) a first polypeptide chain comprising a GDF15 region and a first Fc domain, and (ii) a second polypeptide chain comprising a second Fc domain.

9. 9. The dimer of claim 8, wherein the first Fc domain comprises a sequence selected from the group consisting of SEQ ID NOs: 16, 22, 28, 29, 33, 35, 38, 48, 85, 91, 106, 132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213, 220, 227, 233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 and 302.

10. 10. The dimer of claim 9, wherein the second Fc domain comprises a sequence selected from the group consisting of SEQ ID NOs: 16, 22, 28, 29, 33, 35, 38, 48, 85, 91, 106, 132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213, 220, 227, 233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 and 302.

11. The dimer of claim 9 , wherein the GDF15 region comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 25, 52 and 55.

12. The dimer of claim 9 , wherein the GDF15 region and the Fc domain are linked by a polypeptide linker.

13. The dimer of claim 12, wherein the polypeptide linker is selected from the group consisting of SEQ ID NOs: 18, 30, 34, 40, 58, 61, 64, 69, 72, 75, 78, 113, 116, 119, 122, 125, and 128.

14. The fusion protein is selected from the group consisting of SEQ ID NOs: 46, 24, 27, 32, 37, 20, 42, 50, 54, 57, 60, 63, 66, 68, 71, 74, 77, 82, 84, 88, 93, 96, 98, 100, 102, 104, 108, 134, 137, 139, 143, 146, 150, 153, 269, 272, 276, 280, 282, 284, 286, 288, 290, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 3 9. The dimer of claim 8, comprising a sequence selected from the group consisting of: 76, 279, 157, 160, 164, 167, 171, 174, 178, 181, 185, 188, 194, 197, 201, 204, 208, 211, 215, 218, 222, 225, 229, 232, 233, 238 and 240.

15. The dimer of claim 8 , wherein the first polypeptide chain and the second polypeptide chain are non-covalently bound.

16. The dimer of claim 8 , wherein the first polypeptide chain and the second polypeptide chain are covalently linked.

17. 17. The dimer of claim 16, wherein the first polypeptide chain and the second polypeptide chain are covalently linked via a disulfide bond between their respective Fc domains.

18. 1. A tetramer comprising (i) a first dimer and (ii) a second dimer, wherein the first dimer and the second dimer independently comprise a dimer as described in claim 1, and wherein the first polypeptide chain of the first dimer is linked to the first polypeptide chain of the second dimer via an interchain disulfide bond between their respective GDF15 domains.

19. 20. The tetramer of claim 18, wherein the Fc domain of the first polypeptide of the first dimer comprises a sequence selected from the group consisting of SEQ ID NOs: 16, 22, 28, 29, 33, 35, 38, 48, 85, 91, 106, 132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213, 220, 227, 233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 and 302.

20. 20. The tetramer of claim 19, wherein the GDF15 region of the first polypeptide chain of the first dimer comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 25, 52 and 55.

21. The tetramer of claim 20 , wherein the GDF15 region and the Fc domain are linked by a polypeptide linker.

22. 22. The tetramer of claim 21 , wherein the polypeptide linker is selected from the group consisting of SEQ ID NOs: 18, 30, 34, 40, 58, 61, 64, 69, 72, 75, 78, 113, 116, 119, 122, 125, and 128.

23. The Fc domain of the first polypeptide of the first dimer is selected from the group consisting of SEQ ID NOs: 46, 24, 27, 32, 37, 20, 42, 50, 54, 57, 60, 63, 66, 68, 71, 74, 77, 82, 84, 88, 93, 96, 98, 100, 102, 104, 108, 134, 137, 139, 143, 146, 150, 153 20, 215, 218, 222, 225, 229, 232, 233, 238 and 240.

24. 20. The tetramer of claim 19, wherein the Fc domain of the first polypeptide of the second dimer comprises a sequence selected from the group consisting of SEQ ID NOs: 16, 22, 28, 29, 33, 35, 38, 48, 85, 91, 106, 132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213, 220, 227, 233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 and 302.

25. The Fc domain of the first polypeptide of the second dimer is selected from the group consisting of SEQ ID NOs: 46, 24, 27, 32, 37, 20, 42, 50, 54, 57, 60, 63, 66, 68, 71, 74, 77, 82, 84, 88, 93, 96, 98, 100, 102, 104, 108, 134, 137, 139, 143, 146, 150, 153 25. The tetramer of claim 24, comprising a sequence selected from the group consisting of: 269, 272, 276, 279, 157, 160, 164, 167, 171, 174, 178, 181, 185, 188, 194, 197, 201, 204, 208, 211, 215, 218, 222, 225, 229, 232, 233, 238 and 240.

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