# Patent

## Title
Growth differentiation factor 15 (GDF-15) polypeptides

## Abstract

GDF15 polypeptides, constructs comprising GDF15, and mutants thereof are provided. In various embodiments the GDF15 polypeptides, constructs comprising GDF15, and mutants thereof, can be of use in the treatment 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.

## Background
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 28, 2013, is named A1682WOP.txt and is 253,155 bytes in size.
FIELD OF THE INVENTION
The instant disclosure relates to GDF15 polypeptides, polypeptides comprising GDF15, and the generation and use thereof.
BACKGROUND OF THE INVENTION
Growth differentiation factor 15 (GDF15) is a divergent member of the TGFβ superfamily. It is also called macrophage inhibitory cytokine 1 (MIC1) (Bootcov M R, 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 L N 1997, Gene. 203:17-26), prostate derived factor (PDF) (Paralkar V M 1998, J Biol. Chem. 273:13760-7), and nonsteroidal anti-inflammatory drug-activated gene (NAG-1) (Baek S J 2001, J Biol. Chem. 276: 33384-92).
Human GDF15 gene is located on chromosome 19p13.2-13.1; rat GDF15 gene is located on chromosome 16; and mouse GDF15 gene is located on chromosome 8. The GDF15 open reading frames span 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.).
GDF15 is synthesized as a large precursor protein that is cleaved at the dibasic cleavage site to release the carboxyterminal mature peptide. The mouse and rat GDF15 prepro-peptides both contain 303 amino acids. Human full-length precursor contains 308 amino acids. The rodent mature peptides contain 115 amino acids after processing at the RGRR (SEQ ID NO:1) cleavage site. The human mature peptide contains 112 amino acids after processing at the RGRRRAR (SEQ ID NO:2) cleavage site. Human mature GDF15 peptide shares 66.1% and 68.1% sequence similarity with rat and mouse mature GDF15 peptides (Bottner M 1999, Gene. 237:105-11; Bauskin A R 2000, EMBO J. 19:2212-20; NCBI). There is no glycosylation site in the mature GDF15 peptide.
The mature GDF15 peptide contains the seven conserved cysteine residues required for the formation of the cysteine knot motif (having three intrachain disulfide bonds) and the single interchain disulfide bond that are typical for TGFβ superfamily members. 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.
GDF15 circulating levels have been reported to be elevated in multiple pathological and physiological conditions, most notably pregnancy (Moore A G 2000. J Clin Endocrinol Metab 85: 4781-4788), β-thalassemia (Tanno T 2007, Nat Med 13:1096-101) (Zimmermann M B, 2008 Am J Clin Nutr 88:1026-31), and congenital dyserythropoietic anemia (Tamary H 2008, Blood. 112:5241-4). GDF15 has also been linked to multiple biological activities in literature reports. Studies of GDF15 knockout and transgenic mice suggested that GDF15 may be protective against ischemic/reperfusion- or overload-induced heart injury (Kempf T, 2006, Circ Res. 98:351-60) (Xu J, 2006, Circ Res. 98:342-50), protective against aging-associated motor neuron and sensory neuron loss (Strelau J, 2009, J. Neurosci. 29:13640-8), mildly protective against metabolic acidosis in kidney, and may cause cachexia in cancer patients (Johnen H 2007 Nat. Med. 11:1333-40). Many groups also studied the role of GDF15 in cell apoptosis and proliferation and reported controversial results using different cell culture and xenograft models. Studies on transgenic mice showed that GDF15 is protective against carcinogen or Apc mutation induced neoplasia in intestine and lung (Baek S J 2006, Gastroenterology. 131:1553-60; Cekanova M 2009, Cancer Prev Res 2:450-8).
SUMMARY OF THE INVENTION
Provided herein is a construct comprising a GDF15 polypeptide and one or more Fc sequences. In one embodiment, the construct comprises two or more Fc sequences. In a further embodiment, the one or more of the Fc sequences independently comprise a sequence selected from the group consisting of SEQ ID NOs:18, 19, 85, 86, 89, 90, 91, 99, 100, 108, 109 and 111. In another embodiment, two or more of the Fc sequences are associated. In still a further embodiment, the GDF15 polypeptide and one or more of the Fc sequences form a contiguous sequence. In yet another embodiment, the GDF15 polypeptide and an Fc sequence are joined by a linker. In another embodiment, the construct comprises a sequence selected from the group consisting of SEQ ID NOs:44, 50, 57, 61, 65, 69, 74, 79, 84, 95 and 104. In yet a further embodiment, the construct further comprises a sequence selected from the group consisting of SEQ ID NOs:18, 19, 85, 86, 89, 90, 91, 99, 100, 108, 109 and 110. In another embodiment, a dimer comprising the constructs described herein is provided. In a particular embodiment, the construct comprises: (a) a sequence comprising SEQ ID NO:18 and a sequence comprising SEQ ID NO:44; (b) a sequence comprising SEQ ID NO:86 and a sequence comprising SEQ ID NO:50; (c) a sequence comprising SEQ ID NO:18 and a sequence comprising SEQ ID NO: 57; (d) a sequence comprising SEQ ID NO:86 and a sequence comprising SEQ ID NO:61; (e) a sequence comprising SEQ ID NO:86 and a sequence comprising SEQ ID NO:65; (f) a sequence comprising SEQ ID NO:86 and a sequence comprising SEQ ID NO:69; (g) a sequence comprising SEQ ID NO:86 and a sequence comprising SEQ ID NO:74; (h) a sequence comprising SEQ ID NO:86 and a sequence comprising SEQ ID NO:79; (i) a sequence comprising SEQ ID NO:86 and a sequence comprising SEQ ID NO:84; (j) a sequence comprising SEQ ID NO:91 and a sequence comprising SEQ ID NO:95; or (k) a sequence comprising SEQ ID NO:100 and a sequence comprising SEQ ID NO:104, as well as a dimer comprising the construct.
Also provided herein is a construct comprising a GDF15 polypeptide and two or more Fc sequences, wherein one or more Fc sequences independently comprise a sequence selected from the group consisting of SEQ ID NOs:23, 110, 114 and 115. In one embodiment, two or more Fc sequences are associated. In a further embodiment, the GDF15 polypeptide and one or more of the Fc sequences form a contiguous sequence. In yet another embodiment, the GDF15 polypeptide and an Fc sequence are joined by a linker. In still a further embodiment, the construct comprises SEQ ID NO:113. In another embodiment a dimer comprising the constructs described herein is provided. In a particular embodiment, the construct comprises: a sequence comprising SEQ ID NO:110 and a sequence comprising SEQ ID NO:113, as well as a dimer comprising the construct.
Also provided herein is a construct comprising a GDF15 polypeptide and two or more Fc sequences, wherein the Fc sequences comprise SEQ ID NO:28 and SEQ ID NO:30. In one embodiment the two Fc sequences are joined by a linker. In another embodiment the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 87, 88 and 131. In still another embodiment, the C-terminus of a first Fc sequence is joined to the N-terminus of a second Fc sequence. In yet another embodiment, the GDF15 polypeptide and one or more of the Fc sequences form a contiguous sequence. In still a further embodiment, the GDF15 polypeptide and an Fc sequence are joined by a linker. In another embodiment, the linker comprises SEQ ID NO:31. In still a further embodiment, a construct comprising a dimer of the constructs described herein is provided. In a particular embodiment, the construct comprises a sequence selected from the group consisting of SEQ ID NOs:33, 123 and 128, as well as a dimer comprising the construct.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a series of graphics depicting the arrangement of the charged pair (delHinge) construct DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) (FIG. 1A); the charged pair construct CpmFc(−)-(G4S)4-GDF15:CpmFc(+) (FIG. 1B); and the hemi construct Fc-(G4S)8-Fc-GS(G4S)4-GDF15 (FIG. 1C). FIG. 1 discloses “(G4S)4” (SEQ ID NO: 20), “GS(G4S)4” (SEQ ID NO:31) and “(G4S)8” (SEQ ID NO: 34).
FIG. 2 is a table showing the results of a food intake assay in hyperphagic ob/ob mice in which dimers of the following GDF15 constructs were studied: DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+), DhCpmFc(+)-(G4S)4-GDF15:DhCpmFc(−), DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+), DhCpmFc(+)-(G4S)4-GDF15(H6D):DhCpmFc(−), DhCpmFc(+)-(G4S)4-GDF15 (N3Q):DhCpmFc(−), DhCpmFc(+)-GDF15:DhCpmFc(−), DhCpmFc(+)-G4-GDF15:DhCpmFc(−), DhCpmFc(+)-(G4S)2-GDF15:DhCpmFc(−), DhCpmFc(+)-(G4Q)4-GDF15:DhCpmFc(−), DhCpmFc(+)-(1K)-GDF15:DhCpmFc(−), DhCpmFc(+)(L351C)-G4-GDF15:DhCpmFc(−)(L351C), DhCpmFc(+)(S354C)-G4-GDF15:DhCpmFc(−)(Y349C) CpmFc(+)-(G4S)-4-GDF15:CpmFc(−); Fc-(G4S)8-Fc-GS(G4S)4-GDF15, Fc-(G4S)3-Fc-GS(G4S)4-GDF15, Fc-(G4S)5-Fc-GS(G4S)4-GDF15, GDF15 and GDF15 (H6D) variant.
FIG. 3 is a plot showing the effect on food intake (g food/g body weight (BW) of ob/ob mice as a function of dose (log [g protein/kg BW]) using a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct.
FIG. 4 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)-(G4S)4-GDF15:DhCpmFc(−) construct.
FIG. 5 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct.
FIG. 6 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)-(G4S)4-GDF15(H6D):DhCpmFc(−) construct.
FIG. 7 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)-(G4S)4-GDF15(N3Q):DhCpmFc(−) construct.
FIG. 8 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)-GDF15:DhCpmFc(−) construct.
FIG. 9 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)-G4-GDF15:DhCpmFc(−) construct.
FIG. 10 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)-(G4S)2-GDF15:DhCpmFc(−) construct.
FIG. 11 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)-(G4Q)4-GDF15:DhCpmFc(−) construct.
FIG. 12 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)-(1K)-GDF15:DhCpmFc(−) construct.
FIG. 13 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)(L351C)-G4-GDF15:DhCpmFc(−)(L351C) construct.
FIG. 14 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the DhCpmFc(+)(S354C)-G4-GDF15:DhCpmFc(−)(Y349C) construct.
FIG. 15 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the CpmFc(+)-(G4S)4-GDF15:CpmFc(−) construct.
FIG. 16 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the Fc-G(G4S)-8-Fc-GS(G4S)4-GDF15 construct.
FIG. 17 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the Fc-G(G4S)3-Fc-GS(G4S)4-GDF15 construct.
FIG. 18 is a plot showing the effect on food intake of ob/ob mice as a function of dose using a dimer of the Fc-G(G4S)5-Fc-GS(G4S)4-GDF15 construct.
FIG. 19 is a plot showing the effect on food intake of ob/ob mice as a function of dose using native mature GDF15 dimer.
FIG. 20 is a plot showing the effect on food intake of ob/ob mice as a function of dose using native mature hGDF15 dimer.
FIG. 21 is a plot showing the effect on food intake of ob/ob mice as a function of dose using mature hGDF15(H6D) variant dimer
FIG. 22 is a bar graph of the results of a lipid tolerance assay showing the effect on triglyceride (mg/dL) for native mature GDF15 dimer (1 mg/kg, i.v.), a dimer of the Fc-(G4S)-8-Fc-GS(G4S)4-GDF15 construct (1 mg/kg, i.v.) and control.
FIG. 23 is a plot of the results of a lipid tolerance assay using a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, showing effect on triglyceride (mg/dL) as a function of dose (log [g construct/kg BW]).
FIG. 24 is a plot showing the results of a lipid tolerance assay using a dimer of the native mature hGDF15.
FIG. 25 is a plot showing the results of a lipid tolerance assay using a dimer of the mature hGDF15(H6D) variant.
FIG. 26 is plot showing the results of a two week OGTT performed using a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi) in DIO (diet-induced obese) mice.
FIG. 27 is bar graph summarizing the data of FIG. 26 in the form of AUC data.
FIG. 28 is a plot showing the results of a five week OGTT performed using a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi).
FIG. 29 is a bar graph summarizing the data of FIG. 28 in the form of AUC data.
FIG. 30 is a plot showing the effect on the body weight (g) of DIO mice of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi).
FIG. 31 is a plot showing the effect on the food intake (g food/animal/day) of DIO mice of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi).
FIG. 32 is a bar graph showing the effect on the glucose levels (mg/dL) of DIO mice after 4 hour fast of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi).
FIG. 33 is a plot showing the effect on insulin levels (ng/mL) of DIO mice after 4 hour fast of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi).
FIG. 34 is a bar graph showing the effect on insulin levels (ng/mL) of DIO mice after overnight fast dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 and rosiglitizone (Rosi).
FIG. 35 is a plot showing the effect on the triglyceride levels (mg/dL) of DIO mice after 4 hour fast dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi).
FIG. 36 is a bar graph showing the effect on the triglyceride levels (mg/dL) of DIO mice after overnight fast dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi).
FIG. 37 is a plot showing the effect on the total cholesterol levels (mg/dL) of DIO mice after 4 hour fast dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi).
FIG. 38 is a bar graph showing the effect on the total cholesterol levels (mg/dL) of DIO mice after overnight fast dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and rosiglitizone (Rosi).
FIG. 39 is a plot showing the results of a two week OGTT after 4 hour fast performed on DIO mice dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 40 is a bar graph summarizing the data of FIG. 39 in the form of AUC data.
FIG. 41 is a plot showing the results of a five week OGTT after an overnight fast performed on DIO mice dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 42 is a bar graph summarizing the data of FIG. 41 in the form of AUC data.
FIG. 43 is a plot showing the effect on the body weight of DIO mice dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 44 is a plot showing the effect on food intake of DIO mice dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 45 is a bar graph showing the effect on glucose levels (mg/dL) of DIO mice after 4 hour fast dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 46 is a plot showing the effect on the insulin levels (ng/mL) of DIO mice after 4 hour fast dosed with a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 47 is a bar graph showing the effect on the insulin levels of DIO mice fed ad libitum using a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 48 is a bar graph showing the effect on the insulin levels (ng/ml) of DIO mice after overnight fast of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 49 is a bar graph showing the effect on the triglyceride levels (mg/dL) of DIO mice after 4 hour fast of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 50 is a bar graph showing the effect on the triglyceride levels (mg/dL) of DIO mice fed ad libitum of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 51 is a bar graph showing the effect on the triglyceride levels (mg/dL) of DIO mice after overnight fast of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 52 is a bar graph showing the effect on the total cholesterol levels (mg/dL) of DIO mice after 4 hour fast of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 53 is a plot showing the effect on the total cholesterol levels (mg/dL) of DIO mice fed ad libitum of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 54 is a bar graph showing the effect on the total cholesterol levels (mg/dL) of DIO mice after overnight fast of a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct and rosiglitizone (Rosi).
FIG. 55 is a diagram graphically depicting the study design for a five-week treatment performed in obese cynomolgous monkeys using a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct.
FIG. 56 is a plot showing the serum levels (ng/mL) of a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct in the cynomolgous monkeys studied.
FIG. 57 is a bar graph showing the results of an acclimation OGTT following an overnight fast performed on cynomolgous monkeys using a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle, in the form of glucose AUC data.
FIG. 58 is a bar graph showing the results of OGTT following an overnight fast performed on cynomolgous monkeys after 2 week administration of a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle, in the form of glucose AUC data.
FIG. 59 is a bar graph showing the results of a OGTT following an overnight fast performed on cynomolgous monkeys after 5 week administration of a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle, in the form of glucose AUC data.
FIG. 60 is a bar graph showing the results of an OGTT following an overnight fast performed on cynomolgous monkeys after 4 week of wash out using a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle in the form of glucose AUC data.
FIG. 61 is a bar graph showing the results of an acclimation OGTT following an overnight fast performed on cynomolgous monkeys using a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle, in the form of insulin AUC data.
FIG. 62 is a bar graph showing the results of OGTT following an overnight fast performed on cynomolgous monkeys after 2 week administration of a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle, in the form of insulin AUC data.
FIG. 63 is a bar graph showing the results of a OGTT following an overnight fast performed on cynomolgous monkeys after 5 week administration of a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle, in the form of insulin AUC data.
FIG. 64 is a bar graph showing the results of a OGTT following an overnight fast performed on cynomolgous monkeys 4 week of wash out of a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhFc(+) construct and vehicle in the form of insulin AUC data.
FIG. 65 is a plot of body weight (kg) as a function of time (days) collected in the month preceding, during a 30 day treatment and subsequent washout period performed on cynomolgous monkeys using a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle.
FIG. 66 is a plot of daily food intake (g) as a function of time (days) collected in the week preceding, during a 5 week treatment and subsequent 4 week washout period performed on cynomolgous monkeys using a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle.
FIG. 67 is a plot of fasting triglyceride (mg/dL) as a function of time (days) collected in the week preceding, during a 5 week treatment and subsequent 4 week washout period performed on cynomolgous monkeys using a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle.
FIG. 68 is a plot of fasting insulin (ng/dL) as a function of time (days) collected in the week preceding, during a 5 week treatment and subsequent 4 week washout period performed on cynomolgous monkeys using a dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and vehicle.



DETAILED DESCRIPTION OF THE INVENTION
The instant disclosure provides GDF15 polypeptides and constructs comprising GDF15 polypeptides. Also provided is the generation and uses of the disclosed molecules, for example in treating a metabolic disorder, such as Type 2 diabetes, elevated glucose levels, elevated insulin levels, dyslipidemia or obesity.
Recombinant polypeptide and nucleic acid methods used herein, including in the Examples, are generally those set forth 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), both of which are incorporated herein by reference for any purpose.
I. GENERAL DEFINITIONS
Following convention, as used herein “a” and “an” mean “one or more” unless specifically indicated otherwise.
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 the naturally occurring amino acids.
The terms “naturally occurring amino acid” and “naturally encoded amino acid” are used interchangeably and refer to an amino acid that is encoded by the genetic code, as well as those amino acids that are encoded by the genetic code that are modified after synthesis, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.
An “amino acid analog” is a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. 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.
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 in a manner similar to a naturally occurring amino acid. Examples include a methacryloyl or acryloyl derivative of an amide, β-, γ-, δ-imino acids (such as piperidine-4-carboxylic acid) and the like.
The terms “non-naturally occurring amino acid” and “non-naturally encoded amino acid” are used interchangeably and refer to a compound that has the same basic chemical structure as a naturally occurring amino acid, but is not incorporated into a growing polypeptide chain by the translation complex. “Non-naturally occurring amino acid” also includes, but is not limited to, amino acids that occur by modification (e.g., posttranslational modifications) of a naturally encoded amino acid (including but not limited to, the 20 common amino acids) but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. A non-limiting lists of examples of non-naturally occurring amino acids that can be inserted into a polypeptide sequence or substituted for a wild-type residue in polypeptide sequence include β-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains. Examples include (in the L-form or D-form; abbreviated as 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-methylarginine (NMeR), Nα-methylleucine (Nα-MeL or NMeL), N-methylhomolysine (NMeHoK), Nα-methylglutamine (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-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine (abbreviated “K(Nε-glycyl)” or “K(glycyl)” or “K(gly)”), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or Amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad), Nα-methyl valine (NMeVal), N-α-methyl leucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionoic 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 (Aib), beta-alanine, beta-aminopropionic acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-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.
Also included in the definition of “non-naturally occurring amino acid” is any amino acid comprising the structure




wherein the R group is any substituent other than the one used in the twenty natural amino acids.
In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group. In some embodiments, the non-naturally encoded amino acid has the structure:




wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, or substituted aryl; R2 is H, an alkyl, aryl, substituted alkyl, and substituted aryl; and R3 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R4 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

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.
In some embodiments, the non-naturally encoded amino acid residue comprises an azide group. In some embodiments, the non-naturally encoded amino acid has the structure:




wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, substituted aryl or not present; X is O, N, S or not present; m is 0-10; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises an alkyne group. In some embodiments, the non-naturally encoded amino acid has the structure:




wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, or substituted aryl; X is O, N, S or not present; m is 0-10, R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

The term “substituted” means that a hydrogen atom on a molecule or group is replaced with a group or atom, which is referred to as a substituent. Typical substitutents include: halogen, C1-8alkyl, hydroxyl, C1-8alkoxy, —NRxRx, nitro, cyano, halo or perhaloC1-8alkyl, C2-8alkenyl, C2-8alkynyl, —SRx, —S(═O)2Rx, —C(═O)ORx, —C(═O)Rx, wherein each Rx is independently hydrogen or C1-C8 alkyl. It is noted that when the substituent is —NRxRx, the Rx groups may be joined together with the nitrogen atom to form a ring.
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 alkyl groups having from 1 to 8 carbon atoms, which groups are commonly represented as C1-8alkyl.
The term “alkoxy” means an alkyl group bonded to an oxygen atom. Representative examples of alkoxy groups include methoxy, ethoxy, tert-butoxy, propoxy and isobutoxy. Common alkoxy groups are C1-8alkoxy.
The term “halogen” or “halo” means chlorine, fluorine, bromine or iodine.
The term “alkenyl” means a branched or straight chain hydrocarbon having one or more carbon-carbon double bonds. Representative examples alkenyl groups include ethenyl, propenyl, allyl, butenyl and 4-methylbutenyl. Common alkenyl groups are C2-8alkenyl.
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 are C2-8 alkynyl.
The term “cycloalkyl” means a cyclic, nonaromatic hydrocarbon. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. A cycloalkyl group can contain one or more double bond. Examples of cycloalkyl groups that contain double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl and cyclobutadienyl. Common cycloalkyl groups are C3-8 cycloalkyl groups.
The term “perfluoroalkyl” means an alkyl group in which all of the hydrogen atoms have been replaced with fluorine atoms. Common perfluoroalkyl groups are C1-8 perfluoroalkyl. An example of a common perfluoroalkyl group is —CF3.
The term “acyl” means a group derived from an organic acid by removal of the hydroxy group (—OH). For example, the acyl group CH3C(═O)— is formed by the removal of the hydroxy group from CH3C(═O)OH.
The term “aryl” means a cyclic, aromatic hydrocarbon. Examples of aryl groups include phenyl and naphthyl. Common aryl groups are six to thirteen membered rings.
The term “heteroatom” as used herein means an oxygen, nitrogen or sulfur atom.
The term “heteroaryl” means a cyclic, aromatic hydrocarbon in which one or more carbon atoms of an aryl group have been replaced with a heteroatom. If the heteroaryl group contains more than one heteroatom, the heteroatoms may be the same or different. Examples of heteroaryl groups 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 five to thirteen membered rings that contain from 1 to 4 heteroatoms. Heteroaryl groups that are five and six membered rings that contain 1 to 3 heterotaoms are particularly common.
The term “heterocycloalkyl” means a cycloalkyl group in which one or more of the carbon atoms has been replaced with 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 also possible for the heterocycloalkyl group to have one or more double bonds, but is not aromatic. Examples of heterocycloalkyl groups containing double bonds include dihydrofuran. Common heterocycloalkyl groups are three to ten membered rings containing from 1 to 4 heteroatoms. Heterocycloalkyl groups that are five and six membered rings that contain 1 to 2 heterotaoms are particularly common.
It is also noted that the cyclic ring groups, i.e., aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, can comprise more than one ring. For example, the naphthyl group is a fused bicyclic ring system. It is also intended that the present invention include ring groups that have bridging atoms, or ring groups that have a spiro orientation.
Representative examples of five to six 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.
Representative examples of partially saturated, fully saturated or fully unsaturated five to eight membered rings, optionally having one to three heteroatoms, are cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and phenyl. Further exemplary five 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-oxadizaolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4oxadiazolyl, 1,2,3-triazolyl, 1,2,4-trizaolyl, 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-oxathiolyl.
Further exemplary six membered rings are 2H-pyranyl, 4H-pyranyl, pyridinyl, piperidinyl, 1,2-dioxinyl, 1,3-dioxinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, pyndazinyl, 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-(3 oxathiazinyl, and 1,4,2-oxadiazinyl.
Further exemplary seven membered rings are azepinyl, oxepinyl, thiepinyl and 1,2,4-triazepinyl.
Further exemplary eight membered rings are cyclooctyl, cyclooctenyl and cyclooctadienyl.
Exemplary bicyclic rings consisting of two fused partially saturated, fully saturated or fully unsaturated five and/or six membered rings, optionally having one to four 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, phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, indenyl, isoindenyl, naphthyl, tetralinyl, decalinyl, 2H-1-benzopyranyl, 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.
A cyclic ring group may be bonded to another group in more than one way. If no particular bonding arrangement is specified, then 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.
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., a GDF15 nucleic acid sequence provided herein), or an analog thereof, that has been separated from at least about 50 percent of polypeptides, peptides, lipids, carbohydrates, polynucleotides or other materials with which the nucleic acid is naturally found when total nucleic acid is isolated from the source cells. Preferably, an isolated nucleic acid molecule is substantially free from any other contaminating nucleic acid molecules or other molecules that are found in the natural environment of the nucleic acid that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.
The term “isolated polypeptide” refers to a polypeptide (e.g., a GDF15 polypeptide sequence provided herein) that has been separated from at least about 50 percent of polypeptides, peptides, lipids, carbohydrates, polynucleotides, or other materials with which the polypeptide is naturally found when isolated from a source cell. Preferably, the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.
The term “encoding” refers to a polynucleotide sequence encoding one or more amino acids. The term does not require a start or stop codon. An amino acid sequence can be encoded in any one of the different reading frames provided by a polynucleotide sequence.
The terms “identical” and percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) can be addressed by a particular mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of the 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.
In calculating percent identity, the sequences being compared are aligned in a way that gives the largest 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, Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3× the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., (1978) Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.
Recommended parameters for determining percent identity for polypeptides or nucleotide sequences using the GAP program are the following:
Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;
Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;
Gap Penalty: 12 (but with no penalty for end gaps)
Gap Length Penalty: 4
Threshold of Similarity: 0
Certain alignment schemes for aligning two amino acid sequences can result in matching of only a short region of the two sequences, and this small aligned region can have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (e.g., the GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide.
The terms “GDF15 polypeptide” and “GDF15 protein” are used interchangeably and mean a naturally-occurring wild-type polypeptide expressed in a mammal, such as a human or a mouse. For purposes of this disclosure, the term “GDF15 polypeptide” can be used interchangeably to refer to any full-length GDF15 polypeptide, e.g., SEQ ID NO:4, which consists of 308 amino acid residues and which is encoded by the nucleotide sequence SEQ ID NO:3; any form comprising the active and prodomains of the polypeptide, e.g., SEQ ID NO:8, which consists of 279 amino acid residues and which is encoded by the nucleotide sequence SEQ ID NO:7, and in which the 29 amino acid residues at the amino-terminal end of the full-length GDF15 polypeptide (i.e., which constitute the signal peptide) have been removed; and any form of the polypeptide comprising the active domain from which the prodomain and signal sequence have been removed, e.g., SEQ ID NO:12, which consists of 112 amino acid residues and which is encoded by the nucleotide sequence SEQ ID NO:11, in which the signal sequence and the prodomain have been removed. GDF15 polypeptides can but need not comprise an amino-terminal methionine, which may be introduced by engineering or as a result of a bacterial expression process.
The term “GDF15 mutant polypeptide” encompasses a GDF15 polypeptide in which a naturally occurring GDF15 polypeptide sequence (e.g., SEQ ID NOs:4, 8 or 12) has been modified. Such modifications include, but are not limited to, one or more amino acid substitutions, including substitutions with non-naturally occurring amino acids non-naturally-occurring amino acid analogs and amino acid mimetics.
In one aspect, the term “GDF15 mutant polypeptide” refers to a GDF15 polypeptide sequence (e.g., SEQ ID NOs:4, 8 or 12) in which at least one residue normally found at a given position of a native GDF15 polypeptide is deleted or is replaced by a residue not normally found at that position in the native GDF15 sequence. In some cases it will be desirable to replace a single residue normally found at a given position of a native GDF15 polypeptide with more than one residue that is not normally found at the position; in still other cases it may be desirable to maintain the native GDF15 polypeptide sequence 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.”
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 NOs:4, 8 or 12). In other embodiments, a GDF15 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 amino acid sequence (e.g., SEQ ID NOs:4, 8 or 12). Such GDF15 mutant polypeptides preferably, but need not, possess at least one activity of a wild-type GDF15 mutant polypeptide, such as the ability to lower blood glucose, insulin, triglyceride, or cholesterol levels; the ability to reduce body weight; or the ability to improve glucose tolerance, energy expenditure, or insulin sensitivity. The present invention also encompasses nucleic acid molecules encoding such GDF15 mutant polypeptide sequences.
As stated herein, a GDF15 mutant polypeptide can comprise a signal sequence (residues 1-29 of SEQ ID NO:4) or it can have the signal sequence removed (providing SEQ ID NO:8). In other embodiments, a GDF15 mutant polypeptide can have the signal sequence removed and can also be cleaved at residue 196, separating the primary sequence of the prodomain (residues 30-196 of SEQ ID NO:4) from the primary sequence of the active domain. The naturally-occurring biologically active form of a GDF15 mutant polypeptide is a homodimer comprising the processed mature peptide (SEQ ID NO:12; residues 197-308 of SEQ ID NO:4). Although the GDF15 polypeptides and GDF15 mutant polypeptides, and the constructs comprising such polypeptides are primarily disclosed in terms of human GDF15, the invention is not so limited and extends to GDF15 polypeptides and GDF15 mutant polypeptides and the constructs comprising such polypeptides where the GDF15 polypeptides and GDF15 mutant polypeptides are derived from other species (e.g., cynomolgous monkeys, mice and rats). In some instances, a GDF15 polypeptide or a GDF15 mutant polypeptide can be used to treat or ameliorate a metabolic disorder in a subject is a mature form of a GDF15 mutant polypeptide that is derived from the same species as the subject.
A GDF15 mutant polypeptide is preferably biologically active. In various respective embodiments, a GDF15 polypeptide or a GDF15 mutant polypeptide has a biological activity that is equivalent to, greater to or less than that of the naturally occurring form of the mature GDF15 protein or GDF15 mutant polypeptide from which the signal peptide has been removed from the N-terminus of a full length GDF15 mutant polypeptide sequence and in which the prodomain has been cleaved (but not necessarily removed from) the active domain. Examples of biological activities include the ability to lower blood glucose, insulin, triglyceride, or cholesterol levels; the ability to reduce body weight; or the ability to improve glucose tolerance, lipid tolerance, or insulin sensitivity; the ability to lower urine glucose and protein excretion.
The terms “therapeutically effective dose” and “therapeutically effective amount,” as used herein, means an amount of GDF15 or GDF15 mutant polypeptide that elicits a biological or medicinal response in a tissue system, animal, or human being sought by a researcher, physician, or other clinician, which includes alleviation or amelioration of the symptoms of the disease or disorder being treated, i.e., an amount of GDF15 or GDF15 mutant polypeptide that supports an observable level of one or more desired biological or medicinal response, for example lowering blood glucose, insulin, triglyceride, or cholesterol levels; reducing body weight; or improving glucose tolerance, energy expenditure, or insulin sensitivity.
II. GDF15 POLYPEPTIDES AND CONSTRUCTS COMPRISING GDF15, INCLUDING MUTANT FORMS THEREOF, AND POLYNUCLEOTIDES
A range of GDF15 polypeptides and constructs comprising a GDF15 polypeptide are provided herein. Some of these molecules were studied in a variety of assays, as described in the Examples presented herein below. Some of the GDF15 polypeptides and constructs comprising a GDF15 polypeptide provided herein include those described below.
II.A. Native Mature GDF15 and Variants
II.A.1 Native Mature GDF15
Bearing in mind that the biologically active form of GDF15 comprises a homodimer, the construct designated “native mature GDF15 dimer” in the instant disclosure refers to a homodimer comprising two mature GDF15 monomers, each of which comprises SEQ ID NO:12. The monomer that homodimerizes to form the native mature GDF15 dimer is encoded by the nucleic acid sequence:







(SEQ ID NO: 11)


gcgcgcaacggggaccactgtccgctcgggcccgggcgttgct


 


gccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgat


 


tgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtg


 


cccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcc


 


tgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgcc


 


agctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgct


 


ccagacctatgatgacttgttagccaaagactgccactgcatatga






and comprises the amino acid sequence:








(SEQ ID NO: 12)


ARNGDHCPLGPGRCCRLHTVRASLEDLGWAD


 


WVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPA


 


SYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.






Thus, the “native mature GDF15 dimer” comprises two covalently associated monomers comprising SEQ ID NO:12.
In some embodiments, a leader, or signal, sequence may be used to direct secretion of a polypeptide. A signal sequence may be positioned within or directly at the 5′ end of a polypeptide coding region. Many signal sequences have been identified and may be selected based upon the host cell used for expression, e.g., the cleaved VH21 signal sequence (see EP2330197 for discussion of the VH21 signal sequence).
In an embodiment employing the VH21 signal sequence, the monomer that homodimerizes to form the native mature GDF15 dimer is encoded by the nucleic acid sequence (VH21 signal sequence underlined):







(SEQ ID NO: 15)


atggaatggagctgggtctttctcttcttcctgtcagtaacgaccggtgt


 


ccactccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgct


 


gccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgat


 


tgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtg


 


cccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcc


 


tgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgcc


 


agctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgct


 


ccagacctatgatgacttgttagccaaagactgccactgcatatga






and comprises the amino acid sequence (cleaved VH21 signal sequence underlined):








(SEQ ID NO: 16)


MEWSWVFLFFLSVTTGVHSARNGDHCPLGPGRCCRLHTVRASLEDLGWAD


 


WVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPA


 


SYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI






II.A.2 Mature GDF15(H6D) Variant

The GDF15(H6D) variant is a naturally occurring human GDF15 variant due to a C→G polymorphism, resulting in the His to Asp change at residue 202 in full-length peptide (SEQ ID NO:4); residue 6 in mature peptide (SEQ ID NO:12).
The construct designated “mature GDF15(H6D) dimer” in the instant disclosure refers to a homodimer comprising two mature GDF15(H6D) monomers, each of which comprises SEQ ID NO:38. The monomer that homodimerizes to form the mature GDF15(H6D) dimer is encoded by the nucleic acid sequence:







(SEQ ID NO: 37)


gcgcgcaacggggacgattgtccgctcgggcccgggcgttgct


 


gccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgat


 


tgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtg


 


cccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcc


 


tgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgcc


 


agctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgct


 


ccagacctatgatgacttgttagccaaagactgccactgcatatga






and comprises the amino acid sequence:










(SEQ ID NO: 38)



ARNGDDCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQ



 



VTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVP



 



ASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI






Thus, the “mature GDF15(H6D) dimer” comprises two covalently associated monomers comprising SEQ ID NO:38.
In an embodiment employing the VH21 signal sequence, the monomer that homodimerizes to form the mature GDF15(H6D) dimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 35)


atggaatggagctgggtctttctcttcttcctgtcagtaacgaccggtgt


 


ccactccgcgcgcaacggggacgattgtccgctcgggcccgggcgttgct


 


gccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgat


 


tgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtg


 


cccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcc


 


tgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgcc


 


agctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgct


 


ccagacctatgatgacttgttagccaaagactgccactgcatatga






and comprises the amino acid sequence (signal sequence underlined):








(SEQ ID NO: 36)


MEWSWVFLFFLSVTTGVHSARNGDDCPLGPGRCCRLHTVRASLEDLGWAD


 


WVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPA


 


SYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI






II.A.3 Mature GDF15(N3Q) Variant

The GDF15(N3Q) variant is a human GDF15 mutant with Asn at residue 3 of the mature peptide (SEQ ID NO:12) replaced by Gln to avoid potential N deamidation.
The construct designated “mature GDF15(N3Q) dimer” in the instant disclosure refers to a homodimer comprising two mature GDF15(N3Q) monomers, each of which comprises SEQ ID NO:42. The monomer that homodimerizes to form the mature GDF15(N3Q) dimer is encoded by the nucleic acid sequence:







(SEQ ID NO: 41)


gcgcgccagggagaccactgtccgctcgggcccgggcgttgct


 


gccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgat


 


tgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtg


 


cccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcc


 


tgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgcc


 


agctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgct


 


ccagacctatgatgacttgttagccaaagactgccactgcata






and comprises the amino acid sequence:










SEQ ID NO: 42)



ARQGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQ



 



VTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPA



 



SYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI






In an embodiment employing the VH21 signal sequence, the monomer that homodimerizes to form the native mature GDF15 dimer is encoded by the nucleic acid sequence (including the cleaved VH21 signal sequence, which is underlined):







(SEQ ID NO: 39)


atggaatggagctgggtctttctcttcttcctgtcagtaacgaccggtgt


 


ccactccgcgcgccagggagaccactgtccgctcgggcccgggcgttgct


 


gccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgat


 


tgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtg


 


cccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcc


 


tgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgcc


 


agctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgct


 


ccagacctatgatgacttgttagccaaagactgccactgcata






and comprises the amino acid sequence (cleaved VH21 signal sequence underlined):








SEQ ID NO: 40)


MEWSWVFLFFLSVTTGVHSARQGDHCPLGPGRCCRLHTVRASLEDLGWAD


 


WVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPA


 


SYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.






Thus, the “mature GDF15(N3Q) dimer” comprises two covalently associated monomers comprising SEQ ID NO:42.
II.B. Charged Pair (delHinge) Constructs
Constructs designated “charged pair (delHinge)” or “charged pair (delHinge) Fc” in the instant disclosure refer to a construct comprising (i) a “negatively charged” Fc sequence lacking the hinge region and comprising a charged pair mutation and (ii) a “positively charged” Fc sequence lacking the hinge region and comprising a charged pair mutation. Note that use of the terms “positively charged” and “negatively charged” is for ease of reference (i.e., to describe the nature of the charge pair mutations in the Fc sequences) and not to indicate that the overall sequence or construct necessary has a positive or negative charge.
The introduction of an aspartatic acid-to-lysine mutation (E356K) and a glutamic acid-to-lysine mutation (D399K) into the unmodified Fc sequence lacking the hinge region provides the positively charged Fc sequence lacking the hinge region (referred to herein as “DhCpmFc(+)”). The introduction of two lysine-to-aspartate mutations (K392D, K409D) into the unmodified Fc sequence lacking the hinge region provides the negatively charged Fc sequence lacking the hinge region (referred to herein as “DhCpmFc(−)”). The C-terminal lysine (K477) optionally also may be deleted in the negatively charged DhCpmFc(−) sequence, the positively charged DhCpmFc(+) sequence, or both. See, e.g., SEQ ID NOs: 18, 19, 85, 86, 89, 90, 91, 99, 100, 108, 109 and 111 (DhCpmFc sequences).
When incubated together, the aspartate residues associate with the lysine residues through electrostatic force, facilitating formation of Fc heterodimers between the DhCpmFc(+) and DhCpmFc(−) sequences, and reducing or preventing formation of Fc homodimers between the DhCpmFc(+) sequences or between DhCpmFc(−) sequences.
In some embodiments a heterodimer comprises (i) a mature GDF15 sequence linked directly or via a linker to the C-terminus of a DhCpmFc(−) and (ii) a DhCpmFc(+). In other embodiments the heterodimer comprises (i) a mature GDF15 sequence linked directly or via a linker to the C-terminus of a DhCpmFc(+) and (ii) a DhCpmFc(−). In either event, two such heterodimers associate to form tetramer in which the heterodimers are linked via an interchain disulfide bond between the two GDF15 sequences. See FIG. 1 for a graphic depiction of a tetramer comprising two heterodimers linked via an interchain disulfide bond between the two human GDF15 sequences, in which each heterodimer is the charged pair (delHinge) construct designated “DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(−)” in the instant disclosure (i.e., where each heterodimer comprises (i) a first monomer comprising a mature GDF15 polypeptide linked via a (G4S)4 (SEQ ID NO:20) linker to the C-terminus of a DhCpmFc(−) sequence and (ii) a second monomer comprising a DhCpmFc(+) sequence).
II.B.1 DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+)
The charged pair (delHinge) construct designated “DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15 polypeptide linked via a (G4S)4 (SEQ ID NO:20) linker to the C-terminus of a DhCpmFc(−) sequence and (ii) a second monomer comprising a DhCpmFc(+) sequence. The negatively charged DhCpmFc(−)-(G4S)4-hGDF15 chain associates with the negatively charged DhCpmFc(−) chain to form the heterodimer. Two such heterodimers associate to form a tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15 sequences.
More particularly, in a specific embodiment, the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc sequence comprising the sequence:







(SEQ ID NO: 18)


APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD


 


GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA


 


PIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPGK,






(b) two chains (one each heterodimer) of an engineered negatively charged Fc sequence comprising the sequence:







(SEQ ID NO: 19)


APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD


 


GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA


 


PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPG,






and

(c) two chains (one each heterodimer) of a native mature human GDF15 polypeptide comprising SEQ ID NO:12.
The GDF15 polypeptide is fused via a linker comprising the sequence:









(SEQ ID NO: 20)



GGGGSGGGGSGGGGSGGGGS






at the N-terminus of the GDF15 polypeptide via peptide bond to the negatively charged Fc sequence.

In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer, is encoded by the nucleic acid sequence:







(SEQ ID NO: 43)


gcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (linker double underlined):








(SEQ ID NO: 44)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI.






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 21)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (signal sequence single underlined, linker sequence double underlined):








(SEQ ID NO: 22)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI.






The second monomer comprising the heterodimer is encoded by the nucleic acid sequence:







(SEQ ID NO: 46)


gcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc


 


ctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtaaatga






and comprises the amino acid sequence of SEQ ID NO:18.

In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 17)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc


 


ctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtaaatga






and comprises the amino acid sequence (signal sequence underlined):








(SEQ ID NO: 45)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.






The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct) comprises two monomers comprising SEQ ID NO:18 and two monomers comprising SEQ ID NO:44.
II.B.2 DhCpmFc (+)-(G4S)4-GDF15:DhCpmFc(−)
The charged pair (delHinge) construct designated “DhCpmFc(+)-(G4S)4-GDF15:DhCpmFc(−)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15 polypeptide linked via a (G4S)4 (SEQ ID NO:20) linker to the C-terminus of a DhCpmFc(+) sequence and (ii) a second monomer comprising a DhCpmFc(−) sequence. The positively charged DhCpmFc(+)-(G4S)4-hGDF15 chain associates with the negatively charged DhCpmFc(−) chain to form the heterodimer. Two such heterodimers associate to form tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15 sequences.
More particularly, in a specific embodiment, the DhCpmFc(+)-(G4S)4-GDF15:DhCpmFc(−) tetramer comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc sequence comprising the sequence:







(SEQ ID NO: 85)


APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD


 


GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA


 


PIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPG






(b) two chains (one each heterodimer) of an engineered negatively charged Fc sequence comprising the sequence







(SEQ ID NO: 86)


APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD


 


GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA


 


PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPGK,






and

(c) two chains (one each heterodimer) of a native mature human GDF15 polypeptide comprising SEQ ID NO:12.
The GDF15 polypeptide is fused via a linker comprising SEQ ID NO:20 at the N-terminus of the GDF15 polypeptide via peptide bond to the positively charged Fc sequence.
In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer, is encoded by the nucleic acid sequence:







(SEQ ID NO: 49)


gcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc


 


ctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (linker double underlined):








(SEQ ID NO: 50)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI.






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 47)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc


 


ctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (signal sequence single underlined, linker sequence double underlined):








(SEQ ID NO: 48)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI.






The second monomer comprising the heterodimer is encoded by the nucleic acid sequence:







(SEQ ID NO: 53)


gcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtaaatga






and comprises the amino acid sequence of SEQ ID NO:86.

In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 51)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtaaatga






and comprises the amino acid sequence (signal sequence underlined):








(SEQ ID NO: 52)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.






The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(+)-(G4S)4-GDF15:DhCpmFc(−) construct) comprises two monomers comprising SEQ ID NO:86 and two monomers comprising SEQ ID NO:50.
II.B.3. DhCpmFc(−)-(G4S)4-GDF15(H6D):DhCpmFc(+)
The charged pair (delHinge) construct designated “DhCpmFc(−)-(G4S)4-GDF15(H6D):DhCpmFc(+)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15(H6D) polypeptide linked via a (G4S)4 (SEQ ID NO:20) linker to the C-terminus of a DhCpmFc(−) sequence and (ii) a second monomer comprising a DhCpmFc(+) sequence. The negatively charged DhCpmFc(−)-(G4S)4-GDF15(H6D) chain associates with the positively charged DhCpmFc(+) chain. Two such heterodimers associate to form a tetramer in which the heterodimers linked via an interchain disulfide bond between the two human GDF15(H6D) sequences.
More particularly, in a specific embodiment, the DhCpmFc(−)-(G4S)4-GDF15(H6D):DhCpmFc(+) tetramer comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc sequence comprising SEQ ID NO:18,
(b) two chains (one each heterodimer) of an engineered negatively charged Fc sequence comprising SEQ ID NO:19, and
(c) two chains (one each heterodimer) of a mature human GDF15(H6D) polypeptide comprising SEQ ID NO:38.
The GDF15(H6D) polypeptide is fused via a linker comprising SEQ ID NO:20 at the N-terminus of the GDF15(H6D) polypeptide via peptide bond to the negatively charged Fc sequence.
In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer, is encoded by the nucleic acid sequence:







(SEQ ID NO: 56)


gcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgcaacggagacgactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (linker double underlined):








(SEQ ID NO: 57)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARNGDDCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 54)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgcaacggagacgactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (signal sequence underlined, linker double underlined):








(SEQ ID NO: 55)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARNGDDCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI






The second monomer comprising the heterodimer is encoded by the nucleic acid sequence of SEQ ID NO:46 and comprises the amino acid sequence of SEQ ID NO:18.
In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence of SEQ ID NO:17 and comprises the amino acid sequence of SEQ ID NO: 45.
The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(−)-(G4S)4-GDF15(H6D):DhCpmFc(+) construct) comprises two monomers comprising SEQ ID NO:18 and two monomers comprising SEQ ID NO:57.
II.B.4. DhCpmFc(+)-(G4S)4-GDF15(H6D):DhCpmFc(−)
The charged pair (delHinge) construct designated “DhCpmFc(+)-(G4S)4-GDF15(H6D):DhCpmFc(−)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15(H6D) polypeptide linked via a (G4S)4 (SEQ ID NO:20) linker to the C-terminus of a DhCpmFc(+) sequence and (ii) a second monomer comprising a DhCpmFc(−) sequence. The positively charged DhCpmFc(+)-(G4S)4-GDF15(H6D) chain associates with the negatively charged DhCpmFc(−) chain. Two such heterodimers associate to form a tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15(H6D) sequences.
More particularly, in a specific embodiment, the DhCpmFc(+)-(G4S)4-GDF15(H6D):DhCpmFc(−) tetramer comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc sequence comprising SEQ ID NO:85,
(b) two chains (one each heterodimer) of an engineered negatively charged Fc sequence comprising SEQ ID NO:86, and
(c) two chains (one each heterodimer) of a mature human GDF15(H6D) polypeptide comprising SEQ ID NO:38.
The GDF15(H6D) polypeptide is fused via a linker comprising SEQ ID NO:20 at the N-terminus of the GDF15(H6D) polypeptide via peptide bond to the positively charged Fc sequence.
In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer, is encoded by the nucleic acid sequence:







(SEQ ID NO: 60)


gcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc


 


ctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgcaacggagacgactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (linker double underlined):








(SEQ ID NO: 61)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARNGDDCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI.






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 58)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc


 


ctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgcaacggagacgactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (signal sequence single underlined, linker double underlined):








(SEQ ID NO: 59)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARNGDDCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI.






The second monomer comprising the heterodimer is encoded by the nucleic acid of SEQ ID NO:53 and comprises the amino acid sequence of SEQ ID NO:86.
In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence of SEQ ID NO:51 and comprises the amino acid sequence of SEQ ID NO:52.
The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(+)-(G4S)4-GDF15(H6D):DhCpmFc(−) construct) comprises two monomers comprising SEQ ID NO:86 and two monomers comprising SEQ ID NO:61.
II.B.5. DhCpmFc (+)-(G4S)4-GDF15(N3Q):DhCpmFc(−)
The charged pair (delHinge) construct designated “DhCpmFc(+)-(G4S)4-GDF15(N3Q):DhCpmFc(−)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15(N3Q) polypeptide linked via a (G4S)4 (SEQ ID NO:20) linker to the C-terminus of a DhCpmFc(+)”) sequence and (ii) a second monomer comprising a DhCpmFc(−) sequence. The positively charged DhCpmFc(+)-(G4S)4-GDF15(N3Q) chain then associates with the negatively charged DhCpmFc(−) chain. Two such heterodimers associate to form a tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15(N3Q) sequences.
More particularly, in a specific embodiment, the DhCpmFc(+)-(G4S)4-GDF15(N3Q):DhCpmFc(−) tetramer comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc sequence comprising SEQ ID NO:85
(b) two chains (one each heterodimer) of an engineered negatively charged Fc sequence comprising SEQ ID NO:86, and
(c) two chains (one each heterodimer) of a mature human GDF15(N3Q) polypeptide comprising SEQ ID NO:42.
The GDF15(N3Q) polypeptide is fused via a linker comprising SEQ ID NO:20 at the N-terminus of the GDF15(N3Q) polypeptide via peptide bond to the positively charged Fc sequence.
In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer, is encoded by the nucleic acid sequence:







(SEQ ID NO: 64)


gcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc


 


ctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgccagggagaccactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (linker sequence double underlined):








(SEQ ID NO: 65)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARQGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 62)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc


 


ctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatct


 


ggaggcggtggaagcgcgcgccagggagaccactgtccgctcgggcccgg


 


gcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggct


 


gggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatc


 


ggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaa


 


gacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcg


 


tgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggg


 


gtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat


 


atga






and comprises the amino acid sequence (signal sequence single underlined, linker sequence double underlined):








(SEQ ID NO: 63)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS


 


GGGGSARQGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI






The second monomer comprising the heterodimer is encoded by the nucleic acid of SEQ ID NO: 53 and comprises the amino acid sequence of SEQ ID NO:86.
In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence of SEQ ID NO:51 and comprises the amino acid sequence of SEQ ID NO:52.
The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(+)-(G4S)4-GDF15(N3Q):DhCpmFc(−) construct) comprises two monomers comprising SEQ ID NO:86 and two monomers comprising SEQ ID NO:65.
II.B.6. DhCpmFc(+)-GDF15:DhCpmFc(−)
The charged pair (delHinge) construct designated “DhCpmFc(+)-GDF15:DhCpmFc(−)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a one mature human GDF15 polypeptide linked to the C-terminus of a DhCpmFc(+) sequence and (ii) a second monomer comprising a DhCpmFc(−) sequence. The positively charged DhCpmFc(+)-GDF15 chain associates with the negatively charged DhCpmFc(−) chain to form the heterodimer. Two such heterodimers associate to form a tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15 sequences.
More particularly, in a specific embodiment, the DhCpmFc(+)-GDF15:DhCpmFc(−) tetramer comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc sequence comprising SEQ ID NO:85
(b) two chains (one each heterodimer) of an engineered negatively charged Fc sequence comprising SEQ ID NO:86, and
(c) two chains (one each heterodimer) of a native mature human GDF15 polypeptide comprising SEQ ID NO:12.
The GDF15 polypeptide is fused at the N-terminus of the GDF15 polypeptide via peptide bond to the positively charged Fc sequence.
In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer, is encoded by the nucleic acid sequence:







(SEQ ID NO: 68)


gccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacaccctgcctccgagcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggagcacggaacggggaccattgtcccctgggacctggtcggtgctgc


 


cggcttcacaccgtcagagcctctctggaggaccttggatgggctgattg


 


ggtgctgagccctcgggaggtgcaagtcaccatgtgcatcggggcctgcc


 


ctagccagttccgcgcagccaacatgcacgctcagatcaaaacctctctt


 


cacagactgaagcccgacaccgtgccagcaccttgctgtgtgccggcctc


 


ttataaccccatggtcctcattcagaaaaccgacaccggagtgtcacttc


 


agacttacgatgacctcctggccaaggactgccactgtatttga






and comprises the amino acid sequence:








(SEQ ID NO: 69)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCC


 


RLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSL


 


HRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 66)


atggagtggtcttgggtctttctgttcttcctctccgtcaccaccggtgt


 


gcattctgccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacaccctgcctccgagcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggagcacggaacggggaccattgtcccctgggacctggtcggtgctgc


 


cggcttcacaccgtcagagcctctctggaggaccttggatgggctgattg


 


ggtgctgagccctcgggaggtgcaagtcaccatgtgcatcggggcctgcc


 


ctagccagttccgcgcagccaacatgcacgctcagatcaaaacctctctt


 


cacagactgaagcccgacaccgtgccagcaccttgctgtgtgccggcctc


 


ttataaccccatggtcctcattcagaaaaccgacaccggagtgtcacttc


 


agacttacgatgacctcctggccaaggactgccactgtatttga






and comprises the amino acid sequence (signal sequence underlined,):








(SEQ ID NO: 67)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCC


 


RLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSL


 


HRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI.






The second monomer comprising the heterodimer is encoded by the nucleic acid of SEQ ID NO:53 and comprises the amino acid sequence of SEQ ID NO:86.
In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence of SEQ ID NO:51 and comprises the amino acid sequence of SEQ ID NO:52. The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(+)-GDF15:DhCpmFc(−) construct) comprises two monomers comprising SEQ ID NO:19 and two monomers comprising SEQ ID NO:69.
II.B.7. DhCpmFc(+)-G4-GDF15:DhCpmFc(−)
The charged pair (delHinge) construct designated “DhCpmFc(+)-G4-GDF15:DhCpmFc(−)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15 polypeptide linked via G4 (SEQ ID NO:70) linker to the C-terminus of a DhCpmFc(+) sequence and (ii) a second monomer comprising aDhCpmFc(−) sequence. The positively charged DhCpmFc(+)-G4-GDF15 chain associates with the negatively charged DhCpmFc(−) chain. Two such heterodimers associate to form a tetramer in which the heterodimers linked via an interchain disulfide bond between the two human GDF15 sequences.
More particularly, in a specific embodiment, the DhCpmFc(+)-G4-GDF15:DhCpmFc(−) tetramer comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc sequence comprising SEQ ID NO:85,
(b) two chains (one each heterodimer) of an engineered negatively charged Fc sequence comprising SEQ ID NO:86, and
(c) two chains (one each heterodimer) of a native mature human GDF15 polypeptide comprising SEQ ID NO:12.
The GDF15 polypeptide is fused via a linker comprising the sequence









(SEQ ID NO: 70)



GGGG






at the N-terminus of the GDF15 polypeptide via peptide bond to the positively charged Fc sequence.

In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer, is encoded by the nucleic acid sequence:







(SEQ ID NO: 73)


gccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacaccctgcctccgagcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggcggaggtggaggagcacggaacggggaccattgtcccctgggacct


 


ggtcggtgctgccggcttcacaccgtcagagcctctctggaggaccttgg


 


atgggctgattgggtgctgagccctcgggaggtgcaagtcaccatgtgca


 


tcggggcctgccctagccagttccgcgcagccaacatgcacgctcagatc


 


aaaacctctcttcacagactgaagcccgacaccgtgccagcaccttgctg


 


tgtgccggcctcttataaccccatggtcctcattcagaaaaccgacaccg


 


gagtgtcacttcagacttacgatgacctcctggccaaggactgccactgt


 


atttga






and comprises the amino acid sequence (linker sequence double underlined):








(SEQ ID NO: 74)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARNGDHCPLGP


 


GRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQI


 


KTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHC


 


I.






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (cleaved signal sequence underlined):







(SEQ ID NO: 71)


atggagtggtcttgggtctttctgttcttcctctccgtcaccaccggtgt


 


gcattctgccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacaccctgcctccgagcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggcggaggtggaggagcacggaacggggaccattgtcccctgggacct


 


ggtcggtgctgccggcttcacaccgtcagagcctctctggaggaccttgg


 


atgggctgattgggtgctgagccctcgggaggtgcaagtcaccatgtgca


 


tcggggcctgccctagccagttccgcgcagccaacatgcacgctcagatc


 


aaaacctctcttcacagactgaagcccgacaccgtgccagcaccttgctg


 


tgtgccggcctcttataaccccatggtcctcattcagaaaaccgacaccg


 


gagtgtcacttcagacttacgatgacctcctggccaaggactgccactgt


 


atttga






and comprises the amino acid sequence (signal sequence single underlined, linker sequence double underlined):








(SEQ ID NO: 72)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARNGDHCPLGP


 


GRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQI


 


KTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHC


 


I.






The second monomer comprising the heterodimer is encoded by the nucleic acid of SEQ ID NO:53 and comprises the amino acid sequence of SEQ ID NO:86.
In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence of SEQ ID NO:51 and comprises the amino acid sequence of SEQ ID NO:52.
The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(+)-G4-GDF15:DhCpmFc(−) construct) comprises two monomers comprising SEQ ID NO:86 and two monomers comprising SEQ ID NO:74.
II.B.8. DhCpmFc (+)-(G4S)2-GDF15:DhCpmFc(−)
The charged pair (delHinge) construct designated “DhCpmFc(+)-(G4S)2-GDF15:DhCpmFc(−)” in the instant disclosure refers to a construct comprising heterodimer, which comprises (i) a first monomer comprising a mature human GDF15 polypeptide linked via a (G4S)2 (SEQ ID NO:75) linker to the C-terminus of a DhCpmFc(+) sequence and (ii) a second monomer comprising aDhCpmFc(−) sequence. The positively charged DhCpmFc(+)-(G4S)2-GDF15 chain associates with the negatively charged DhCpmFc(−) chain. Two such heterodimers associate to form a tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15 sequences.
More particularly, in a specific embodiment, the DhCpmFc(+)-(G4S)2-GDF15:DhCpmFc(−) tetramer comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc sequence comprising SEQ ID NO:85,
(b) two chains (one each heterodimer) of an engineered negatively charged Fc sequence comprising SEQ ID NO:86 and
(c) two chains (one each heterodimer) of a native mature human GDF15 polypeptide comprising SEQ ID NO:12.
The GDF15 polypeptide is fused via a linker comprising the sequence









(SEQ ID NO: 75)



GGGGSGGGGS






at the N-terminus of the GDF15 polypeptide via peptide bond to the positively charged Fc.

In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer, is encoded by the nucleic acid sequence:







(SEQ ID NO: 78)


gcgccggaactgctgggcggcccgagcgtgtttctgtttccgc


 


cgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgc


 


gtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggta


 


tgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaac


 


agtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcag


 


gattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgct


 


gccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcg


 


aaccgcaggtgtataccctgccgccgagccgcaaagaaatgaccaaaaac


 


caggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgc


 


ggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccc


 


cgccggtgctgaaaagcgatggcagcttttttctgtatagcaaactgacc


 


gtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgat


 


gcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcc


 


cgggcggcggcggcggcagcggcggcggcggcagcgcgcgcaacggcgat


 


cattgcccgctgggcccgggccgctgctgccgcctgcataccgtgcgcgc


 


gagcctggaagatctgggctgggcggattgggtgctgagcccgcgcgaag


 


tgcaggtgaccatgtgcattggcgcgtgcccgagccagtttcgcgcggcg


 


aacatgcatgcgcagattaaaaccagcctgcatcgcctgaaaccggatac


 


cgtgccggcgccgtgctgcgtgccggcgagctataacccgatggtgctga


 


ttcagaaaaccgataccggcgtgagcctgcagacctatgatgatctgctg


 


gcgaaagattgccattgcatttga






and comprises the amino acid sequence (linker sequence double underlined):








(SEQ ID NO: 79)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSARNGD


 


HCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAA


 


NMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLL


 


AKDCHCI.






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 76)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcgccggaactgctgggcggcccgagcgtgtttctgtttccgc


 


cgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgc


 


gtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggta


 


tgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaac


 


agtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcag


 


gattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgct


 


gccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcg


 


aaccgcaggtgtataccctgccgccgagccgcaaagaaatgaccaaaaac


 


caggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgc


 


ggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccc


 


cgccggtgctgaaaagcgatggcagcttttttctgtatagcaaactgacc


 


gtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgat


 


gcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcc


 


cgggcggcggcggcggcagcggcggcggcggcagcgcgcgcaacggcgat


 


cattgcccgctgggcccgggccgctgctgccgcctgcataccgtgcgcgc


 


gagcctggaagatctgggctgggcggattgggtgctgagcccgcgcgaag


 


tgcaggtgaccatgtgcattggcgcgtgcccgagccagtttcgcgcggcg


 


aacatgcatgcgcagattaaaaccagcctgcatcgcctgaaaccggatac


 


cgtgccggcgccgtgctgcgtgccggcgagctataacccgatggtgctga


 


ttcagaaaaccgataccggcgtgagcctgcagacctatgatgatctgctg


 


gcgaaagattgccattgcatttga






and comprises the amino acid sequence (signal sequence single underlined, linker sequence double underlined):








(SEQ ID NO: 77)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSARNGD


 


HCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAA


 


NMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLL


 


AKDCHCI.






The second monomer comprising the heterodimer is encoded by the nucleic acid of SEQ ID NO:53 and comprises the amino acid sequence of SEQ ID NO:86.
In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence of SEQ ID NO:51 and comprises the amino acid sequence of SEQ ID NO:52.
The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(+)-(G4S)2-GDF15:DhCpmFc(−) construct) comprises two monomers comprising SEQ ID NO:86 and two monomers comprising SEQ ID NO:79.
II.B.9. DhCpmFc(+)-(G4Q)2-GDF15:DhCpmFc(−)
The charged pair (delHinge) construct designated “DhCpmFc(+)-(G4Q)4-GDF15:DhCpmFc(−)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15 polypeptide linked via a (G4Q)4 (SEQ ID NO:80) linker to the C-terminus of a DhCpmFc(+) sequence and (ii) a second monomer comprising a DhCpmFc(−) sequence. The positively charged DhCpmFc(+)-(G4Q)4-GDF15 chain associates with the negatively charged DhCpmFc(−) chain. Two such heterodimers associate to form a tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15 sequences.
More particularly, in a specific embodiment, the DhCpmFc(+)-(G4Q)4-GDF15:DhCpmFc(−) tetramer comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc sequence comprising SEQ ID NO:85,
(b) two chains (one each heterodimer) of an engineered negatively charged Fc sequence comprising SEQ ID NO:86, and
(c) two chains (one each heterodimer) of a native mature human GDF15 polypeptide comprising SEQ ID NO:12.
The GDF15 polypeptide is fused via a linker comprising the sequence









(SEQ ID NO: 80)



GGGGQGGGGQGGGGQGGGGQ






at the N-terminus of the GDF15 polypeptide via peptide bond to the positively charged Fc.

In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer is encoded by the nucleic acid sequence:







(SEQ ID NO: 83)


gccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacaccctgcctccgagcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggaggtggcggagggcagggtggtggaggtcagggaggcggaggacag


 


ggaggaggtggacaagcacggaacggggaccattgtcccctgggacctgg


 


tcggtgctgccggcttcacaccgtcagagcctctctggaggaccttggat


 


gggctgattgggtgctgagccctcgggaggtgcaagtcaccatgtgcatc


 


ggggcctgccctagccagttccgcgcagccaacatgcacgctcagatcaa


 


aacctctcttcacagactgaagcccgacaccgtgccagcaccttgctgtg


 


tgccggcctcttataaccccatggtcctcattcagaaaaccgacaccgga


 


gtgtcacttcagacttacgatgacctcctggccaaggactgccactgtat


 


ttga






and comprises the amino acid sequence (linker sequence double underlined):








(SEQ ID NO: 84)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGQGGGGQGGGGQ


 


GGGGQARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI.






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 81)


atggagtggtcttgggtctttctgttcttcctctccgtcaccaccggtgt


 


gcattctgccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacaccctgcctccgagcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggaggtggcggagggcagggtggtggaggtcagggaggcggaggacag


 


ggaggaggtggacaagcacggaacggggaccattgtcccctgggacctgg


 


tcggtgctgccggcttcacaccgtcagagcctctctggaggaccttggat


 


gggctgattgggtgctgagccctcgggaggtgcaagtcaccatgtgcatc


 


ggggcctgccctagccagttccgcgcagccaacatgcacgctcagatcaa


 


aacctctcttcacagactgaagcccgacaccgtgccagcaccttgctgtg


 


tgccggcctcttataaccccatggtcctcattcagaaaaccgacaccgga


 


gtgtcacttcagacttacgatgacctcctggccaaggactgccactgtat


 


ttga






and comprises the amino acid sequence (signal sequence single underlined, linker sequence double underlined):








(SEQ ID NO: 82)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGQGGGGQGGGGQ


 


GGGGQARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI


 


GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG


 


VSLQTYDDLLAKDCHCI.






The second monomer comprising the heterodimer is encoded by the nucleic acid of SEQ ID NO:53 and comprises the amino acid sequence of SEQ ID NO:86.
In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence of SEQ ID NO:51 and comprises the amino acid sequence of SEQ ID NO:52.
The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(+)-(G4Q)4-GDF15:DhCpmFc(−) construct) comprises two monomers comprising SEQ ID NO:86 and two monomers comprising SEQ ID NO:84.
II.B.10. DhCpmFc(+)(L351C)-G4-GDF15:DhCpmFc(−)(L351C)
The charged pair (delHinge) construct designated “DhCpmFc(+)(L351C)-G4-GDF15:DhCpmFc(−)(L351C)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15 polypeptide linked via a G4 (SEQ ID ID:70) linker to the C-terminus of a positively charged Fc monomer lacking the hinge region (referred to herein as “DhCpmFc(+)(L351C)”) and one negatively charged Fc monomer lacking the hinge region (referred to herein as “DhCpmFc(−)(L351C)”).
As discussed above, the introduction of an aspartatic acid-to-lysine mutation (E356K) and a glutamic acid-to-lysine mutation (D399K) in the unmodified Fc sequence lacking the hinge region provides the positively charged DhCpmFc(+) sequence. The introduction of two lysine-to-aspartate mutations (K392D, K409D) provides the negatively charged DhCpmFc(−) sequence. The C-terminal lysine (K477) optionally may also deleted in the negatively charged DhCpmFc(−) sequence, the positively charged DhCpmFc(+) sequence, or both. When incubated together, the aspartate residues associate with the lysine residues through electrostatic force, facilitating formation of Fc heterodimers between the DhCpmFc(+) and DhCpmFc(−) sequences, and reducing or preventing formation of Fc homodimers between DhCpmFc(+) sequences or between DhCpmFc(−) sequences.
Introduction of a leucine-to-cysteine mutation (L351C) in the positively charged DhCpmFc(+) sequence (referred to herein as “DhCpmFc(+)(L351C)”) and a leucine-to-cysteine mutation (L351C) in the negatively charged DhCpmFc(−) sequence (referred to herein as “DhCpmFc(−)(L351C)”) further enhances the formation of Fc heterodimers between the DhCpmFc(+)(L351C) and DhCpmFc(−) (L351C) sequences by the formation of a disulfide bond (or “cysteine clamp”) between them. The positively charged DhCpmFc(+)(L351C)-G4-GDF15 chain associates with the negatively charged DhCpmFc(−)(L351C) chain. Two such heterodimers associate to form a tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15 sequences.
More particularly, in a specific embodiment, the DhCpmFc(+)(L351C)-G4-GDF15:DhCpmFc(−)(L351C) tetramer comprises:
(a) two chains (one each heterodimer) of an engineered positively charged Fc(L351) sequence comprising:







(SEQ ID NO: 90


APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD


 


GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA


 


PIEKTISKAKGQPREPQVYTCPPSRKEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPG






(b) two chains (one each heterodimer) of an engineered negatively charged Fc(L351) sequence:







(SEQ ID NO: 91)


APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD


 


GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA


 


PIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPGK






and.

(c) two chains (one each heterodimer) of a native mature human GDF15 polypeptide comprising SEQ ID NO:12.
The GDF15 polypeptide is fused via a linker comprising SEQ ID NO:70 at the N-terminus of the GDF15 polypeptide via peptide bond to the positively charged Fc(L351C) sequence.
In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer is encoded by the nucleic acid sequence:







(SEQ ID NO: 94)


gccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacacctgtcctccgagcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggcggaggtggaggagcacggaacggggaccattgtcccctgggacct


 


ggtcggtgctgccggcttcacaccgtcagagcctctctggaggaccttgg


 


atgggctgattgggtgctgagccctcgggaggtgcaagtcaccatgtgca


 


tcggggcctgccctagccagttccgcgcagccaacatgcacgctcagatc


 


aaaacctctcttcacagactgaagcccgacaccgtgccagcaccttgctg


 


tgtgccggcctcttataaccccatggtcctcattcagaaaaccgacaccg


 


gagtgtcacttcagacttacgatgacctcctggccaaggactgccactgc


 


atatga






and comprises the amino acid sequence (linker sequence double underlined):








(SEQ ID NO: 95)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARNGDHCPLGP


 


GRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQI


 


KTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHC


 


I.






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 92)


atggaatggagctgggtctttctgttcttcctctccgtcaccaccggtgt


 


gcattctgccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacacctgtcctccgagcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggcggaggtggaggagcacggaacggggaccattgtcccctgggacct


 


ggtcggtgctgccggcttcacaccgtcagagcctctctggaggaccttgg


 


atgggctgattgggtgctgagccctcgggaggtgcaagtcaccatgtgca


 


tcggggcctgccctagccagttccgcgcagccaacatgcacgctcagatc


 


aaaacctctcttcacagactgaagcccgacaccgtgccagcaccttgctg


 


tgtgccggcctcttataaccccatggtcctcattcagaaaaccgacaccg


 


gagtgtcacttcagacttacgatgacctcctggccaaggactgccactgc


 


atatga






and comprises the amino acid sequence (signal sequence single underlined, linker sequence double underlined):








(SEQ ID NO: 93)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARNGDHCPLGP


 


GRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQI


 


KTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHC


 


I.






The second monomer comprising the heterodimer is encoded by the nucleic acid sequence:







(SEQ ID NO: 98)


gcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacacctgtcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtaaatga






and comprises the amino acid sequence of SEQ ID NO:91.

In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 96)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtacacctgtcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtaaatga






and comprises the amino acid sequence (signal sequence underlined):








(SEQ ID NO: 97)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK






The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(+)(L351C)-G4-GDF15:DhCpmFc(−)(L351C) construct comprises two monomers comprising SEQ ID NO:91 and two monomers comprising SEQ ID NO:95.
II.B.11. DhCpmFc(+)(S354C)-G4-GDF15:DhCpmFc(−)(Y349C)
The charged pair (delHinge) construct designated “DhCpmFc(+)(S354C)-G4-GDF15:DhCpmFc(−)(Y349C)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15 polypeptide linked via a G4 (SEQ ID NO:70) linker to the C-terminus of a positively charged Fc monomer lacking the hinge region (referred to herein as “DhCpmFc(+)(S354C)”) and one negatively charged Fc monomer lacking the hinge region (referred to herein as “DhCpmFc(−)(Y349C)”).
As discussed above, the introduction of an aspartatic acid-to-lysine mutation (E356K) and a glutamic acid-to-lysine mutation (D399K) in the unmodified Fc sequence lacking the hinge region provides the positively charged DhCpmFc(+) sequence. The introduction of two lysine-to-aspartate mutations (K392D, K409D) provides the negatively charged DhCpmFc(−) sequence. The C-terminal lysine (K477) optionally may also deleted in the negatively charged DhCpmFc(−) sequence, the positively charged DhCpmFc(+) sequence, or both. When incubated together, the aspartate residues associate with the lysine residues through electrostatic force, facilitating formation of Fc heterodimers between the DhCpmFc(+) and DhCpmFc(−) sequences, and reducing or preventing formation of Fc homodimers between DhCpmFc(+) sequences or between DhCpmFc(−) sequences.
Introduction of a leucine-to-cysteine mutation (S354C) in the positively charged DhCpmFc(+) sequence (referred to herein as “DhCpmFc(+)(S354C)”) and a leucine-to-cysteine mutation (Y349C) in the negatively charged DhCpmFc(−) sequence (referred to herein as “DhCpmFc(−)(L351C)”) further enhances the formation of Fc heterodimers between the two DhCpmFc(+)(S354C) and DhCpmFc(−) (Y349C) sequences by the formation of a disulfide bond (or “cysteine clamp”) between them. The positively charged DhCpmFc(+)(S354C)-G4-GDF15 chain associates with the negatively charged DhCpmFc(−)(Y349C) chain. Two such heterodimers associate to form a tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15 sequences.
More particularly, in a specific embodiment, the DhCpmFc(+)(S354C)-G4-GDF15:DhCpmFc(−)(Y349C) tetramer comprises
(a) two chains (one each heterodimer) of an engineered positively charged Fc(S354C) comprising the sequence:







(SEQ ID NO: 99)


APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD


 


GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA


 


PIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPG,






(b) two chains (one each heterodimer) of an engineered negatively charged Fc(Y349C) comprising the sequence:







(SEQ ID NO: 100


APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD


 


GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA


 


PIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPGK






and

(c) two chains (one each) of a native mature human GDF15 polypeptide comprising SEQ ID NO:12.
The GDF15 polypeptide is fused via a linker comprising SEQ ID NO:70 at the N-terminus of the GDF15 polypeptide via peptide bond to the positively charged Fc(S354C) sequence.
In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer is encoded by the nucleic acid sequence:







(SEQ ID NO: 103)


gccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacaccctgcctccgtgcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggcggaggtggaggagcacggaacggggaccattgtcccctgggacct


 


ggtcggtgctgccggcttcacaccgtcagagcctctctggaggaccttgg


 


atgggctgattgggtgctgagccctcgggaggtgcaagtcaccatgtgca


 


tcggggcctgccctagccagttccgcgcagccaacatgcacgctcagatc


 


aaaacctctcttcacagactgaagcccgacaccgtgccagcaccttgctg


 


tgtgccggcctcttataaccccatggtcctcattcagaaaaccgacaccg


 


gagtgtcacttcagacttacgatgacctcctggccaaggactgccactgc


 


atatga






and comprises the amino acid sequence (linker sequence double underlined):








(SEQ ID NO: 104)


APELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARNGDHCPLGP


 


GRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQI


 


KTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHC


 


I






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 101)


atggaatggagctgggtctttctgttcttcctctccgtcaccaccggtgt


 


gcattctgccccagagctgcttggtggaccatccgtgttcctgtttcctc


 


caaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgc


 


gtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggta


 


tgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaac


 


agtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccag


 


gactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccct


 


ccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaaggg


 


aacctcaagtgtacaccctgcctccgtgcaggaaggagatgaccaagaac


 


caggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgc


 


tgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccc


 


cacccgtgctgaaatctgatgggtccttcttcctttactccaagctgacc


 


gtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgat


 


gcatgaggctctccataatcactacactcagaagtcactgtccctgtcac


 


ctggcggaggtggaggagcacggaacggggaccattgtcccctgggacct


 


ggtcggtgctgccggcttcacaccgtcagagcctctctggaggaccttgg


 


atgggctgattgggtgctgagccctcgggaggtgcaagtcaccatgtgca


 


tcggggcctgccctagccagttccgcgcagccaacatgcacgctcagatc


 


aaaacctctcttcacagactgaagcccgacaccgtgccagcaccttgctg


 


tgtgccggcctcttataaccccatggtcctcattcagaaaaccgacaccg


 


gagtgtcacttcagacttacgatgacctcctggccaaggactgccactgc


 


atatga






and comprises the amino acid sequence (signal sequence single underlined, linker sequence double underlined):








(SEQ ID NO: 102)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARNGDHCPLGP


 


GRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQI


 


KTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHC


 


I






The second monomer comprising the heterodimer is encoded by the nucleic acid sequence:







(SEQ ID NO: 107)


gcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtaaatga






and comprises the amino acid sequence of SEQ ID NO:100.

In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 105)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgcacctgaactcctggggggaccgtcagtcttcctcttccccc


 


caaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc


 


gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta


 


cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc


 


agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag


 


gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct


 


cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag


 


aaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaac


 


caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc


 


cgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgc


 


ctcccgtgctggactccgacggctccttcttcctctatagcgacctcacc


 


gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat


 


gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc


 


cgggtaaatga






and comprises the amino acid sequence (signal sequence underlined):








(SEQ ID NO: 106)


MEWSWVFLFFLSVTTGVHSAPELLGGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLT


 


VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK






The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the DhCpmFc(+)(S354C)-G4-GDF15:DhCpmFc(−)(Y349C) construct) comprises two monomers comprising SEQ ID NO:100 and two monomers comprising SEQ ID NO:104.
II.C. Charged Pair Construct
Constructs designated “charged pair” or “charged pair Fc” in the instant disclosure refer to a construct comprising a “negatively charged” Fc sequence comprising a charge pair mutation and (ii) a positively charged Fc sequence comprising a charged pair mutation. Note that use of the terms “positively charged” and “negatively charged” is for ease of reference (i.e., to describe the nature of the charge pair mutations in the Fc sequences) and not to indicate that the overall sequence or construct necessarily has a positive or negative charge.
The introduction of an aspartatic acid-to-lysine mutation (E356K) and a glutamic acid-to-lysine mutation (D399K) in the unmodified Fc sequence provides the positively charged Fc sequence (referred to herein as “CpmFc(+)”). The introduction of two lysine-to-aspartate mutations (K392D, K409D) into the unmodified Fc sequence provides the negatively charged Fc sequence (referred to herein as “CpmFc(−)”). The C-terminal lysine (K477) optionally also may be deleted in the negatively charged CpmFc(−) sequence, the positively charged CpmFc(+) sequence, or both. See, e.g., SEQ ID NOs: 23, 110, 114 and 115 (CpmFc sequences). When incubated together, the aspartate residues associate with the lysine residues, facilitating forming of Fc heterodimers between the CpmFc(+) and CpmFc(−) sequences, and reducing or preventing forming of Fc homodimers between the CpmFc(+) and CpmFc(−) sequences.
In some embodiments a heterodimer comprises (i) a mature GDF15 sequence linked directly or via a linker to the C-terminus of a CpmFc(−) and (ii) a CpmFc(+). In other embodiments the heterodimer comprises (i) a mature GDF15 sequence linked directly or via a linker to the C-terminus of a CpmFc(+) and (ii) a CpmFc(−). In either event, two such heterodimers associate to form tetramer in which the heterodimers are linked via an interchain disulfide bond between the two GDF15 sequences. See FIG. 1 for a graphic depiction of a tetramer comprising two heterodimers linked via an interchain disulfide bond between two human GDF15 sequences, in which each heterodimer is the charged pair construct designated “CpmFc(−)-(G4S)4-GDF15 CpmFc(−)” in the instant disclosure (i.e., where each heterodimer comprises (i) a first monomer comprising a mature GDF15 polypeptide linked via a (G4S)4 (SEQ ID NO:20) linker to the C-terminus of a CpmFc(−) sequence and (ii) a second monomer comprising a CpmFc(+) sequence).
CpmFc(−)-(G4S)4-GDF15:CpmFc(+)
The charged pair construct designated “CpmFc(−)-(G4S)4-GDF15:CpmFc(+)” in the instant disclosure refers to a construct comprising a heterodimer, which comprises (i) a first monomer comprising a mature human GDF15 polypeptide linked via a (G4S)4 (SEQ ID NO:20) linker to the C-terminus of a CpmFc(−) sequence and (ii) a second monomer comprising a CpmFc(+) sequence. Although the hinge region is present, the negatively charged CpmFc(−)-(G4S)4-hGDF15 chain associates with the negatively charged CpmFc(−) chain to form the heterodimer. Two such heterodimers associate to form a tetramer in which the heterodimers are linked via an interchain disulfide bond between the two human GDF15 sequences.
More particularly, in a specific embodiment, the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct comprises:
(a) two chains (one each heterodimer) of an engineered positively charge Fc sequence comprising the sequence (with the hinge region indicated by parentheses):







(SEQ ID NO: 110)


(DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH


 


EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE


 


YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQ


 


QGNVFSCSVMHEALHNHYTQKSLSLSPGK,






(b) two chains (one each) of an engineered negatively charged Fc sequence comprising the sequence (with the hinge region indicated by parentheses):







(SEQ ID NO: 23


(DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH


 


EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE


 


YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQ


 


QGNVFSCSVMHEALHNHYTQKSLSLSPG,






and

(c) two chains of a native mature human GDF15 polypeptide comprising SEQ ID NO:12.
The GDF15 polypeptide is fused via a linker comprising SEQ ID NO:20 at the N-terminus of the GDF15 polypeptide via peptide bond to the negatively charged Fc monomer.
In its final form, the first monomer comprising the heterodimer, that with another such heterodimer forms the tetramer, is encoded by the nucleic acid sequence:







(SEQ ID NO: 112)


gacaaaactcacacatgcccaccgtgcccagcacctgaactcc


 


tggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctc


 


atgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcca


 


cgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgc


 


ataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt


 


gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagga


 


gtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaa


 


ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg


 


cccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcct


 


ggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatg


 


ggcagccggagaacaactacgacaccacgcctcccgtgctggactccgac


 


ggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggca


 


gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacc


 


actacacgcagaagagcctctccctgtctccgggtggaggtggtggatcc


 


ggaggcggtggaagcggaggtggtggatctggaggcggtggaagcgcgcg


 


caacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcaca


 


cggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcg


 


ccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagtt


 


ccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctga


 


agcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatccc


 


atggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatga


 


tgacttgttagccaaagactgccactgcatatga






and comprises the amino acid sequence (hinge region indicated by parentheses and linker sequence double underlined):








(SEQ ID NO: 113)


(DKTHTCPPCP)APELLGGPSVFLFPPKPKD


 


TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST


 


YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY


 


TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLD


 


SDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGG


 


GSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWV


 


LSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY


 


NPMVLIQKTDTGVSLQTYDDLLAKDCHCI






In an embodiment employing the VH21 signal sequence, in its final form, the first monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 24)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgacaaaactcacacatgcccaccgtgcccagcacctgaactcc


 


tggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctc


 


atgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcca


 


cgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgc


 


ataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt


 


gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagga


 


gtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaa


 


ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg


 


cccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcct


 


ggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatg


 


ggcagccggagaacaactacgacaccacgcctcccgtgctggactccgac


 


ggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggca


 


gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacc


 


actacacgcagaagagcctctccctgtctccgggtggaggtggtggatcc


 


ggaggcggtggaagcggaggtggtggatctggaggcggtggaagcgcgcg


 


caacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcaca


 


cggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcg


 


ccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagtt


 


ccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctga


 


agcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatccc


 


atggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatga


 


tgacttgttagccaaagactgccactgcatatga






and comprises the amino acid sequence (signal sequence single underlined, hinge region in parentheses and linker sequence double underlined):








(SEQ ID NO: 25)


MEWSWVFLFFLSVTTGVHS(DKTHTCPPCP)APELLGGPSVFLFPPKPKD


 


TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST


 


YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY


 


TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLD


 


SDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGG


 


GSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWV


 


LSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY


 


NPMVLIQKTDTGVSLQTYDDLLAKDCHCI.






The second monomer comprising the heterodimer is encoded by the nucleic acid sequence:







(SEQ ID NO: 116)


gacaaaactcacacatgcccaccgtgcccagcacctgaactcc


 


tggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctc


 


atgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcca


 


cgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgc


 


ataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt


 


gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagga


 


gtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaa


 


ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg


 


cccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcct


 


ggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatg


 


ggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgac


 


ggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggca


 


gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacc


 


actacacgcagaagagcctctccctgtctccgggtaaatga






and comprises the amino acid sequence of SEQ ID NO:110.

In an embodiment employing the VH21 signal sequence, the second monomer comprising the heterodimer is encoded by the nucleic acid sequence (signal sequence underlined):







(SEQ ID NO: 26)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgacaaaactcacacatgcccaccgtgcccagcacctgaactcc


 


tggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctc


 


atgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcca


 


cgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgc


 


ataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt


 


gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagga


 


gtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaa


 


ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg


 


cccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcct


 


ggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatg


 


ggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgac


 


ggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggca


 


gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacc


 


actacacgcagaagagcctctccctgtctccgggtaaatga






and comprises the amino acid sequence (signal sequence underlined, hinge region in parentheses):








(SEQ ID NO: 27)


MEWSWVFLFFLSVTTGVHS(DKTHTCPPCP)APELLGGPSVFLFPPKPKD


 


TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST


 


YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY


 


TLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLK


 


SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.






The first and second monomers associate to form the heterodimer. Two such heterodimers associate to form the final tetramer. Accordingly, the resulting tetramer (a dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct) comprises two monomers comprising SEQ ID NO:110 and two monomers comprising SEQ ID NO:113.
II.D. HemiFc Constructs
Constructs designated “hemi” or “hemiFc” in the instant disclosure refer to a construct comprising two Fc sequences joined in tandem by a linker that connects the N-terminus of a first Fc sequence (e.g., SEQ ID NOs: 28 or 30) to the C-terminus of a second Fc sequence (e.g., SEQ ID NOs: 28 or 30). In some embodiments, a monomer comprises a mature GDF15 sequence linked to the first Fc sequence by a first linker that connects the N-terminus of the GDF15 sequence to the C-terminus of the first Fc sequence, wherein the first Fc sequence is linked to the second Fc sequence by a second linker that connects the N-terminus of the first Fc sequence to the C-terminus of the second Fc sequence. The first and second Fc sequences also are associated by the Fc hinge regions. Two such monomers associate to form a dimer in which the monomers are linked via an interchain disulfide bond between the two GDF15 sequences. See FIG. 1 for a graphic depiction of a hemi construct “Fc-(G4S)8-Fc-GS(G4S)4-GDF15” (i.e., two monomers, each comprising a mature GDF15 polypeptide linked via a GS(G4S)4 linker to the C-terminus of a first Fc sequence, the N-terminus of which is linked via a (G4S)8 linker to the C-terminus of a second Fc sequence; and where the two mature GDF15 sequences are linked via an interchain disulfide bond).
II.D.1 Fc-(G4S)8-Fc-GS(G4S)4-GDF15
The hemiFc construct designated “Fc-(G4S)8-Fc-GS(G4S)4-GDF15” in the instant disclosure refers to a construct comprising a monomer, which comprises a mature human GDF15 sequence linked to a first Fc sequence via a first linker comprising SEQ ID NO:31 that connects the N-terminus of the human GDF15 sequence to the C-terminus of the first Fc sequence and a second linker comprising the amino acid sequence:









(SEQ ID NO: 34)



GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.






that connects the N-terminus of the first Fc sequence to the C-terminus of the second Fc sequence. The first and second Fc sequences also are associated by the Fc hinge regions. Two such monomers associate to form a homodimer in which the monomers are linked via an interchain disulfide bond between the two GDF15 sequences.

More particularly, in a specific embodiment, the hemi construct Fc-(G4S)8-Fc-GS(G4S)4-GDF15 comprises a monomer comprising a second Fc chain comprising the sequence (hinge region in parentheses):







(SEQ ID NO: 28)


GGG(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV


 


DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL


 


NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS


 


LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK


 


SRWQQGNVFSCSVMHEALHNHYTQKSLSLSP






Joined by the linker:










(SEQ ID NO: 29)



GGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS






to a first Fc chain comprising the sequence (hinge region in parentheses):








(SEQ ID NO: 30)


(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS


 


HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK


 


EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC


 


LVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW


 


QQGNVFSCSVMHEALHNHYTQKSLSLSP






to the C-terminus of which, the GDF15 polypeptide comprising SEQ ID NO:12 is joined by a linker having the sequence:










(SEQ ID NO: 31)



GSGGGGSGGGGSGGGGSGGGGS






In its final form, the monomer is encoded by the nucleic acid sequence:







(SEQ ID NO: 32)


ggaggtggagagcgcaaatcttctgtcgagtgcccaccgtgcccagcacc


 


acctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggaca


 


ccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtg


 


agccacgaagaccccgaggtccagttcaactggtacgtggacggcgtgga


 


ggtgcataatgccaagacaaaaccacgggaggagcagttcaacagcacgt


 


tccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggc


 


aaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcga


 


gaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtaca


 


ccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacc


 


tgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagag


 


caatgggcagccggagaacaactacaagaccacacctcccatgctggact


 


ccgacggctccttcttcctctacagcaagctcaccgtggacaagagcagg


 


tggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgca


 


caaccactacacgcagaagagcctctccctgtctccgggtggaggtggcg


 


gtagcggtggcggaggttcaggtggtggcggttctggcggaggtggcagt


 


ggcggtggcggatcaggtggcggtggcagcggtggcggcggaagcggtgg


 


aggaggttcagagcggaaatccagcgttgaatgtcctccgtgccctgctc


 


cacccgtcgcggggcctagtgtcttccttttccctccaaaaccaaaggat


 


acactgatgatcagccggacccccgaggttacgtgcgtcgtcgtcgatgt


 


ctcccacgaggatccagaggtccaattcaactggtacgtggacggggtcg


 


aggtgcataatgcaaagacaaagccacgggaagagcagtttaactctact


 


ttccgcgtggtttctgtgctgaccgtggtgcaccaagattggctcaacgg


 


caaggagtacaagtgcaaggtaagcaataaggggctccctgcccccattg


 


agaagactatctccaagacaaagggacagccacgcgagccacaagtctat


 


acactccccccttcccgcgaagaaatgaccaagaatcaggttagcctgac


 


atgcttggttaagggtttctacccctctgacatagccgtggagtgggaga


 


gcaatggacaaccagagaacaactacaagaccaccccacccatgctggat


 


agcgacggttcattctttctgtatagtaagcttaccgtggacaagtcccg


 


gtggcaacaaggaaatgtcttttcatgctctgtgatgcacgaggccttgc


 


ataatcactatactcagaagagcttgagcctcagccccggatctggaggt


 


ggcggatccgggggcggtggaagcggaggtggtggatcgggaggcggtgg


 


aagcgcgcgcaacggcgaccactgtccgctcgggcccggacgttgctgcc


 


gtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgg


 


gtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgccc


 


gagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgc


 


accgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagc


 


tacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctcca


 


gacctatgatgacttgttagccaaagactgccactgcatatga






and comprises the amino acid sequence (hinge regions in parentheses, linkers sequences double underlined):










(SEQ ID NO: 33)



GGG(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVT



 



CVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVL



 



TVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP



 



PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML



 



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS



 



PGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(ERK



 



SSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS



 



HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW



 



LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMT



 



KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFF



 



LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGG



 



SGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWA



 



DWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAP



 



CCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI






In an embodiment employing the VH21 signal sequence, in its final form, the monomer is encoded by the nucleic acid (signal sequence underlined):







(SEQ ID NO: 117)


atggaatggagctgggtctttctcttcttcctgtcagtaacgactg


 


gtgtccactccggaggtggagagcgcaaatcttctgtcgagtgccc


 


accgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttc


 


cccccaaaacccaaggacaccctcatgatctcccggacccctgagg


 


tcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtcca


 


gttcaactggtacgtggacggcgtggaggtgcataatgccaagaca


 


aaaccacgggaggagcagttcaacagcacgttccgtgtggtcagcg


 


tcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaa


 


gtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaacc


 


atctccaaaaccaaagggcagccccgagaaccacaggtgtacaccc


 


tgcccccatcccgggaggagatgaccaagaaccaggtcagcctgac


 


ctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgg


 


gagagcaatgggcagccggagaacaactacaagaccacacctccca


 


tgctggactccgacggctccttcttcctctacagcaagctcaccgt


 


ggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg


 


atgcatgaggctctgcacaaccactacacgcagaagagcctctccc


 


tgtctccgggtggaggtggcggtagcggtggcggaggttcaggtgg


 


tggcggttctggcggaggtggcagtggcggtggcggatcaggtggc


 


ggtggcagcggtggcggcggaagcggtggaggaggttcagagcgga


 


aatccagcgttgaatgtcctccgtgccctgctccacccgtcgcggg


 


gcctagtgtcttccttttccctccaaaaccaaaggatacactgatg


 


atcagccggacccccgaggttacgtgcgtcgtcgtcgatgtctccc


 


acgaggatccagaggtccaattcaactggtacgtggacggggtcga


 


ggtgcataatgcaaagacaaagccacgggaagagcagtttaactct


 


actttccgcgtggtttctgtgctgaccgtggtgcaccaagattggc


 


tcaacggcaaggagtacaagtgcaaggtaagcaataaggggctccc


 


tgcccccattgagaagactatctccaagacaaagggacagccacgc


 


gagccacaagtctatacactccccccttcccgcgaagaaatgacca


 


agaatcaggttagcctgacatgcttggttaagggtttctacccctc


 


tgacatagccgtggagtgggagagcaatggacaaccagagaacaac


 


tacaagaccaccccacccatgctggatagcgacggttcattctttc


 


tgtatagtaagcttaccgtggacaagtcccggtggcaacaaggaaa


 


tgtcttttcatgctctgtgatgcacgaggccttgcataatcactat


 


actcagaagagcttgagcctcagccccggatctggaggtggcggat


 


ccgggggcggtggaagcggaggtggtggatcgggaggcggtggaag


 


cgcgcgcaacggcgaccactgtccgctcgggcccggacgttgctgc


 


cgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccg


 


attgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcgg


 


cgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatc


 


aagacgagcctgcaccgcctgaagcccgacacggtgccagcgccct


 


gctgcgtgcccgccagctacaatcccatggtgctcattcaaaagac


 


cgacaccggggtgtcgctccagacctatgatgacttgttagccaaa


 


gactgccactgcatatga






and comprises the amino acid sequence (signal sequence underlined, hinge region in parentheses and linker sequences double underlined):








(SEQ ID NO: 118)


MEWSWVFLFFLSVTTGVHSGGG(ERKSSVECPPCP)APPVAGPSVF


 


LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA


 


KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE


 


KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV


 


EWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC


 


SVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGS


 


GGGGSGGGGSGGGGS(ERKSSVECPPCP)APPVAGPSVFLFPPKPK


 


DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE


 


QFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK


 


GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ


 


PENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL


 


HNHYTQKSLSLSPGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGP


 


GRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANM


 


HAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD


 


LLAKDCHCI






Two such monomers associate to form the dimer in which the two hGDF15 polypeptides are linked via a disulfide bond between two naturally occurring (i.e., not engineered) cysteine residues. Accordingly, the specific hemiFc dimeric construct Fc-(G4S)8-Fc-GS(G4S)4-GDF15 comprises two monomers comprising SEQ ID NO:33.
II.D.2 Fc-(G4S)3-Fc-GS(G4S)4-GDF15
The hemiFc construct designated “Fc(G4S)3-Fc-GS(G4S)4-GDF15” in the instant disclosure refers to a construct comprising a monomer, which comprises a mature human GDF15 sequence linked to a first Fc sequence via a first linker comprising SEQ ID NO:31 that connects the N-terminus of the human GDF15 sequence to the C-terminus of the first Fc sequence and a second linker comprising the amino acid sequence:









(SEQ ID NO: 87)



GGGGSGGGGSGGGGS.






that connects the N-terminus of the first Fc sequence to the C-terminus of the second Fc sequence. The first and second Fc sequences are also associated by the Fc hinge regions. Two such monomers associate to form a homodimer in which the monomers are linked via an interchain disulfide bond between the two GDF15 sequences.

More particularly, in a specific embodiment, the hemiFc construct Fc(G4S)3-Fc-GS(G4S)4-GDF15 comprises a monomer comprising a second Fc chain comprising the sequence of SEQ ID NO:28 joined by the linker:









(SEQ ID NO: 119)



GGGGGSGGGGSGGGGS






to a first Fc chain comprising the sequence of SEQ ID NO:30 to the C-terminus of which, the GDF15 polypeptide comprising SEQ ID NO:12 is joined by a linker having the sequence of SEQ ID NO:31.

In its final form, the monomer is encoded by the nucleic acid sequence:







(SEQ ID NO: 122)


ggaggaggcgagaggaagagctccgtggagtgtccaccctgccctgc


 


tccgcctgtggctggaccctctgtgttcctgtttccgccgaagccga


 


aagacaccctcatgatcagcaggactcccgaggtcacttgtgtggtc


 


gtggatgtgagccatgaggacccagaggtgcagttcaactggtacgt


 


ggacggcgtggaagtccacaacgccaagaccaagccacgcgaggaac


 


agttcaatagcaccttccgcgtggtcagcgtcctcaccgtggtccac


 


caggattggcttaacggaaaggaatacaaatgcaaggtgtccaacaa


 


ggggcttcctgccccgattgaaaagaccatctccaagaccaagggac


 


agccaagggagccccaagtgtacactctgccacccagccgcgaagaa


 


atgactaagaatcaagtgtctctgacctgtcttgtcaaaggcttcta


 


ccccagcgacatcgctgtcgagtgggaatcaaacgggcagcccgaga


 


acaactacaagaccactcctccaatgctcgactcagatggcagcttt


 


ttcctttactccaagctgaccgtggacaagtcaagatggcaacaggg


 


taacgtgttctcatgctccgtgatgcacgaagccctccataatcact


 


atacccagaaatctctgtctctttccccgggaggaggagggggatct


 


ggtggaggaggctctggtggtggaggtagcgaacggaaatcctcagt


 


ggagtgcccaccatgcccggctcctccagtggctggtccatctgtct


 


ttctttttcctccgaaacccaaggacacccttatgatctctcgcacc


 


cctgaagtgacttgcgtggtcgtcgatgtgtcacatgaagaccctga


 


ggtccagttcaattggtatgtggacggagtcgaggtgcataacgcca


 


aaaccaaacctcgcgaagaacaattcaactctaccttccgggtggtg


 


tctgtgctcactgtcgtccatcaggactggctgaacgggaaggagta


 


caagtgtaaggtgtctaacaaaggcctgccggctcccatcgaaaaga


 


ctatcagcaagactaaggggcaacccagagaaccccaagtctacacc


 


ctgcctccgtcacgggaggagatgaccaagaatcaggtgtccctcac


 


ctgtctggtcaagggtttctaccctagcgacattgctgtggagtggg


 


agagcaatggacagcccgaaaacaattacaagactaccccacccatg


 


ctggactcagacggatcatttttcctctactctaagctcactgtgga


 


caagagccggtggcagcaagggaatgtgttcagctgttcagtgatgc


 


atgaggccctgcataaccactacacccagaagagcctttcactgtca


 


cccgggtctggtggcggtgggtcaggtggcggaggatcaggaggagg


 


tggaagcggcggaggaggatctgccaggaacggtgatcactgccctc


 


tgggccctggtcgctgctgtaggcttcacactgtgcgggcttccctc


 


gaagatctgggatgggccgactgggtgctgagcccaagagaggtgca


 


agtgaccatgtgcatcggggcatgtccctcccaattccgcgctgcaa


 


acatgcatgctcagattaagacttcactgcatagactgaagccagat


 


accgtcccagcaccctgttgtgtgcccgcttcatacaaccccatggt


 


cctgattcaaaagaccgacaccggggtgtctctccagacctatgatg


 


atcttcttgcaaaggactgccactgcatctga






and comprises the amino acid sequence (hinge regions in parentheses, linker sequences double underlined):










(SEQ ID NO: 123)



GGG(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVT



 



CVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVL



 



TVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP



 



PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML



 



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS



 



PGGGGGSGGGGSGGGGS(ERKSSVECPPCP)APPVAGPSVFLFPPK



 



PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR



 



EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK



 



TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN



 



GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE



 



ALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSGGGGSARNGDHCPL



 



GPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAA



 



NMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY



 



DDLLAKDCHCI.






In an embodiment employing the VH21 signal sequence, in its final form, the monomer is encoded by the nucleic acid (signal sequence underlined):







(SEQ ID NO: 120)


atggaatggtcatgggtgttccttttctttctctccgtcactaccggtgt


 


gcactccggaggaggcgagaggaagagctccgtggagtgtccaccctgcc


 


ctgctccgcctgtggctggaccctctgtgttcctgtttccgccgaagccg


 


aaagacaccctcatgatcagcaggactcccgaggtcacttgtgtggtcgt


 


ggatgtgagccatgaggacccagaggtgcagttcaactggtacgtggacg


 


gcgtggaagtccacaacgccaagaccaagccacgcgaggaacagttcaat


 


agcaccttccgcgtggtcagcgtcctcaccgtggtccaccaggattggct


 


taacggaaaggaatacaaatgcaaggtgtccaacaaggggcttcctgccc


 


cgattgaaaagaccatctccaagaccaagggacagccaagggagccccaa


 


gtgtacactctgccacccagccgcgaagaaatgactaagaatcaagtgtc


 


tctgacctgtcttgtcaaaggcttctaccccagcgacatcgctgtcgagt


 


gggaatcaaacgggcagcccgagaacaactacaagaccactcctccaatg


 


ctcgactcagatggcagctttttcctttactccaagctgaccgtggacaa


 


gtcaagatggcaacagggtaacgtgttctcatgctccgtgatgcacgaag


 


ccctccataatcactatacccagaaatctctgtctctttccccgggagga


 


ggagggggatctggtggaggaggctctggtggtggaggtagcgaacggaa


 


atcctcagtggagtgcccaccatgcccggctcctccagtggctggtccat


 


ctgtctttctttttcctccgaaacccaaggacacccttatgatctctcgc


 


acccctgaagtgacttgcgtggtcgtcgatgtgtcacatgaagaccctga


 


ggtccagttcaattggtatgtggacggagtcgaggtgcataacgccaaaa


 


ccaaacctcgcgaagaacaattcaactctaccttccgggtggtgtctgtg


 


ctcactgtcgtccatcaggactggctgaacgggaaggagtacaagtgtaa


 


ggtgtctaacaaaggcctgccggctcccatcgaaaagactatcagcaaga


 


ctaaggggcaacccagagaaccccaagtctacaccctgcctccgtcacgg


 


gaggagatgaccaagaatcaggtgtccctcacctgtctggtcaagggttt


 


ctaccctagcgacattgctgtggagtgggagagcaatggacagcccgaaa


 


acaattacaagactaccccacccatgctggactcagacggatcatttttc


 


ctctactctaagctcactgtggacaagagccggtggcagcaagggaatgt


 


gttcagctgttcagtgatgcatgaggccctgcataaccactacacccaga


 


agagcctttcactgtcacccgggtctggtggcggtgggtcaggtggcgga


 


ggatcaggaggaggtggaagcggcggaggaggatctgccaggaacggtga


 


tcactgccctctgggccctggtcgctgctgtaggcttcacactgtgcggg


 


cttccctcgaagatctgggatgggccgactgggtgctgagcccaagagag


 


gtgcaagtgaccatgtgcatcggggcatgtccctcccaattccgcgctgc


 


aaacatgcatgctcagattaagacttcactgcatagactgaagccagata


 


ccgtcccagcaccctgttgtgtgcccgcttcatacaaccccatggtcctg


 


attcaaaagaccgacaccggggtgtctctccagacctatgatgatcttct


 


tgcaaaggactgccactgcatctga






and comprises the amino acid sequence (signal sequence single underlined, hinge regions in parentheses and linker sequences double underlined):








(SEQ ID NO: 121)


MEWSWVFLFFLSVTTGVHSGGG(ERKSSVECPPCP)APPVAGPSVFLF


 


PPKPKDTLMISRTPEVTCVVVDVISHEDPEVQFNWYVDGVEVHNAKTK


 


PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK


 


TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ


 


PENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN


 


HYTQKSLSLSPGGGGGSGGGGSGGGGS(ERKSSVECPPCP)APPVAGP


 


SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN


 


AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK


 


TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE


 


SNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGP


 


GRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHA


 


QIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK


 


DCHCI.






Two such monomers associate to form the dimer in which the two hGDF15 polypeptides are linked via a disulfide bond between two naturally occurring (i.e., not engineered) cysteine residues. Accordingly, the specific hemiFc dimeric construct Fc-(G4S)3-Fc-GS(G4S)4-GDF15 comprises two monomers comprising SEQ ID NO:123.
II.D.3 Fc-(G4S)5-Fc-GS(G4S)4-GDF15
The hemiFc construct designated “Fc-(G4S)5-Fc-GS(G4S)4-GDF15” in the instant disclosure refers to a construct comprising a monomer, which comprises a mature human GDF15 sequence linked to a first Fc sequence via a first linker comprising SEQ ID NO:31 that connects the N-terminus of the human GDF15 sequence to the C-terminus of the first Fc sequence and a second linker comprising the amino acid sequence:









(SEQ ID NO: 88)



GGGGSGGGGSGGGGSGGGGSGGGGS.






That connects the N-terminus of the first Fc sequence to the C-terminus of the second Fc sequence. The first and second Fc sequences are also associated by the Fc hinge regions. Two such monomers associate to form a homodimer in which the monomers are linked via an interchain disulfide bond between the two GDF15 sequences.

More particularly, in a specific embodiment, the hemiFc construct Fc-(G4S)5-Fc-GS(G4S)4-GDF15 comprises a monomer comprising a second Fc chain comprising the sequence of SEQ ID NO:28 joined by the linker:









(SEQ ID NO: 124)



GGGGGSGGGGSGGGGSGGGGGSGGGGGS






to a first Fc chain comprising the sequence of SEQ ID NO:30 to the C-terminus of which, the GDF15 polypeptide comprising SEQ ID NO:12 is joined by a linker having the sequence of SEQ ID NO:31.

In its final form, the monomer is encoded by the nucleic acid sequence:







(SEQ ID NO: 127)


ggcggtggagagcgcaagtcatctgtcgagtgtccgccctgccccgctc


 


cgccggtggctggaccctcagtgttcctctttccaccgaagccgaagga


 


cacccttatgattagccggaccccagaggtcacttgcgtcgtcgtggac


 


gtgtcccatgaggatcccgaagtgcagtttaactggtatgtggacggag


 


tggaggtccataacgccaagaccaagccaagggaagaacagttcaatag


 


caccttccgggtggtgtccgtgctcaccgtggtgcatcaagactggctg


 


aatggcaaagagtacaaatgtaaggtgtcaaacaaggggctcccagccc


 


ctattgaaaagaccatctcaaagactaagggacagccacgcgaacctca


 


agtgtataccctcccgccttcacgcgaagaaatgactaagaatcaggtc


 


agccttacttgtctggtcaagggcttctacccgagcgacattgcagtcg


 


aatgggagagcaatggtcagccagagaataactacaagaccactcctcc


 


catgcttgatagcgatggaagctttttcctttacagcaagcttactgtg


 


gataagtctcgctggcaacagggaaatgtgttcagctgttcagtgatgc


 


atgaagcactccacaatcattacacccagaagtcactcagcctctcacc


 


cggaggaggaggcggttctggtggaggagggtctggaggtggagggagc


 


ggcggaggcgggtctggcggtggtgggtctgagaggaagtcatcagtgg


 


aatgcccaccatgccctgctcctcccgtggccggtccgagcgtgtttct


 


cttcccacctaagcccaaggacactctgatgatctcacggactccggaa


 


gtgacttgtgtggtggtggacgtgtctcatgaggaccctgaagtgcagt


 


tcaactggtacgtggacggcgtggaggtgcacaatgctaagaccaagcc


 


tagagaggaacagttcaattccacctttcgcgtggtgagcgtcctgacc


 


gtcgtgcaccaggactggcttaacggaaaggaatacaagtgcaaggtgt


 


ccaacaaaggccttccagctcccattgagaaaaccatctctaaaactaa


 


gggtcaaccaagggaaccccaagtctacaccctccctccgtctagagaa


 


gagatgaccaaaaaccaggtgtccctgacctgtctggtgaagggatttt


 


acccctcagacatcgccgtggagtgggaaagcaacggacagcccgaaaa


 


caactataagactacccctcctatgctggactcagacggatctttcttc


 


ctctatagcaagctcactgtggacaaatccagatggcaacaagggaatg


 


tgttctcatgcagcgtgatgcacgaggctcttcacaaccactataccca


 


gaagagcctgtctctttcacctggttccggaggtggtgggagcggaggg


 


ggtggatcaggtggtggagggtccggaggcggaggatccgcacggaatg


 


gcgaccactgtccactgggacccggaagatgttgtcgcctccacaccgt


 


gagggcctctctggaggaccttggctgggccgactgggtcctgtcacct


 


cgggaggtccaagtcaccatgtgtatcggagcctgccccagccaattca


 


gagcagcaaatatgcacgcacagattaagaccagcctgcatcggcttaa


 


acctgatactgtgccggctccttgttgcgtgccagcatcttacaacccg


 


atggtgctgatccagaaaaccgataccggtgtctccctccagacttacg


 


acgacctccttgcaaaggactgccattgcatctga






and comprises the amino acid sequence (hinge regions in parentheses, linker sequences double underlined):








(SEQ ID NO: 128)


GGG(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV


 


VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSR


 


EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG


 


SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGG


 


GSGGGGSGGGGSGGGGSGGGGS(ERKSSVECPP)CPAPPVAGPSVFL


 


FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKT


 


KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI


 


SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES


 


NGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLG


 


PGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANM


 


HAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDL


 


LAKDCHCI






In an embodiment employing the VH21 signal sequence, in its final form, the monomer is encoded by the nucleic acid (signal sequence underlined):







(SEQ ID NO: 125)


atggagtggagctgggtctttcttttctttctgtctgtgactaccggagt


 


ccattcaggcggtggagagcgcaagtcatctgtcgagtgtccgccctgcc


 


ccgctccgccggtggctggaccctcagtgttcctctttccaccgaagccg


 


aaggacacccttatgattagccggaccccagaggtcacttgcgtcgtcgt


 


ggacgtgtcccatgaggatcccgaagtgcagtttaactggtatgtggacg


 


gagtggaggtccataacgccaagaccaagccaagggaagaacagttcaat


 


agcaccttccgggtggtgtccgtgctcaccgtggtgcatcaagactggct


 


gaatggcaaagagtacaaatgtaaggtgtcaaacaaggggctcccagccc


 


ctattgaaaagaccatctcaaagactaagggacagccacgcgaacctcaa


 


gtgtataccctcccgccttcacgcgaagaaatgactaagaatcaggtcag


 


ccttacttgtctggtcaagggcttctacccgagcgacattgcagtcgaat


 


gggagagcaatggtcagccagagaataactacaagaccactcctcccatg


 


cttgatagcgatggaagctttttcctttacagcaagcttactgtggataa


 


gtctcgctggcaacagggaaatgtgttcagctgttcagtgatgcatgaag


 


cactccacaatcattacacccagaagtcactcagcctctcacccggagga


 


ggaggcggttctggtggaggagggtctggaggtggagggagcggcggagg


 


cgggtctggcggtggtgggtctgagaggaagtcatcagtggaatgcccac


 


catgccctgctcctcccgtggccggtccgagcgtgtttctcttcccacct


 


aagcccaaggacactctgatgatctcacggactccggaagtgacttgtgt


 


ggtggtggacgtgtctcatgaggaccctgaagtgcagttcaactggtacg


 


tggacggcgtggaggtgcacaatgctaagaccaagcctagagaggaacag


 


ttcaattccacctttcgcgtggtgagcgtcctgaccgtcgtgcaccagga


 


ctggcttaacggaaaggaatacaagtgcaaggtgtccaacaaaggccttc


 


cagctcccattgagaaaaccatctctaaaactaagggtcaaccaagggaa


 


ccccaagtctacaccctccctccgtctagagaagagatgaccaaaaacca


 


ggtgtccctgacctgtctggtgaagggattttacccctcagacatcgccg


 


tggagtgggaaagcaacggacagcccgaaaacaactataagactacccct


 


cctatgctggactcagacggatctttcttcctctatagcaagctcactgt


 


ggacaaatccagatggcaacaagggaatgtgttctcatgcagcgtgatgc


 


acgaggctcttcacaaccactatacccagaagagcctgtctctttcacct


 


ggttccggaggtggtgggagcggagggggtggatcaggtggtggagggtc


 


cggaggcggaggatccgcacggaatggcgaccactgtccactgggacccg


 


gaagatgttgtcgcctccacaccgtgagggcctctctggaggaccttggc


 


tgggccgactgggtcctgtcacctcgggaggtccaagtcaccatgtgtat


 


cggagcctgccccagccaattcagagcagcaaatatgcacgcacagatta


 


agaccagcctgcatcggcttaaacctgatactgtgccggctccttgttgc


 


gtgccagcatcttacaacccgatggtgctgatccagaaaaccgataccgg


 


tgtctccctccagacttacgacgacctccttgcaaaggactgccattgca


 


tctga






and comprises the amino acid sequence (signal sequence single underlined, hinge regions in parentheses and linker sequences double underlined):








(SEQ ID NO: 126)


MEWSWVFLFFLSVTTGVHS


 


GGG(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV


 


DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL


 


NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS


 


LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK


 


SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGG


 


GSGGGGS(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVT


 


CVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH


 


QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK


 


NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKL


 


TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGG


 


GSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTM


 


CIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTD


 


TGVSLQTYDDLLAKDCHCI 






Two such monomers associate to form the dimer in which the two hGDF15 polypeptides are linked via a disulfide bond between two naturally occurring (i.e., not engineered) cysteine residues. Accordingly, the specific hemiFc dimeric construct Fc-(G4S)5-Fc-GS(G4S)4-GDF15 comprises two monomers comprising SEQ ID NO:128.
III. GDF15 POLYPEPTIDES AND CONSTRUCTS COMPRISING GDF15, INCLUDING MUTANT FORMS THEREOF
As disclosed herein, the GDF15 polypeptides (including the full length and mature forms of human GDF15) and the constructs comprising GDF15 described in the instant disclosure can be engineered and/or produced using standard molecular biology methodology to form a mutant form of the GDF15 polypeptides and constructs provided herein. In various examples, a nucleic acid sequence encoding a mutant form of the GDF15 polypeptides and constructs provided herein, which can comprise all or a portion of SEQ ID NOs: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 and amino acid sequences provided herein according to standard (RT)-PCR amplification techniques. The amplified GDF15 mutant polypeptide nucleic acid can then be cloned into a suitable vector and characterized by DNA sequence analysis.
Oligonucleotides for use as probes in isolating or amplifying all or a portion of a mutant form of the GDF15 polypeptides and constructs provided herein can be designed and generated using standard synthetic techniques, e.g., automated DNA synthesis apparatus, or can be isolated from a longer sequence of DNA.
III.A. GDF15 Polypeptide and Polynucleotide Sequences
In vivo, GDF15 is expressed as a contiguous amino acid sequence comprising a signal sequence, a pro domain and an active domain.
The 308 amino acid sequence of full length human GDF15 is:







(SEQ ID NO: 4)


MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSED


 


SRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGH


 


LHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARP


 


QAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNG


 


DHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRA


 


ANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDL


 


LAKDCHCI 






and is encoded by the DNA sequence:








(SEQ ID NO: 3)


atgcccgggcaagaactcaggacggtgaatggctctcagatgctcctggt


 


gttgctggtgctctcgtggctgccgcatgggggcgccctgtctctggccg


 


aggcgagccgcgcaagtttcccgggaccctcagagttgcactccgaagac


 


tccagattccgagagttgcggaaacgctacgaggacctgctaaccaggct


 


gcgggccaaccagagctgggaagattcgaacaccgacctcgtcccggccc


 


ctgcagtccggatactcacgccagaagtgcggctgggatccggcggccac


 


ctgcacctgcgtatctctcgggccgcccttcccgaggggctccccgaggc


 


ctcccgccttcaccgggctctgttccggctgtccccgacggcgtcaaggt


 


cgtgggacgtgacacgaccgctgcggcgtcagctcagccttgcaagaccc


 


caggcgcccgcgctgcacctgcgactgtcgccgccgccgtcgcagtcgga


 


ccaactgctggcagaatcttcgtccgcacggccccagctggagttgcact


 


tgcggccgcaagccgccagggggcgccgcagagcgcgtgcgcgcaacggg


 


gaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccg


 


cgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacggg


 


aggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcg


 


gcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccga


 


cacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgc


 


tcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttg


 


ttagccaaagactgccactgcatatga.






The 303 amino acid sequence of full length murine GDF15 is:








(SEQ ID NO: 6)


MAPPALQAQPPGGSQLRFLLFLLLLLLLLSWPSQGDALAMPEQRPSGPES


 


QLNADELRGRFQDLLSRLHANQSREDSNSEPSPDPAVRILSPEVRLGSHG


 


QLLLRVNRASLSQGLPEAYRVHRALLLLTPTARPWDITRPLKRALSLRGP


 


RAPALRLRLTPPPDLAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPL


 


GPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHA


 


QIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGVSLQTYDDLVARGC


 


HCA






and is encoded by the DNA sequence:








(SEQ ID NO: 5)


atggccccgcccgcgctccaggcccagcctccaggcggctctcaactgag


 


gttcctgctgttcctgctgctgttgctgctgctgctgtcatggccatcgc


 


agggggacgccctggcaatgcctgaacagcgaccctccggccctgagtcc


 


caactcaacgccgacgagctacggggtcgcttccaggacctgctgagccg


 


gctgcatgccaaccagagccgagaggactcgaactcagaaccaagtcctg


 


acccagctgtccggatactcagtccagaggtgagattggggtcccacggc


 


cagctgctactccgcgtcaaccgggcgtcgctgagtcagggtctccccga


 


agcctaccgcgtgcaccgagcgctgctcctgctgacgccgacggcccgcc


 


cctgggacatcactaggcccctgaagcgtgcgctcagcctccggggaccc


 


cgtgctcccgcattacgcctgcgcctgacgccgcctccggacctggctat


 


gctgccctctggcggcacgcagctggaactgcgcttacgggtagccgccg


 


gcagggggcgccgaagcgcgcatgcgcacccaagagactcgtgcccactg


 


ggtccggggcgctgctgtcacttggagactgtgcaggcaactcttgaaga


 


cttgggctggagcgactgggtgctgtccccgcgccagctgcagctgagca


 


tgtgcgtgggcgagtgtccccacctgtatcgctccgcgaacacgcatgcg


 


cagatcaaagcacgcctgcatggcctgcagcctgacaaggtgcctgcccc


 


gtgctgtgtcccctccagctacaccccggtggttcttatgcacaggacag


 


acagtggtgtgtcactgcagacttatgatgacctggtggcccggggctgc


 


cactgcgcttga.






The amino acid sequence of human GDF15 following cleavage of the 29 residue signal sequence is:







(SEQ ID NO: 8)


LSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTD


 


LVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSP


 


TASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQ


 


LELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWV


 


LSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY


 


NPMVLIQKTDTGVSLQTYDDLLAKDCHCI






and is encoded by the DNA sequence:








(SEQ ID NO: 7)


ctgtctctggccgaggcgagccgcgcaagtttcccgggaccctcagagtt


 


gcactccgaagactccagattccgagagttgcggaaacgctacgaggacc


 


tgctaaccaggctgcgggccaaccagagctgggaagattcgaacaccgac


 


ctcgtcccggcccctgcagtccggatactcacgccagaagtgcggctggg


 


atccggcggccacctgcacctgcgtatctctcgggccgcccttcccgagg


 


ggctccccgaggcctcccgccttcaccgggctctgttccggctgtccccg


 


acggcgtcaaggtcgtgggacgtgacacgaccgctgcggcgtcagctcag


 


ccttgcaagaccccaggcgcccgcgctgcacctgcgactgtcgccgccgc


 


cgtcgcagtcggaccaactgctggcagaatcttcgtccgcacggccccag


 


ctggagttgcacttgcggccgcaagccgccagggggcgccgcagagcgcg


 


tgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtc


 


tgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtg


 


ctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgag


 


ccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcacc


 


gcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctac


 


aatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagac


 


ctatgatgacttgttagccaaagactgccactgcatatga  






The amino acid sequence of murine GDF15 following cleavage of the 32 residue signal sequence is:







(SEQ ID NO: 10)


SQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLHANQSREDSNSEPS


 


PDPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTA


 


RPWDITRPLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELRLRVA


 


AGRGRRSAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQL


 


SMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHR


 


TDSGVSLQTYDDLVARGCHCA 






and is encoded by the DNA sequence:








(SEQ ID NO: 9)


tcgcagggggacgccctggcaatgcctgaacagcgaccctccggccctga


 


gtcccaactcaacgccgacgagctacggggtcgcttccaggacctgctga


 


gccggctgcatgccaaccagagccgagaggactcgaactcagaaccaagt


 


cctgacccagctgtccggatactcagtccagaggtgagattggggtccca


 


cggccagctgctactccgcgtcaaccgggcgtcgctgagtcagggtctcc 


 


ccgaagcctaccgcgtgcaccgagcgctgctcctgctgacgccgacggcc


 


cgcccctgggacatcactaggcccctgaagcgtgcgctcagcctccgggg


 


accccgtgctcccgcattacgcctgcgcctgacgccgcctccggacctgg


 


ctatgctgccctctggcggcacgcagctggaactgcgcttacgggtagcc


 


gccggcagggggcgccgaagcgcgcatgcgcacccaagagactcgtgccc


 


actgggtccggggcgctgctgtcacttggagactgtgcaggcaactcttg


 


aagacttgggctggagcgactgggtgctgtccccgcgccagctgcagctg


 


agcatgtgcgtgggcgagtgtccccacctgtatcgctccgcgaacacgca


 


tgcgcagatcaaagcacgcctgcatggcctgcagcctgacaaggtgcctg


 


ccccgtgctgtgtcccctccagctacaccccggtggttcttatgcacagg


 


acagacagtggtgtgtcactgcagacttatgatgacctggtggcccgggg


 


ctgccactgcgcttga 






The amino acid sequence of the recombinant active form of the human GDF15, which comprises a homodimer comprising nine cysteines in each monomer to form one interchain disulfide bond and four intrachain disulfide bonds (shown with an optional N-terminal methionine residue in parentheses), is:







(SEQ ID NO: 129)


(M)ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGA


 


CPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS


 


LQTYDDLLAKDCHCI 






and is encoded by the DNA sequence (shown with an optional N-terminal methionine codon in parentheses):








(SEQ ID NO: 130)


(atg)gcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgc


 


cgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattg


 


ggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcc


 


cgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctg


 


caccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccag


 


ctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctcc


 


agacctatgatgacttgttagccaaagactgccactgcatataa.






The amino acid sequence of the recombinant active form of the murine GDF15, which comprises a homodimer comprising nine cysteines in each monomer to form one interchain disulfide bond and four intrachain disulfide bonds, is:







(SEQ ID NO: 14)


(M)SAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMC


 


VGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDS


 


GVSLQTYDDLVARGCHCA 






and is encoded by the DNA sequence:








(SEQ ID NO: 13)


(atg)agcgcgcatgcgcacccaagagactcgtgcccactgggtccgggg


 


cgctgctgtcacctggagactgtgcaggcaactcttgaagacttgggctg


 


gagcgactgggtgttgtccccgcgccagctgcagctgagcatgtgcgtgg


 


gcgagtgtccccacctgtatcgctccgcgaacacgcatgcgcagatcaaa


 


gcacgcctgcatggcctgcagcctgacaaggtgcctgccccgtgctgtgt


 


cccctccagctacaccccggtggttcttatgcacaggacagacagtggtg


 


tgtcactgcagacttatgatgacctggtggcccggggctgccactgcgct


 


tga.






As stated herein, the term “GDF15 polypeptide” refers to a GDF polypeptide comprising the human amino acid sequences SEQ ID NOs:4, 8 and 12. The term “GDF15 mutant polypeptide,” however, encompasses polypeptides comprising an amino acid sequence that differs from the amino acid sequence of a naturally occurring GDF polypeptide sequence, e.g., SEQ ID NOs: 4, 8 and 12, by one or more amino acids, such that the sequence is at least 85% identical to SEQ ID NOs: 4, 8 and 12. GDF15 polypeptides can be generated by introducing one or more amino acid substitutions, either conservative or non-conservative and using naturally or non-naturally occurring amino acids, at particular positions of the GDF15 polypeptide, or by deleting particular residues or stretches of residues.
A “conservative amino acid substitution” can involve a substitution of a native amino acid residue (i.e., a residue found in a given position of the wild-type GDF15 polypeptide sequence) with a non-native residue (i.e., a residue that is not found in a given position of the wild-type GDF15 polypeptide sequence) such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues (as defined herein) that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties.
Naturally occurring residues can be divided into classes based on common side chain properties:
(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr;
(3) acidic: Asp, Glu;
(4) basic: Asn, Gln, H is, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro; and
(6) aromatic: Trp, Tyr, Phe.
Additional groups of amino acids can also be formulated using the principles described in, e.g., Creighton (1984) PROTEINS: STRUCTURE AND MOLECULAR PROPERTIES (2d Ed. 1993), W.H. Freeman and Company. In some instances it can be useful to further characterize substitutions based on two or more of such features (e.g., substitution with a “small polar” residue, such as a Thr residue, can represent a highly conservative substitution in an appropriate context).
Conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. Non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.
Synthetic, rare, or modified amino acid residues having known similar physiochemical properties to those of an above-described grouping can be used as a “conservative” substitute for a particular amino acid residue in a sequence. For example, a D-Arg residue may serve as a substitute for a typical L-Arg residue. It also can be the case that a particular substitution can be described in terms of two or more of the above described classes (e.g., a substitution with a small and hydrophobic residue means substituting one amino acid with a residue(s) that is found in both of the above-described classes or other synthetic, rare, or modified residues that are known in the art to have similar physiochemical properties to such residues meeting both definitions).
Nucleic acid sequences encoding a GDF15 mutant polypeptide provided herein, including those degenerate to SEQ ID NOs: 3, 7, 11 and 15, and those encoding polypeptide variants of SEQ ID NOs:4, 8 and 12, form other aspects of the instant disclosure.
III.B. Vectors Useful for Expressing GDF15 Polypeptides and Constructs Comprising GDF15, Including Mutant Forms Thereof
In order to express the nucleic acid sequences encoding the GDF15 polypeptides and construct comprising GDF15 provided herein, the appropriate coding sequences, e.g., SEQ ID NOs:3, 7, 11, 15, 17, 21, 24, 26 and 32, can be cloned into a suitable vector and after introduction in a suitable host, the sequence can be expressed to produce the encoded polypeptide according to standard cloning and expression techniques, which are known in the art (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The invention also relates to such vectors comprising a nucleic acid sequence according to the invention.
A “vector” refers to a delivery vehicle that (a) promotes the expression of a polypeptide-encoding nucleic acid sequence; (b) promotes the production of the polypeptide therefrom; (c) promotes the transfection/transformation of target cells therewith; (d) promotes the replication of the nucleic acid sequence; (e) promotes stability of the nucleic acid; (f) promotes detection of the nucleic acid and/or transformed/transfected cells; and/or (g) otherwise imparts advantageous biological and/or physiochemical function to the polypeptide-encoding nucleic acid. A vector can be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors.
A recombinant expression vector can be designed for expression of a GDF15 mutant polypeptide in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells, using baculovirus expression vectors, yeast cells, or mammalian cells). Representative host cells include those hosts typically used for cloning and expression, including Escherichia coli strains TOP10F′, TOP10, DH10B, DH5a, HB101, W3110, BL21(DE3) and BL21 (DE3)pLysS, BLUESCRIPT (Stratagene), mammalian cell lines CHO, CHO-K1, HEK293, 293-EBNA pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264: 5503-5509 (1989); pET vectors (Novagen, Madison Wis.). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase and an in vitro translation system. Preferably, the vector contains a promoter upstream of the cloning site containing the nucleic acid sequence encoding the polypeptide. Examples of promoters, which can be switched on and off, include the lac promoter, the T7 promoter, the trc promoter, the tac promoter and the trp promoter.
Thus, provided herein are vectors comprising a nucleic acid sequence encoding a GDF15 polypeptide or construct comprising a GDF15 polypeptide, including mutant forms thereof, that facilitate the expression of the polypeptide or construct of interest. In various embodiments, the vectors comprise an operably linked nucleotide sequence which regulates the expression of a GDF15 polypeptide construct comprising a GDF15 polypeptide or a mutant form thereof. A vector can comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e.g., a human CMV IE promoter/enhancer, an RSV promoter, SV40 promoter, SL3-3 promoter, MMTV promoter, or HIV LTR promoter, EF1alpha promoter, CAG promoter), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and/or a convenient cloning site (e.g., a polylinker). Vectors also can comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE. In one aspect, a nucleic acid comprising a sequence encoding a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, which is operatively linked to a tissue specific promoter which promotes expression of the sequence in a metabolically-relevant tissue, such as liver or pancreatic tissue is provided.
III.C. Host Cells
In another aspect of the instant disclosure, host cells comprising the nucleic acids and vectors disclosed herein are provided. In various embodiments, the vector or nucleic acid is integrated into the host cell genome, which in other embodiments the vector or nucleic acid is extra-chromosomal.
Recombinant cells, such as yeast, bacterial (e.g., E. coli), and mammalian cells (e.g., immortalized mammalian cells) comprising such a nucleic acid, vector, or combinations of either or both thereof are provided. In various embodiments, cells comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, are provided.
A vector comprising a nucleic acid sequence encoding a GDF15 mutant polypeptide provided herein can be introduced into a host cell by transformation or by transfection. Methods of transforming a cell with an expression vector are well known.
A nucleic acid encoding a GDF15 polypeptide-, construct comprising a GDF15 polypeptide or a mutant form thereof can be positioned in and/or delivered to a host cell or host animal via a viral vector. Any suitable viral vector can be used in this capacity. A viral vector can comprise any number of viral polynucleotides, alone or in combination with one or more viral proteins, which facilitate delivery, replication, and/or expression of the nucleic acid of the invention in a desired host cell. The viral vector can be a polynucleotide comprising all or part of a viral genome, a viral protein/nucleic acid conjugate, a virus-like particle (VLP), or an intact virus particle comprising viral nucleic acids and a GDF15 mutant polypeptide-encoding nucleic acid. A viral particle viral vector can comprise a wild-type viral particle or a modified viral particle. The viral vector can be a vector which requires the presence of another vector or wild-type virus for replication and/or expression (e.g., a viral vector can be a helper-dependent virus), such as an adenoviral vector amplicon. Typically, such viral vectors consist of a wild-type viral particle, or a viral particle modified in its protein and/or nucleic acid content to increase transgene capacity or aid in transfection and/or expression of the nucleic acid (examples of such vectors include the herpes virus/AAV amplicons). Typically, a viral vector is similar to and/or derived from a virus that normally infects humans. Suitable viral vector particles in this respect, include, for example, adenoviral vector particles (including any virus of or derived from a virus of the adenoviridae), adeno-associated viral vector particles (AAV vector particles) or other parvoviruses and parvoviral vector particles, papillomaviral vector particles, flaviviral vectors, alphaviral vectors, herpes viral vectors, pox virus vectors, retroviral vectors, including lentiviral vectors.
III.D. Isolation of a GDF15 Polypeptide, Construct Comprising a GDF15 Polypeptide or a Mutant Form Thereof
A GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof expressed as described herein can be isolated using standard protein purification methods. A GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof can be isolated from a cell that has been engineered to express a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, for example a cell that does not naturally express native GDF15.
Protein purification methods that can be employed to isolate a GDF15 mutant polypeptide, as well as associated materials and reagents, are known in the art. Exemplary methods of purifying a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof are provided in the Examples herein below. Additional purification methods that may be useful for isolating a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof can be found in references such as Bootcov M R, 1997, Proc. Natl. Acad. Sci. USA 94:11514-9, Fairlie W D, 2000, Gene 254: 67-76.
IV. PHARMACEUTICAL COMPOSITIONS COMPRISING A GDF15 POLYPEPTIDE, CONSTRUCT COMPRISING A GDF15 POLYPEPTIDE OR A MUTANT FORM THEREOF
Pharmaceutical compositions comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof are provided. Such polypeptide pharmaceutical compositions can comprise a therapeutically effective amount of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to one or more formulation agents suitable for accomplishing or enhancing the delivery of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof into the body of a human or non-human subject. The term includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In some cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. Pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof polypeptide can also act as, or form a component of, a carrier. Acceptable pharmaceutically acceptable carriers are preferably nontoxic to recipients at the dosages and concentrations employed.
A pharmaceutical composition can contain formulation agent(s) for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation agents include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides—preferably sodium or potassium chloride—or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 19th edition, (1995); Berge et al., J. Pharm. Sci., 6661), 1-19 (1977). Additional relevant principles, methods, and agents are described in, e.g., Lieberman et al., PHARMACEUTICAL DOSAGE FORMS-DISPERSE SYSTEMS (2nd ed., vol. 3, 1998); Ansel et al., PHARMACEUTICAL DOSAGE FORMS & DRUG DELIVERY SYSTEMS (7th ed. 2000); Martindale, THE EXTRA PHARMACOPEIA (31st edition), Remington's PHARMACEUTICAL SCIENCES (16th-20th and subsequent editions); The Pharmacological Basis Of Therapeutics, Goodman and Gilman, Eds. (9th ed.—1996); Wilson and Gisvolds' TEXTBOOK OF ORGANIC MEDICINAL AND PHARMACEUTICAL CHEMISTRY, Delgado and Remers, Eds. (10th ed., 1998). Principles of formulating pharmaceutically acceptable compositions also are described in, e.g., Aulton, PHARMACEUTICS: THE SCIENCE OF DOSAGE FORM DESIGN, Churchill Livingstone (New York) (1988), EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP (1998), incorporated herein by reference for any purpose).
The optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage (see, e.g., Remington's PHARMACEUTICAL SCIENCES, supra). Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof.
The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with free serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the present invention, compositions comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or an aqueous solution. Furthermore, a product comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof can be formulated as a lyophilizate using appropriate excipients such as sucrose.
The polypeptide pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.
The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
When parenteral administration is contemplated, the therapeutic compositions for use in this invention can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising a desired GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
In one embodiment, a pharmaceutical composition can be formulated for inhalation. For example, a GDF15 mutant polypeptide can be formulated as a dry powder for inhalation. GDF15 polypeptide inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions can be nebulized. Pulmonary administration is further described in International Publication No. WO 94/20069, which describes the pulmonary delivery of chemically modified proteins.
It is also contemplated that certain formulations can be administered orally. In one embodiment of the present invention, a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof that is administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
Another pharmaceutical composition can involve an effective quantity of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
Additional pharmaceutical compositions comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof will be evident to those skilled in the art, including formulations comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art (see, e.g., International Publication No. WO 93/15722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions, and Wischke & Schwendeman, 2008, Int. J. Pharm. 364: 298-327, and Freiberg & Zhu, 2004, Int. J. Pharm. 282: 1-18, which discuss microsphere/microparticle preparation and use). As described herein, a hydrogel is an example of a sustained- or controlled-delivery formulation.
Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and European Patent No. 0 058 481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22: 547-56), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid (European Patent No. 0 133 988). Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Epstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82: 3688-92; and European Patent Nos. 0 036 676, 0 088 046, and 0 143 949.
A pharmaceutical composition comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, which is to be used for in vivo administration typically should be sterile. This can be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
In a specific embodiment, the present invention is directed to kits for producing a single-dose administration unit. The kits can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).
The effective amount of pharmaceutical composition comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, which is to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage can range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg, 10 μg/kg, 15 μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45 μg/kg, 50 μg/kg, 55 μg/kg, 60 μg/kg, 65 μg/kg, 70 μg/kg, 75 μg/kg, 100 μg/kg, 200 μg/kg or up to about 10 mg/kg.
The frequency of dosing will depend upon the pharmacokinetic parameters of the GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems (which may also be injected); or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
Alternatively or additionally, the composition can be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
In order to deliver drug, e.g., a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, at a predetermined rate such that the drug concentration can be maintained at a desired therapeutically effective level over an extended period, a variety of different approaches can be employed. In one example, a hydrogel comprising a polymer such as a gelatin (e.g., bovine gelatin, human gelatin, or gelatin from another source) or a naturally-occurring or a synthetically generated polymer can be employed. Any percentage of polymer (e.g., gelatin) can be employed in a hydrogel, such as 5, 10, 15 or 20%. The selection of an appropriate concentration can depend on a variety of factors, such as the therapeutic profile desired and the pharmacokinetic profile of the therapeutic molecule.
Examples of polymers that can be incorporated into a hydrogel include polyethylene glycol (“PEG”), polyethylene oxide, polyethylene oxide-co-polypropylene oxide, co-polyethylene oxide block or random copolymers, polyvinyl alcohol, poly(vinyl pyrrolidinone), poly(amino acids), dextran, heparin, polysaccharides, polyethers and the like.
Another factor that can be considered when generating a hydrogel formulation is the degree of crosslinking in the hydrogel and the crosslinking agent. In one embodiment, cross-linking can be achieved via a methacrylation reaction involving methacrylic anhydride. In some situations, a high degree of cross-linking may be desirable while in other situations a lower degree of crosslinking is preferred. In some cases a higher degree of crosslinking provides a longer sustained release. A higher degree of crosslinking may provide a firmer hydrogel and a longer period over which drug is delivered.
Any ratio of polymer to crosslinking agent (e.g., methacrylic anhydride) can be employed to generate a hydrogel with desired properties. For example, the ratio of polymer to crosslinker can be, e.g., 8:1, 16:1, 24:1, or 32:1. For example, when the hydrogel polymer is gelatin and the crosslinker is methacrylate, ratios of 8:1, 16:1, 24:1, or 32:1 methyacrylic anhydride:gelatin can be employed.
V. THERAPEUTIC USES OF A GDF15 POLYPEPTIDE, CONSTRUCT COMPRISING A GDF15 POLYPEPTIDE OR A MUTANT FORM THEREOF
A GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, can be used to treat, diagnose or ameliorate, a metabolic condition or disorder. In one embodiment, the metabolic disorder to be treated is diabetes, e.g., type 2 diabetes. In another embodiment, the metabolic condition or disorder is obesity. In other embodiments the metabolic condition or disorder is dyslipidemia, elevated glucose levels, elevated insulin levels or diabetic nephropathy. For example, a metabolic condition or disorder that can be treated or ameliorated using a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, includes a state in which a human subject has a fasting blood glucose level of 125 mg/dL or greater, for example 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or greater than 200 mg/dL. Blood glucose levels can be determined in the fed or fasted state, or at random. The metabolic condition or disorder can also comprise a condition in which a subject is at increased risk of developing a metabolic condition. For a human subject, such conditions include a fasting blood glucose level of 100 mg/dL. Conditions that can be treated using a pharmaceutical composition comprising a GDF15 mutant polypeptide can also be found in the American Diabetes Association Standards of Medical Care in Diabetes Care-2011, American Diabetes Association, Diabetes Care Vol. 34, No. Supplement 1, S11-S61, 2010, incorporated herein by reference.
In application, a metabolic disorder or condition, such as Type 2 diabetes, elevated glucose levels, elevated insulin levels, dyslipidemia, obesity or diabetic nephropathy, can be treated by administering a therapeutically effective dose of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, to a patient in need thereof. The administration can be performed as described herein, such as by IV injection, intraperitoneal (IP) injection, subcutaneous injection, intramuscular injection, or orally in the form of a tablet or liquid formation. In some situations, a therapeutically effective or preferred dose of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, can be determined by a clinician. A therapeutically effective dose of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, will depend, inter alia, upon the administration schedule, the unit dose of agent administered, whether the GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof is administered in combination with other therapeutic agents, the immune status and the health of the recipient. The term “therapeutically effective dose,” as used herein, means an amount of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, that elicits a biological or medicinal response in a tissue system, animal, or human being sought by a researcher, medical doctor, or other clinician, which includes alleviation or amelioration of the symptoms of the disease or disorder being treated, i.e., an amount of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, that supports an observable level of one or more desired biological or medicinal response, for example lowering blood glucose, insulin, triglyceride, or cholesterol levels; reducing body weight; or improving glucose tolerance, energy expenditure, or insulin sensitivity.
It is noted that a therapeutically effective dose of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, can also vary with the desired result. Thus, for example, in situations in which a lower level of blood glucose is indicated a dose of a GDF15 mutant polypeptide will be correspondingly higher than a dose in which a comparatively lower level of blood glucose is desired. Conversely, in situations in which a higher level of blood glucose is indicated at a dose of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, will be correspondingly lower than a dose in which a comparatively higher level of blood glucose is desired.
In various embodiments, a subject is a human having a blood glucose level of 100 mg/dL or greater can be treated with a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof.
In one embodiment, a method of the instant disclosure comprises first measuring a baseline level of one or more metabolically-relevant compounds such as glucose, insulin, cholesterol, lipid in a subject. A pharmaceutical composition comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, is then administered to the subject. After a desired period of time, the level of the one or more metabolically-relevant compounds (e.g., blood glucose, insulin, cholesterol, lipid) in the subject is again measured. The two levels can then be compared in order to determine the relative change in the metabolically-relevant compound in the subject. Depending on the outcome of that comparison another dose of the pharmaceutical composition comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, can be administered to achieve a desired level of one or more metabolically-relevant compound.
It is noted that a pharmaceutical composition comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, can be co-administered with another compound. The identity and properties of compound co-administered with the GDF15 mutant polypeptide will depend on the nature of the condition to be treated or ameliorated. A non-limiting list of examples of compounds that can be administered in combination with a pharmaceutical composition comprising a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof, include rosiglitizone, pioglitizone, repaglinide, nateglitinide, metformin, exenatide, stiagliptin, pramlintide, glipizide, glimeprirideacarbose, and miglitol.
VI. KITS
Also provided are kits for practicing the disclosed methods. Such kits can comprise a pharmaceutical composition such as those described herein, including nucleic acids encoding the peptides or proteins provided herein, vectors and cells comprising such nucleic acids, and pharmaceutical compositions comprising such nucleic acid-containing compounds, which can be provided in a sterile container. Optionally, instructions on how to employ the provided pharmaceutical composition in the treatment of a metabolic disorder can also be included or be made available to a patient or a medical service provider.
In one aspect, a kit comprises (a) a pharmaceutical composition comprising a therapeutically effective amount of a GDF15 polypeptide, construct comprising a GDF15 polypeptide or a mutant form thereof; and (b) one or more containers for the pharmaceutical composition. Such a kit can also comprise instructions for the use thereof; the instructions can be tailored to the precise metabolic disorder being treated. The instructions can describe the use and nature of the materials provided in the kit. In certain embodiments, kits include instructions for a patient to carry out administration to treat a metabolic disorder, such as elevated glucose levels, elevated insulin levels, obesity, type 2 diabetes, dyslipidemia or diabetic nephropathy.
Instructions can be printed on a substrate, such as paper or plastic, etc, and can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, such as over the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded.
Often it will be desirable that some or all components of a kit are packaged in suitable packaging to maintain sterility. The components of a kit can be packaged in a kit containment element to make a single, easily handled unit, where the kit containment element, e.g., box or analogous structure, may or may not be an airtight container, e.g., to further preserve the sterility of some or all of the components of the kit.
EXAMPLES
The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.
Example 1
Preparation of Native GDF15 Polypeptides
CHO—S stable cell line growth and transfection was carried out using six-well plates containing a transfection medium (CD-CHO, 4×L-Glutamine, 1×HT, 1×P/S/G). The day before transfection, cultured cells were split to a concentration of 5e5 viable cells (vc)/ml. A transfection complex formation was made using 4 μg Linear DNA/500 Optimem, 10 μl LF-LTX/500 μl Optimem, and a 20 minute incubation period. Cells were then prepared by centrifugation at 1e6 vc/well, washed in DPBS, and resuspend in 1 ml Optimem. Next, 1 ml of transfection complex was added to 1e6 cells in the 6-well plates and incubated for 6 hours with shaking followed by addition 2 ml of MIX-6 Media (w/o antibiotics). Selection was started on the second day using MIX-6 Media with 6 μg/ml Puromycin and replacing media every 2-3 days for 10-12 days by centrifugation of the cells and re-suspension in media.
Cells that were transformed with a GDF15 expression vector constructed with an affinity tag were grown to an optical density of 9 at 600 nm and then induced and harvested at an optical density of 63 by centrifugation 6 hours later. Frozen cell paste was thawed and re-suspended into buffer at 15% (wt./vol.) with a low shear homogenizer until the slurry was homogeneous. The cells were then subjected to high shear homogenization to break open and release product-containing inclusion bodies. The resulting homogenate was then centrifuged at 5,000×g for an hour at 5 C to harvest the inclusion bodies as a pellet, leaving the cytoplasmic contaminants in the discarded supernatant. The residual cytoplasm is washed from the inclusion bodies by homogeneously re-suspending the pellet to the original homogenate volume using chilled water and a low shear homogenizer followed by centrifugation as before. The resulting pellet, washed inclusion bodies (WIBS), is then frozen at −80 C.
A sufficient amount of WIBS and guanidine hydrochloride (GnHCl) was used at pH 8.5 in a reducing-solubilization to result in approximately 25 mg/ml reduced product and 6 M GnHCl final concentrations. The solubilization was then rapidly diluted 25-fold with stirring into a refolding buffer containing redox reagents, chaotrope and co-solvents at alkaline pH. The refold solution was allowed to gently stir and air oxidize at 6 C for 72 hours or until the solution was negative to Ellman's reagent. The refold solution at 5 C was then clarified by depth filtration to allow for a 10-fold ultra-filtration concentration and subsequent diafiltration into a buffer containing 50 mM sodium phosphate and low chaotrope concentration at pH 8.5. The subsequent retentate was warmed to 25 C and then the pH lowered into the acidic range to cause precipitation of contaminants. The precipitate was removed by centrifugation at 5,000×g for 30 min at 25 C and the resulting supernatant further clarified by 0.45 um filtration. The filtrate (AP) was then adjusted to pH 8.5, and low salt concentration to permit the first step of purification involving immobilized metal affinity chromatography (IMAC).
Following protein folding and AP, the GDF15 was purified using a two-step chromatography train. The adjusted AP was applied to an IMAC column that is equilibrated with buffered chaotrope containing a low salt concentration at pH 8.5. The column was next washed with equilibration buffer until a baseline ultraviolet (UV) level is obtained. Product and contaminants are eluted by step-wise increases in displacer concentration and the elutions were collected and subsequently assayed by Coomasie-stained SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) to identify which eluate fractions contained a polypeptide that migrates at the predicted molecular weight of GDF15. After the IMAC was completed, the pooled fraction containing product is adjusted to pH 7.2 and 5 mM EDTA at 25 C. The product was converted into the mature length GDF15 by adding a low concentration of an enzyme to cleave off the affinity tag at 25 C for several hours. The cleavage reaction mixture was adjusted with an organic modifier and acidic pH by the addition of acetic acid and organic solvent. This allowed for the final chromatography step consisting of a linear gradient elution of product from a reverse phase column conducted at 25 C. The elution from the chromatography was collected as fractions and then assayed by SDS-PAGE to determine the appropriate fractions to pool for homogeneous product. The resulting pool was buffer exchanged by diafiltration into a weakly acidic buffer, concentrated by ultra-filtration, sterile filtered, and finally stored at 5 C or frozen.
Example 2
Suppression of Food Intake in Hyperphagic Ob/Ob Mice by Fc Fusion GDF15 Proteins
GDF15 reduces food intake in hyperphagic ob/ob mice, and a food intake assay was used to evaluate efficacy of different forms of GDF15 analogs. As the half-life of human GDF15 polypeptide in mouse was observed to be approximately 3 hours, an Fc fusion strategy was used to extend protein half-life. Different Fc fusion GDF15 polypeptides were generated and analyzed for in vivo GDF15 activity, by introducing the Fc fusion GDF15 and wild type GDF15 polypeptides into hyperphagic leptin-deficient ob/ob mice, and measuring the ability of a particular Fc fusion GDF15 polypeptide to suppress food intake in these animals. The Fc fusion GDF15 polypeptide to be tested was injected subcutaneously into a 7-8 week old ob/ob mouse (Jackson Laboratory) between 4-5 pm on day 0. Animals were transferred after injection to cages where food had been premeasured, and food intake was measured between 9-10 AM the next day.
The results of representative experiments for wild type GDF15 and each Fc fusion GDF15 polypeptide are provided in FIGS. 3-21, which show dose response curves in the food intake assay for the dimers of the charged pair (delHinge) constructs: DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+); DhCpmFc(+)-(G4S)4-GDF15:DhCpmFc(−); DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+); DhCpmFc(+)-(G4S)4-GDF15(H6D):DhCpmFc(−); DhCpmFc(+)-(G4S)4-GDF15 (N3Q):DhCpmFc(−); DhCpmFc(+)-GDF15:DhCpmFc(−); DhCpmFc(+)-G4-GDF15:DhCpmFc(−); DhCpmFc(+)-(G4S)2-GDF15:DhCpmFc(−); DhCpmFc(+)-(G4Q)4-GDF15:DhCpmFc(−); DhCpmFc(+)-(1K)-GDF15:DhCpmFc(−); DhCpmFc(+)(L351C)-(G4S)4-GDF15:DhCpmFc(−)(L351C); and DhCpmFc(+)(S354C)-(G4S)4-GDF15:DhCpmFc(−) (Y349C); dimers of the charged pair construct DhCpmFc(+)(S354C)-(G4S)4-GDF15:DhCpmFc(−)(Y349C); dimers of the hemiFc constructs Fc-(G4S)8-Fc-GS(G4S)4-GDF15; Fc-(G4S)3-Fc-GS(G4S)4-GDF15; and Fc-(G4S)5-Fc-GS(G4S)4-GDF15; native mature hGDF15 homodimer; and mature hGDF15(H6D) variant homodimer.
These experiments demonstrated that the dimers of the charged pair (delHinge), charged pair and hemiFc constructs exhibit a decrease in food intake in ob/ob mice, with greater potency than those of the native mature hGDF15 homodimer The food intake measurement was taken 17 hours after a single injection of the polypeptides, and the stronger potency and efficacy may have resulted from an extended half-life of the Fc fusion polypeptides.
Example 3
Improvement of Lipid Tolerance by Fc Fusion GDF15 Constructs
Dimers of charged pair (delHinge) and hemiFc constructs were analyzed for in vivo GDF15 activity, by introducing these constructs, as well as native mature hGDF15 homodimer, into obese B6D2F1 mice (obesity was induced by feeding the mice a 60% high fat diet), and measuring the ability of each particular polypeptide or construct to improve oral lipid tolerance in these animals.
Male B6D2F1 mice were fed 60% high fat diet (Research Diets D12492i) at 5 weeks old for 6-10 weeks and were stratified by body weight and 3 hour fasting serum triglyceride levels 2-4 days before study. On the day of the study, a dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct (see Section II.B.1), a dimer of Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct (see Section II.D.1), and native mature hGDF15 homodimer were injected intravenously into mice that had been fasted for 3 hours. In the dose response studies for native mature hGDF15 or or mature hGDF15(H6D) variant, proteins were injected subcutaneously. Three hours after protein injection, 20% Intralipid was orally administered at 20 ml/kg. Another 90 min later, blood samples were collected through tail bleeding and serum triglyceride levels were measured.
The results of representative experiments involving the native mature hGDF15 homodimer, the mature hGDF15(H6D) variant homodimer, the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct, and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct are shown in FIGS. 22-25. In FIGS. 23-25, the native mature hGDF15 homodimer, the mature hGDF15(H6D) variant and the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct improved lipid tolerance in a dose dependent fashion. In FIG. 22, the native mature hGDF15 homodimer and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct are shown to be efficacious in the same assay at 1 mg/kg IV dose.
Example 4
Chronic Efficacy of hGDF15 and Constructs Comprising hGDF15 in DIO Mice
GDF15 lowers blood glucose, insulin, triglyceride, or cholesterol levels; reduces body weight; or improves glucose tolerance and insulin sensitivity. To assess the ability of hGDF15 and various Fc fusion GDF15 constructs to improve these parameters, chronic studies were performed.
Example 4.1 Study Design
Two separate studies were conducted to evaluate the chronic efficacy of different Fc fusion GDF15 polypeptides. The first study (Study #1) included the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct (see Section II.B.1) and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct (see Section II.D.1). In this study, the dimer of the charged pair (delHinge) construct and the dimer of the hemiFc constructs were administered chronically and subcutaneously into male C57Bl/6 mice (HAR) fed 60 Kcal % high fat diet (Research Diets) at 4 weeks of age for 16 weeks. Animals were acclimated to single housing conditions for 6 weeks. Weekly handling and subcutaneous injection of saline were performed for 3 weeks prior to administration of any test compound. Mice were divided into 7 groups of 12 and one group of 6 based on weekly food intake, and body weight, 4 hr fasting blood glucose levels, 4 hr fasting serum insulin levels, triglyceride and cholesterol levels 2 days before the first protein injections. Three different dose levels were selected: 10, 1, 0.1 nmol/kg, which are equivalent to 1.29, 0.129, 0.0129 mg/kg for the dimer of the charged pair (delhinge) construct, or 1.35, 0.135, 0.0135 mg/kg for the dimer of the hemiFc construct. The dosing regimen of weekly dosing was selected based on mouse PK data. One group of 12 animals was subcutaneously injected with vehicle buffer weekly as control group. One group of 6 animals was subcutaneously injected with vehicle buffer and given diet containing 0.014% rosiglitazone to target 10 mg/kg oral daily dose as positive control. Studies were carried out for 5 weeks, with the last dose on day 28. The results are shown in FIGS. 26-38.
The second study (Study#2) included the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct (see Section II.B.1) and the dimer of the CpmFc(−)-(G4S)4-GDF15:CpmFc(+) construct (see Section II.C). In this study, the dimer of the charged pair (delHinge) construct and the dimer of the charged pair construct were administered chronically and subcutaneously into male C57Bl/6 mice (HAR) fed 60 Kcal % high fat diet (Research Diets) at 4 weeks of age for 15 weeks. Animals were acclimated to single housing conditions for 5 weeks. Weekly handling and subcutaneous injection of saline were performed for 3 weeks prior to administration of any test compound. Mice were divided into groups of 12 based on weekly food intake, and body weight, 4 hr fasting blood glucose levels, 4 hr fasting serum insulin, triglyceride and cholesterol levels 4 days before the first protein injection. Three different dose levels were selected at 10, 1, 0.1 nmol/kg, equivalent to 1.25, 0.125, 0.0125 mg/kg for the dimer of the charged pair construct. Two different dose levels were selected at 10 and 1 nmol/kg, equivalent to 1.29 and 0.129 mg/kg, for the dimer of the charged pair (delHinge) construct. Dosing regimen of weekly dosing was selected based on mouse PK data. One group of 12 animals was subcutaneously injected with vehicle buffer weekly as control group. One group of 6 animals was subcutaneously injected with vehicle buffer and given diet containing 0.014% rosiglitazone to target 10 mg/kg oral daily dose as positive control. Studies were carried for 5 weeks with the last dose on day 28. The results are shown in FIGS. 39-54.
Body weight and food intake were measured weekly throughout the study. One oral glucose tolerance tests (OGTT) was performed 2 weeks after the first protein injection in animals fasted for 4 hours. One oral glucose tolerance tests (OGTT) was performed 5 weeks after the first protein injection in animals fasted for 16 hours. Blood samples were collected from 4 hr fasted animals at 2 and 4 weeks after first protein injection, from animals with free access to food 3 weeks after the first protein injection, and from animals fasted for 16 hr 5 weeks after the first protein injection. Serum samples were used to measure insulin, triglyceride and cholesterol levels, as well as the levels of test compound.
Example 4.2 Effect of Test Compounds on Body Weight
Body weight was followed weekly throughout the study, both pre- and post-administration of test compounds. Body weight from baseline of the vehicle animals increased with time, whereas body weight of animals treated with 10 or 1 nmol/kg of the dimer of the charged pair (delHinge) construct, the dimer of the charged pair construct and the dimer of the hemiFc construct decreased over the course of the 5 week treatment period and started to come back during the compound wash-out phase, as shown in FIGS. 30 and 43. The change of body weight in animals treated with 1 nmol/kg of the dimer of the charged pair (delHinge) construct, the dimer of the charged pair construct and the dimer of the hemiFc constructs did not reach statistical significance.
Example 4.3 Effect of Test Compounds on Food Intake
Food intake was measured every week. Average daily food intake was calculated by dividing weekly food intake by 7. At 10 nmol/kg, the dimer of the charged pair (delHinge) construct, the dimer of the charged pair construct and the dimer of the hemiFc construct lowered food intake, with statistical significance in most weekly measurements, as demonstrated in FIGS. 31 and 44. The effect at 1 nmol/kg dose was less significant, but a trend was clear. The food intake in animals treated with 1 nmol/kg of each of the three constructs (i.e., the dimer of the charged pair (delHinge) construce, the dimer of the charged pair construct and the dimer of the hemiFc constructs) were lower compared to food intake in animals treated with vehicle buffer.
Example 4.4 Effect of Test Compounds on Glucose and OGTT
OGTTs (OGTT1 and OGTT2) were performed after treatment was initiated. OGTT1 was conducted 14 days after the first protein injection, in animals fasted for 4 hr from 6 am. 4 hr fasting glucose levels before the oral glucose challenge were compared with the baseline 4 hr fasting glucose levels measured 2 days or 4 days before protein injection (FIGS. 32 and 45). The glucose profile of OGTT1 is shown in FIGS. 26, 27, 39 and 40. Animals treated with 10 and 1 nmol/kg of the dimer of the charged pair (delHinge) construct, the dimer of the charged pair Fc-hGDF15 construct or the dimer of the hemiFc constructs exhibited improved glucose levels and oral glucose tolerance when compared to vehicle treated animals, as demonstrated by glucose levels before and during OGTT and glucose AUC during the OGTT. Animals treated with 0.1 nmol/kg of the dimer of the charged pair (delHinge) construct, the dimer of the charged pair construct and the dimer of the hemiFc construct exhibited improved glucose tolerance, but the improvement in glucose levels in these animals was not as significant. The improved glucose tolerance in 0.1 nmol/kg dimer of the hemiFc construct treated animals 2 weeks after the first protein injection is indicative of improved glucose tolerance independently from body weight or food intake changes, since these animals have similar body weight and food intake to vehicle treated animals.
OGTT2 was conducted 35 days after the first protein injection, 7 days after the last protein injection, in animals fasted for 16 hr from 5 pm the day before the oral glucose challenge. The glucose profile of OGTT2 is shown in FIGS. 28, 29, 41 and 42. Treatment with 10, 1, or 0.1 nmol/kg of each of the three constructs (i.e., the dimer of the charged pair (delHinge) construct, the dimer of the charged pair construct and the dimer of the hemiFc construct) for 5 weeks significantly improved oral glucose tolerance, as demonstrated by glucose levels and glucose curve AUC during the OGTT.
Example 4.5 Effect of Test Compounds on Insulin
Insulin levels were measured in blood samples that had been collected from 4 hr fasted animals on day 4, day 14 and day 28, from animals with free access to food on day 21, and from animals fasted for 16 hr on day 35. Mice consume majority of their food during the dark cycle. The protocol of 4 hr fasting from 6 am was used for longitudinal comparison within the same group to remove variation induced by nibbling of food during the light cycle. Observed insulin levels are shown in FIGS. 33, 34 and 46-48.
The 4 hr fasting insulin levels in vehicle treated animals increased over time, as insulin resistance further progresses in these animals. Treatment with 10 or 1 nmol/kg of the dimer of the charged pair (delHinge) construct, the dimer of the charged pair construct or the dimer of the hemiFc construct brought insulin levels to levels lower than where the animals started before the treatment was initiated, indicative of a reversal of high fat diet-induced insulin resistance. 0.1 nmol/kg dosage prevented the increase of insulin levels over time.
All three Fc fusion GDF15 constructs also demonstrated similar dose-dependent improvement in insulin levels in animals fasted overnight on day 35. Data collected for Study #2 demonstrated that the dimer of the charged pair (delHinge) construct and the dimer of the charged pair construct lower insulin levels in animals with free access to food.
Example 4.6 Effect of Test Compounds on Triglyceride Levels
Triglyceride levels were measured in blood samples that had been collected from 4 hr fasted animals at day −4, day 14 and day 28, from animals with free access to food on day 21, and from animals fasted for 16 hr on day 35. Mice consume majority of their food during the dark cycle. The protocol of 4 hr fasting from 6 am was used for longitudinal comparison within the same group to remove variation induced by nibbling of food during the light cycle.
The effect of the dimer of the charged pair (delHinge) construct, the dimer of the charged pair construct and the dimer of the hemiFc constructs on triglyceride levels in animals fasted for 4 hr or 16 hr were not robust but statistically significant, as demonstrated in FIGS. 35, 36, 49 and 51. At 10 nmol/kg, the dimer of the charged pair (delHinge) Fc-construct consistently lowered serum triglyceride levels at day 28 and day 35. Data collected from Study #2 demonstrated that in animals with free access to food, the effect of 10 and 1 nmol/kg of both the dimer of the charged pair (delHinge) construct and the dimer of the charged pair construct were statistically significant, as shown in FIG. 50. We have demonstrated in earlier sections that GDF15 improves oral lipid tolerance. The observation of more potent efficacy in lowering serum triglyceride levels in animals with free access food may indicate improvement of postprandial lipid profile and may have resulted from the improvement in oral/diet lipid tolerance.
Example 4.7 Effect of Test Compounds on Cholesterol Levels
Total cholesterol levels were measured in blood samples that had been collected from 4 hr fasted animals at day −4, day 14 and day 28, from animals with free access to food on day 21, and from animals fasted for 16 hr on day 35. Mice consume majority of their food during the dark cycle. The protocol of 4 hr fasting from 6 am was used for longitudinal comparison within the same group to remove variation induced by nibbling of food during the light cycle.
All three constructs (i.e., the dimer of the charged pair (delHinge) construct, the dimer of the charged pair construce and the dimer of the hemiFc construct) dose-dependently lowered total cholesterol levels, and this effect was impacted by the feeding state of the animals, as demonstrated in FIGS. 37, 38 and 52-54.
Conclusions From Example 4
All three constructs, namely the dimer of the charged pair (delHinge) construct, the dimer of the charged pair construct and the dimer of the hemiFc construct, demonstrated efficacy in improving various metabolic parameters, including body weight, blood glucose levels and glucose tolerance, serum insulin levels, serum cholesterol levels, serum triglyceride levels and oral lipid tolerance. The polypeptides were injected once per week, and efficacy was dose-dependent, with 10 nom/kg dosage demonstrating the most robust efficacy.
Example 5
Chronic Efficacy of hGDF15 and Constructs Comprising hGDF15 in Obese Cynomolgous Monkey
The charged pair (delHinge) construct “DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+)” and the hemiFc construct “Fc-(G4S)8-Fc-GS(G4S)4-GDF15” were analyzed for in vivo GDF15 activity by introducing these constructs, as well as vehicle (A4.5Su) into obese cynomolgous monkeys.
Example 5.1 Study Design
The study was conducted in cynomolgous monkeys. The monkeys were 11-14 years old. Their body weights ranged from 7-10 kg and BMI ranged from 38-58 kg/m2. 40 monkeys were acclimated for 6 weeks prior to the initiation of compound administration. During the acclimation period, monkeys were trained 4 times a week for 6 weeks to familiarize the procedures including chair-restrain, subcutaneous injection (PBS, 0.1 ml/kg), gavage (Water, 10 ml/kg), blood drawn for non OGTT and OGTT samples. During 6 weeks of training, baseline OGTT and plasma metabolic parameters were measured. 30 out of 40 monkeys were selected and randomized into three treatment groups to achieve similar baseline levels of body weight, OGTT AUC response, and plasma glucose, insulin and triglyceride levels.
The study was conducted in a blind fashion. Vehicle (n=10), dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct (n=10) and dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct (n=10) were labeled as compound A, B and C and administered once a week via subcutaneous injection. Compounds were given at 1.5 mg/kg/week. After 6 weeks of compound treatments, animals were monitored for additional 5 weeks for compound washout and recovery from treatments. Food intake, body weight, clinical chemistry and OGTT were monitored throughout the study. Food intake was measured at every meal. Body weight was measured weekly. Blood samples were collected weekly 6 days post each injection to measure glucose, triglyceride. OGTTs were conducted on day 13, 34 and 55 after the initiation of treatments. The day starting the treatment is designated as 0 and the detailed study design is shown in FIG. 55.
The results shown in this example are data collected at the end of 6 weeks treatment and during the washout.
Example 5.2 Compounds Exposure
Compounds (dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct) exposure measurements were performed on samples collected after an overnight fast and the day after in non fasted animals. These measurements were made every week during the treatment and washout phase. The dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct showed a greater exposure than the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct. Two out of ten animals in the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct group presented antibody whereas five out of ten animals were identified in the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct group. The loss of pharmacodynamic responses in suspected animals with antibody correlates with loss of drug exposure. The exposure levels for each compound are shown in FIG. 56.
Example 5.3 Effect of Test Compounds on Oral Glucose Tolerance Test (OGTT)
OGTTs were conducted before and after initiation of treatments. FIGS. 57-64 show pre- and post-OGTT area under the OGTT curve (AUC). Acclimation OGTT was performed before the treatment was initiated, post-dose OGTTs were performed on week 2 and week 5 and washout OGTT was performed on week 8 (3 weeks after the last dose). Glucose and insulin (AUC) were measured for glucose (FIGS. 57-60) and insulin (FIGS. 61-64). The dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct significantly improved OGTT glucose AUC on week 2 and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct on week 5. OGTT Glucose AUC was still improved on week 8 (during washout phase with the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15) construct. OGTT insulin AUC was significantly ameliorated on week 2 and 5 for the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct and on week 2 only for the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct.
Example 5.4 Effect of Test Compounds on Fasting Insulin Levels
Blood was collected from overnight fasted animals. The blood drawn was conducted weekly at 6 days post each injection. Both the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct reduced fasting blood insulin levels. This effect was not seen during the washout period. FIG. 68 shows the levels of fasting insulin during the course of the study.
Example 5.5 Effect of Test Compounds on Food Intake
Animals were fed twice a day and food intake was recorded at each meal. The feeding times were from 8:00 AM to 9:00 AM (±30 minutes) and then from 4:00 PM to 5:00 PM (±30 minutes). Apple (150 g) was supplied to each animal at 1:30-2:30 PM (±30 minutes) every day.
Compared with vehicle, both the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct reduced food intake in monkeys (FIG. 66). However, the effect diminished and the food intake returned close to baseline or control levels after about 30 days of treatment.
Example 5.6 Effect of Test Compounds on Triglyceride Levels
Blood was collected from overnight fasted animals. The blood drawn was conducted weekly at 6 days post each injection. Triglyceride levels were significantly reduced in animals treated with the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct throughout the treatment phase. This effect diminished during the washout period. FIG. 67 shows the levels of fasting plasma triglycerides during the course of the study.
Example 5.7 Effect of Test Compounds on Body Weight
Body weight was monitored weekly throughout the study. Over the course of the 6 week treatments, the body weight of animals treated with vehicle remained constant or slightly increased while body weight of animals treated with the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct progressively decreased as shown in FIG. 65.
Conclusions from Example 5
In a study conducted in male obese cynomolgous monkeys, animals treated with the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct showed improved metabolic parameters. Body weight was reduced. Short-term reduction of food intake was observed and the effect diminished and the food intake recovered to baseline or control levels at the mid term of the study. Fasting triglyceride levels were also reduced by both compounds, the dimer of the DhCpmFc(−)-(G4S)4-GDF15:DhCpmFc(+) construct and the dimer of the Fc-(G4S)8-Fc-GS(G4S)4-GDF15 construct. OGTT glucose and insulin AUC were improved.
While the present invention has been described in terms of various embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the invention as claimed. In addition, the section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
All references cited in this application are expressly incorporated by reference herein for any purpose.

## Claims

1. A fusion protein comprising:
(a) a GDF15 polypeptide comprising the amino acid sequence of SEQ ID NOs: 12, 14 or a variant thereof, wherein the variant is a GDF15(H6D) variant comprising the amino acid sequence of SEQ ID NO: 38, or a GDF15(N3Q) variant comprising the amino acid sequence of SEQ ID NO: 42: and
(b) a negatively charged Fc sequence comprising two lysine-to-aspartate mutations.

2. The fusion protein of claim 1, wherein the Fc sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 23, 86, 91, 100, 108, 111, and 115.

3. A dimer comprising the fusion protein of claim 1, and a second Fc sequence comprising a charged pair mutation, wherein the second Fc sequence is a positively charged Fc sequence.

4. The fusion protein of claim 1, fused to a second Fc sequence comprising a charged pair mutation.

5. The fusion protein of claim 1, wherein the GDF15 polypeptide and the Fc sequence are joined by a linker.

6. The fusion protein of claim 5, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 57, and 113.

7. The fusion protein of claim 4, wherein the second Fc sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:18, 19, 23, 85, 86, 89, 90, 91, 99, 100, 108, 109, 110, 111, 114 and 115.

8. The dimer of claim 3, wherein the second Fc sequence comprises an amino acid sequence the selected from the group consisting of SEQ ID NOs: 18, 85, 89, 90, 99, 109, 110, and 114.

9. The dimer of claim 8, comprising:
(a) an Fc sequence comprising the amino acid sequence of SEQ ID NO: 18 and a fusion protein comprising the amino acid sequence of SEQ ID NO: 44;
(b) an Fc sequence comprising the amino acid sequence of SEQ ID NO: 18 and a fusion protein comprising the amino acid sequence of SEQ NO: 113: or
(c) an Fc sequence comprising the amino acid sequence of SEQ ID NO: 18 and a fusion protein comprising the amino acid sequence of SEQ ID NO: 57.

10. A tetramer comprising the dimer of claim 3.

11. The dimer of claim 3, wherein the dimer comprises an Fc sequence comprising the amino acid sequence of SEQ ID NO: 110 and a fusion protein comprising the amino acid sequence of SEQ ID NO: 113.

12. A tetramer comprising the dimer of claim 11.

13. The fusion protein of claim 5, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 31, 34, 80, 87, 88 and 131.

14. The fusion protein of claim 1, wherein the C-terminus of the GDF polypeptide is joined to the N-terminus of the Fc sequence.

15. The fusion protein of claim 14, further comprising a second Fc sequence, wherein the C-terminus of the fusion protein is joined to the C-terminus of the second Fc sequence.

16. The fusion protein of claim 15, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 123, and 128.

17. The fusion protein of claim 3, wherein the charged pair mutation is an aspartic acid to lysine mutation or a glutamic acid to lysine mutation.

18. The fusion protein of claim 3, wherein the wherein the second Fc sequence comprises an aspartic acid to lysine mutation and a glutamic acid to lysine mutation.