BR112017005986B1 - COMPLEX COMPRISING A FIRST HETERODIMER AND A SECOND HETERODIMER, PHARMACEUTICAL COMPOSITION AND USE OF THE COMPLEX - Google Patents

Abstract

COMPOSITIONS AND METHODS OF USE FOR THE TREATMENT OF METABOLIC DISORDERS. It is a complex that comprises a GDF15 polypeptide. Methods for treating individuals with a metabolic disorder, such as a glucose metabolism disorder and/or a body weight disorder, and compositions associated therewith are provided.

Description

INCORPORATION BY REFERENCE FROM THE SEQUENCE LISTING

[001] A sequence listing is provided here as a text file, “NGMB-142_SeqList.txt” created on October 28, 2015 and having a size of 188 KB. The contents of the text file are incorporated herein by reference in their entirety.

CROSS REFERENCE TO RELATED ORDERS

[002] This application claims priority benefit from the US Provisional Application. 62/073,737, filed October 31, 2014, and U.S. Provisional Patent Application Serial No. 62/244,604 filed October 21, 2015, which applications are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[003] The present invention relates, among other things, to polypeptide complex and compositions thereof that are useful in the treatment of disorders related to metabolism.

INTRODUCTION

[004] Obesity is most commonly caused by excessive food intake, coupled with limited energy expenditure and/or lack of physical exercise. Obesity increases the risk of developing several diseases, such as diabetes mellitus, hypertension, atherosclerosis, coronary artery disease, sleep apnea, gout, rheumatism and arthritis. Furthermore, the risk of mortality is directly correlated with obesity, such that, for example, a body mass index greater than 40 results in a decrease in average life expectancy of more than 10 years.

[005] Current pharmacological treatment modalities include appetite suppressants targeting receptor classes (e.g., CB1, 5-HT2C and NPY); regulators of appetite circuits in the hypothalamus and the molecular actions of ghrelin; and lipase-targeted nutrient absorption inhibitors. Unfortunately, none of the current modalities have been shown to effectively treat obesity without causing adverse effects, some of which can be very serious.

[006] High blood glucose levels stimulate insulin secretion by pancreatic beta cells. Insulin, in turn, stimulates the entry of glucose into fat cells and muscles, leading to the storage of glycogen and triglycerides and the synthesis of proteins. Activation of insulin receptors in various cell types decreases circulating glucose levels by increasing glucose uptake and utilization and reducing hepatic glucose production. Disruptions within this regulatory network can result in diabetes and associated pathological syndromes that affect a large and growing percentage of the human population.

[007] Patients who have a glucose metabolism disorder may suffer from hyperglycemia, hyperinsulinemia and/or glucose intolerance. An example of a disorder that is often associated with aberrant glucose and/or insulin levels is insulin resistance, in which liver, fat and muscle cells lose their ability to respond to normal blood insulin levels.

[008] In view of the prevalence and severity of obesity, diabetes and associated metabolic and non-metabolic disorders, treatment modalities that modulate, for example, appetite, glucose and/or insulin levels and increase biological response to fluctuating glucose levels in a patient remain of interest.

[009] Wild-type GDF15, also known as MIC-1 (macrophage inhibitory cytokine-1) has been linked to the regulation of body weight (Tsai VW, et al., PLoS One 2013; 8 (2): e55174; US8 ,192,735).

SUMMARY

[010] Modified GDF15 polypeptides for the treatment of metabolic disorders are provided. The modified GDF15 polypeptides may be present in a complex. A complex of the present disclosure may include two GDF15 polypeptides.

[011] In certain cases, a complex of the present invention comprises a first polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH 3 sequence comprising at least one engineered protuberance; and a second polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH 3 sequence comprising at least one engineered cavity, wherein the first polypeptide dimerizes with the second polypeptide through positioning of the bulge of the first polypeptide in the cavity of the second polypeptide and wherein the C-terminus of the first polypeptide or the C-terminus of the second polypeptide is conjugated to the N-terminus of a GDF15 mutein comprising at least one N-linked glycosylation consensus site.

[012] In certain cases, a complex of the present invention comprises a first heterodimer and a second heterodimer, each of the first heterodimer and the second heterodimer comprising a first polypeptide and a second polypeptide, the first polypeptide comprising an IgG Fc sequence , the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered protuberance; and the second polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered pocket; wherein the first polypeptide dimerizes with the second polypeptide by positioning the protuberance of the first polypeptide in the cavity of the second polypeptide, wherein the C-terminus of the first polypeptide or the C-terminus of the second polypeptide is conjugated to the N-terminus of a GDF15 mutein comprising at least one N-linked glycosylation consensus site, and wherein the GDF15 mutein in the first heterodimer dimerizes with the GDF15 mutein in the second heterodimer thereby forming the complex comprising the first heterodimer and the second heterodimer .

[013] In exemplary embodiments, the C-terminus of the first polypeptide can be conjugated to the N-terminus of the GDF15 mutein. In other cases, the C-terminus of the second polypeptide may be conjugated to the N-terminus of the GDF15 mutein.

[014] Also contemplated herein is a first polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered protuberance, wherein the first polypeptide dimerizes with a second polypeptide comprising a IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered pocket; and a GDF15 mutein comprising at least one N-linked glycosylation consensus site, wherein the C-terminus of the first polypeptide is conjugated to the N-terminus of the GDF15 mutein. The first polypeptide may be present in a complex that may also include the second polypeptide.

[015] Also contemplated herein is a first polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered pocket, wherein the first polypeptide dimerizes with a second polypeptide comprising a IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered protuberance; and a GDF15 mutein comprising at least one N-linked glycosylation consensus site, wherein the C-terminus of the first polypeptide is conjugated to the N-terminus of the GDF15 mutein. The first polypeptide may be present in a complex that may also include the second polypeptide.

[016] In certain cases, the GDF15 mutein in the complex may comprise a contiguous amino acid sequence that is at least 90% identical to the wild-type GDF15 amino acid sequence (SEQ ID NO:1). For example, the GDF15 mutein may include at least one substitution of the corresponding amino acid in SEQ ID NO:1, which creates the N-linked glycosylation consensus site, for example, the substitution may be D5T or D5S. In other cases, the replacement may be R21N.

[017] In exemplary cases, the GDF15 mutein can include at least one of the following pairs of substitutions of the corresponding amino acids in SEQ ID NO:1 that create the consensus site of glycosylation linked to N:R16N and H18T/S; S23N and E25T/S; L24N and D26T/S; S50N and F52T/S; F52N and A54T/S; Q51N and R53T/S; R53N and A55T/S; S64N and H66T/S; L65N and R67T/S; S82N and N84T/S; K91N and D93T/S; D93N and G95T/S; T94N and V96T/S; V96N and L98T/S; S97N and Q99T/S; and A106N and D108T/S.

[018] In exemplary cases, the GDF15 mutein can include at least one of the following pairs of substitutions of the corresponding amino acids in SEQ ID NO:1 that create the consensus site of glycosylation linked to N:R16N and H18T; S23N and E25T; L24N and D26T; S50N and F52T; F52N and A54T; Q51N and R53T; R53N and A55T; S64N and H66T; L65N and R67T; S82N and N84T; K91N and D93T; D93N and G95T; T94N and V96T; V96N and L98T; S97N and Q99T; and A106N and D108T.

[019] In some cases, the GDF15 mutein may include at least one of the following pairs of substitutions of the corresponding amino acids in SEQ ID NO:1 that create the consensus site of glycosylation linked to N:R16N and H18S; S23N and E25S; L24N and D26S; S50N and F52S; F52N and A54S; Q51N and R53S; R53N and A55S; S64N and H66S; L65N and R67S; S82N and N84S; K91N and D93S; D93N and G95S; T94N and V96S; V96N and L98S; S97N and Q99S; and A106N and D108S.

[020] In certain embodiments, the GDF15 mutein may include at least one of the following pairs of substitutions of the corresponding amino acids in SEQ ID NO:1:S23N and E25T/S; R53N and A55T/S; S64N and H66T/S; K91N and D93T/S; D93N and G95T/S; S97N and Q99T/S; and A106N and D108T/S.

[021] In certain embodiments, the GDF15 mutein may include at least one of the following pairs of substitutions of the corresponding amino acids in SEQ ID NO:1:S23N and E25T; R53N and A55T; S64N and H66T; K91N and D93T; D93N and G95T; S97N and Q99T; and A106N and D108S.

[022] In certain embodiments, the GDF15 mutein may include at least one of the following pairs of substitutions of the corresponding amino acids in SEQ ID NO:1:S23N and E25S; R53N and A55S; S64N and H66S; K91N and D93S; D93N and G95S; S97N and Q99S; and A106N and D108S.

[023] In certain embodiments, the GDF15 mutein may include at least one of the following pairs of substitutions of the corresponding amino acids in SEQ ID NO:1:S64N and H66T/S; K91N and D93T/S; D93N and G95T/S; and S97N and Q99T/S. For example, the GDF15 mutein may include at least one of the following pairs of corresponding amino acid substitutions in SEQ ID NO:1:S64N and H66T; K91N and D93T; D93N and G95T; and S97N and Q99T; or S64N and H66S; K91N and D93S; D93N and G95S; and S97N and Q99S.

[024] In other embodiments, the GDF15 mutein may include at least one of the following pairs of substitutions of the corresponding amino acids in SEQ ID NO: 1: K91N and D93T or D93S; and D93N and G95T or G95S.

[025] In other embodiments, the GDF15 mutein in the complex may comprise a contiguous amino acid sequence that may be at least 98 amino acids in length and may be at least 90% identical to the amino acid sequence of SEQ ID NO:1, wherein the C-terminal amino acid of the GDF15 mutein corresponds to isoleucine at position 112 in SEQ ID NO:1.

[026] In other embodiments, the contiguous amino acid sequence that may be at least 98 amino acids in length and may be at least 95% identical to the amino acid sequence of SEQ ID NO:1, in which the C-terminal amino acid of the mutein of GDF15 corresponds to isoleucine at position 112 in SEQ ID NO:1.

[027] The exemplary GDF15 mutein present in the complex disclosed herein includes a contiguous amino acid sequence that is at least 98 amino acids in length, at least 90% identical to the amino acid sequence of SEQ ID NO:1, and has amino acid deletions in in relation to SEQ ID NO:1. For example, polypeptides may have an N-terminal truncation relative to SEQ ID NO: 1. The N-terminal truncation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or more amino acids in relation to SEQ ID NO: 1, for example, 1 to 14 amino acids, 2 to 14 amino acids, 3 to 14 amino acids, 2 to 3 amino acids, 3 to 5 amino acids or 4 to 6 amino acids .

[028] Exemplary complexes disclosed herein include the GDF15 mutein which includes a contiguous amino acid sequence of at least 98 amino acids in length and at least 95% identical to the amino acid sequence of SEQ ID NO:1, in which the amino acid C- The terminus of the polypeptide corresponds to Isoleucine at position 112 in SEQ ID NO:1.

[029] In certain cases, the contiguous amino acid sequence in the GDF15 mutein is at least 98 amino acids in length and does not include the first amino acid that corresponds to the first amino acid present at the N-terminus of SEQ ID NO:1, in which the amino acid C-terminus corresponds to Isoleucine at position 112 in SEQ ID NO:1.

[030] In certain cases, the contiguous amino acid sequence in the GDF15 mutein is at least 98 amino acids in length and does not include the first two amino acids that correspond to the first two amino acids present at the N-terminus of SEQ ID NO:1, wherein the C-terminal amino acid corresponds to Isoleucine at position 112 in SEQ ID NO:1.

[031] In certain cases, the contiguous amino acid sequence is at least 98 amino acids in length and does not include the first three amino acids that correspond to the first three amino acids present at the N-terminus of SEQ ID NO: 1, in which the amino acid C- terminal corresponds to Isoleucine at position 112 in SEQ ID NO:1.

[032] In certain cases, the contiguous amino acid sequence is at least 98 amino acids in length and does not include the first six amino acids that correspond to the first six amino acids present at the N-terminus of SEQ ID NO: 1, in which the amino acid C- terminal corresponds to Isoleucine at position 112 in SEQ ID NO:1.

[033] In certain cases, the contiguous amino acid sequence is at least 98 amino acids in length and does not include the first fourteen amino acids that correspond to the first fourteen amino acids present at the N-terminus of SEQ ID NO:1.

[034] In certain cases, the C-terminus of the first polypeptide (e.g., Fc-button) or the second polypeptide (e.g., Fc-hole) is conjugated to the N-terminus of the GDF15 mutein through a linker. Exemplary linkers include the sequence (G4S)n, where n = 1 to 10, for example, 1 to 5 or 2 to 5, for example, 2, 3, 4 or 5.

[035] In certain cases, the IgG Fc comprises a contiguous amino acid sequence at least 90% identical to the amino acid sequence in SEQ ID NO:2 (human IgG1 Fc sequence). The engineered protuberance may include at least one corresponding amino acid substitution in a human IgG1 Fc sequence, wherein the substitution is at a position selected from the group consisting of amino acid residues 347, 366 and 394, in accordance with the numbering of the HUH. For example, at least one replacement is selected from the group consisting of Q347W/Y, T366W/Y, and T394W/Y, according to EU numbering.

[036] In certain cases, the engineered cavity comprises at least one corresponding amino acid substitution in a human IgG1 Fc sequence, wherein the substitution is at a position selected from the group consisting of amino acid residues 366, 368, 394, 405 and 407, according to EU numbering. For example, at least one replacement is selected from the group consisting of T366S, L368A, T394S, F405T/V/A, and Y407T/V/A, according to EU numbering.

[037] In certain cases, the protuberance may include the T366W/Y substitution and the cavity may include the T366S, L368A and Y407T/V/A substitutions, in accordance with EU numbering.

[038] For example, the protuberance may include the T366W/Y substitution and the cavity may include the Y407T/V/A substitution, in accordance with EU numbering. In other cases, the bulge may include the T366Y substitution and the cavity may include the Y407T substitution, in accordance with EU numbering. In other examples, the protuberance may include substitution T366W and the cavity may include substitution Y407A, in accordance with EU numbering. In other examples, the bulge may include substitution T394Y and the cavity may include substitution Y407T, in accordance with EU numbering.

[039] In certain embodiments, the IgG Fc sequences of the first and second polypeptides may each include a hinge region that forms at least one disulfide bond between the first and second polypeptides. In certain embodiments, the IgG Fc sequences of the first and second polypeptides may each include a hinge region, a CH2 region, and a CH3 region, wherein the hinge regions form at least one disulfide bond between the first and the second polypeptides.

[040] Also provided herein is a nucleic acid molecule encoding the first and second polypeptides described above. The nucleic acid molecule can be operably linked to an expression control element that confers expression of the nucleic acid molecule encoding the polypeptides in vitro or in vivo. A vector that includes the nucleic acid molecule is also contemplated. The vector may be a viral vector. In certain cases, a first nucleic acid encoding the first polypeptide and a second nucleic acid encoding the second polypeptide are provided. Each of the nucleic acids is operably linked to an expression control element that confers expression of the first and second polypeptides from the first and second nucleic acids, respectively. A first vector comprising the first nucleic acid and a second vector comprising the second nucleic acid are also disclosed. As noted here, the vector may be a viral vector.

[041] Some embodiments include transformed host cells that express one or more of the previously mentioned polypeptides. For example, a host cell is provided that includes the first and second nucleic acids. The host cell expresses the first polypeptide and the second polypeptide.

[042] In particular embodiments of the present disclosure, one or more of the aforementioned complexes are formulated to yield a pharmaceutical composition, wherein the composition also includes one or more pharmaceutically acceptable diluents, carriers or excipients. In certain embodiments, a pharmaceutical composition also includes at least one additional prophylactic or therapeutic agent.

[043] Also provided is a composition (e.g., pharmaceutical composition) of one or more of the above-mentioned complexes for the treatment or prevention of a body weight disorder in a subject; for the treatment or prevention of a disorder of glucose metabolism in a subject. The composition may include an amount of complex that is effective for treating or preventing a body weight disorder in a subject. The composition may include an amount of complex that is effective for treating or preventing a disorder of glucose metabolism in a subject.

[044] Still other embodiments of the present disclosure comprise an antibody that specifically binds to one of the first and second polypeptides mentioned above.

[045] Additionally, the present disclosure contemplates pharmaceutical compositions comprising an antibody, as described above, formulated with at least one pharmaceutically acceptable excipient, carrier or diluent. Such pharmaceutical compositions may also contain at least one additional prophylactic or therapeutic agent.

[046] Certain embodiments of the present disclosure contemplate a sterile container that contains one of the pharmaceutical compositions mentioned above and, optionally, one or more additional components. By way of example, but not limitation, the sterile container may be a syringe. In still other embodiments, the sterile container is a component of a kit; the kit may also contain, for example, a second sterile container that contains at least one prophylactic or therapeutic agent.

[047] Also disclosed herein is a method for manufacturing the aforementioned polypeptides and complexes. The method may include culturing a host cell that expresses the polypeptides; and isolation of the complex that includes the expressed polypeptides.

[048] The present disclosure also contemplates a method of treating or preventing a glucose metabolism disorder in a subject (e.g., a human being), by administering to the subject a therapeutically effective amount of the aforementioned complex. In some methods, the treatment or prevention results in a reduction in plasma glucose in the subject, a reduction in plasma insulin in the subject, a reduction in body weight and/or food intake, or an increase in glucose tolerance in the subject. In particular embodiments, the glucose metabolism disorder is diabetes mellitus.

[049] A method of treating or preventing a body weight disorder in a subject is also disclosed. The method may include administering to the subject the complex of the present disclosure, wherein the complex is administered in an amount effective in treating or preventing the body weight disorder in the subject. In some methods, treatment or prevention results in a reduction in body weight and/or food intake in the subject.

[050] In some embodiments, the subject is obese and/or has a body weight disorder.

[051] Although not limited to any particular route of administration or dosage regimen, in some embodiments administration is by parenteral injection (e.g., subcutaneous).

BRIEF DESCRIPTION OF THE DRAWINGS

[052] Figure 1 represents a schematic drawing of a homodimeric complex of heterodimers of molecules (Fc/Fc) -GDF15 in which the Fc/Fc polypeptides are Fc button-in-hole (A-F) pairs and the incorporation of N-linked glycans in the GDF15 molecule (E,F) to increase the expression and assembly of the homodimeric complex of heterodimers.

[053] Figure 2A represents the recoveries of Expi 293F transient expression of engineered (Fc/Fc)-GDF15 complexes. Recoveries are as follows: (0 = aggregates/no expression, <25 mg/l, 25 mg/l to 49.9 mg/l, 50 mg/l to 74.9 mg/l, 75 mg/l to 99.0 mg /l, > 100 mg /l). The addition of an N-linked glycan to the GDF15 sequence in (Fc/Fc)-GDF15 provides a significant increase to overall recoveries after purification. Figure 2B provides recoveries from Expi 293F transient expression of wild-type GDF15 and GDF15 glycosylation mutants (glycomuteins) that were not conjugated to Fc.

[054] Figure 3 represents the reduction of body weight in a diet-induced obese (DIO) mouse model through the subcutaneous delivery of 0.4 nmol/kg of (Fc/Fc)-GDF15 complexes, once a week for 4 weeks, followed by a 14-day recovery period. The B13a/B13b variant significantly improved efficacy compared to the B9A/B9b and B11a/B11b variants.

[055] Figure 4 represents the percentage of body weight reduction in the DIO mouse model upon subcutaneous delivery of 0.4 nmol/kg of (Fc/Fc)-GDF15 complex, once a week for 4 weeks, followed by a 14-day recovery period. The B13a/B13b variant has a % change in vehicle subtracted % in body weight greater than 20% after 14 days of post-dosing recovery.

[056] Figure 5 represents the reduction of body weight in the DIO mouse model through the subcutaneous distribution of 4.0 nmol/kg of (Fc/Fc)-GDF15 complexes, once a week for 4 weeks, followed by a 14 day recovery period.

[057] Figure 6 represents the percentage of body weight reduction in the DIO mouse model upon subcutaneous delivery of 4.0 nmol/kg of (Fc/Fc)-GDF15 complex, once a week for 4 weeks, followed by a 14-day recovery period.

[058] Figures 7 and 8 summarize the body weight reductions observed (including SEM and p-values) for each group of DIO mice (n = 6) for dose groups of 0.4 nmol/kg and 4.0 nmol/kg. kg represented in Figures 3 and 5. For all groups, (* = p < 0.05, ** = p < 0.01 and *** = p < 0.001) using the unpaired t-test.

[059] Figure 9 summarizes the percentage of weight reductions (including SEM and p-values) for each group of DIO mice (n = 6) for the 0.4 nmol/kg and 4.0 nmol/kg dose groups. kg represented in Figures 4 and 6. For all groups, (* = p < 0.05, ** = p < 0.01 and *** = p < 0.001) using the unpaired t-test.

DETAILED DESCRIPTION

[060] Before the methods and compositions of the present disclosure are further described, it should be understood that the disclosure is not limited to the particular embodiments presented herein, and it should also be understood that the terminology used herein is for the purpose of describing embodiments only. particular, and is not intended to be limiting.

[061] Where a range of values is provided, it is understood that each intervening value, to the tenth of a unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of the range and any other stated value or intervening in this declared range are covered by the invention. The upper and lower limits of these minor ranges may be independently included in the minor ranges and are also encompassed by the invention, subject to any limit specifically excluded in the established range. Where the indicated range includes one or both limits, ranges excluding either or both included limits are also included in the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

[062] It should be noted that as used in this document and the appended claims, the singular forms “a”, “an” and “the” include plural referents, unless the context clearly dictates otherwise. Thus, for example, reference to “the complex” includes reference to one or more complexes, and so on. It should also be noted that claims will be written to exclude any optional elements. As such, this statement is intended to serve as an antecedent basis for the use of such exclusive terminology as “exclusively”, “sole” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[063] The publications discussed in this document are provided solely for dissemination prior to the filing date of this application. Nothing in this document should be construed as an admission that the present invention is not entitled to anticipate such publication by virtue of the prior invention. In addition, the publication dates provided may differ from the actual publication dates which may need to be independently confirmed.

DEFINITIONS

[064] The terms “polypeptide”, “peptide” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified amino acids or derivatives and polypeptides having modified polypeptide backbones. Terms include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically marked proteins; and the like. In specific embodiments, the terms refer to a polymeric form of amino acids of any length, which include genetically encoded amino acids. In particular embodiments, the terms refer to a polymeric form of amino acids of any length, which include genetically encoded amino acids fused to a heterologous amino acid sequence. In particular embodiments, the terms refer to an amino acid 98 to 112 amino acids in length, optionally fused to a heterologous sequence. In specific embodiments, as appropriate, when referring to the proteins and molecules disclosed and described herein, the terms “polypeptide”, “peptide” and “protein” refer to polypeptides, as defined herein.

[065] The term “complex”, as used herein, refers to a protein complex comprising at least two polypeptides, each of which polypeptides comprises an N-terminus and a C-terminus. At least two polypeptides can be associated with each other through one or both of a covalent bond and a non-covalent interaction (e.g., electrostatic, π effects, van der Waals forces, and hydrophobic effects). At least two polypeptides may be the same, that is, have identical amino acid sequences, or they may be different, that is, not have identical amino acid sequences. A complex with two polypeptides, in which both polypeptides are identical, is referred to as a homodimer. A complex with two polypeptides, in which the polypeptides are different, is referred to as a heterodimer. A three-polypeptide complex, in which the three polypeptides are identical, is referred to as a homotrimer. A three-polypeptide complex, in which at least one of the three polypeptides is different from the other polypeptide(s), is referred to as a heterotrimer. A four-polypeptide complex, in which the four polypeptides are identical, is referred to as a homotetramer. A four-polypeptide complex, in which at least one of the four polypeptides is different from the other polypeptide(s), is referred to as a heterotetramer. An exemplary complex of four polypeptides-two molecules of a first polypeptide and two molecules of a second polypeptide, wherein the first polypeptide dimerizes with the second polypeptide to form a heterodimer and wherein two such heterodimers dimerize to form the complex may be mentioned as a homodimeric complex of the two heterodimers.

[066] The present disclosure contemplates complexes, as defined above, including, but not limited to, a heterodimer that has a first polypeptide associated with a second polypeptide, wherein the first polypeptide is an Fc “button” and the second polypeptide is a “hole” Fc, and wherein the first polypeptide or second polypeptide is fused to an amino acid sequence of GDF15 (or GDF15 mutein, such as a GDF15 mutein described herein). The first and second polypeptides can be physically associated with each other through a noncovalent interaction (e.g., hydrophobic effects, such as hydrophobic interaction between the button and hole regions of the Fc), a covalent bond (e.g., a disulfide bond , such as one or two or more disulfide bonds between the Fc hinge regions in the first and second polypeptides), or both.

[067] The present disclosure also contemplates a complex that includes two heterodimers associated with each other, each heterodimer having a first polypeptide and a second polypeptide, wherein the first polypeptide is an Fc “button” and the second polypeptide is an Fc “hole ”, and wherein the first polypeptide or the second polypeptide is fused to an amino acid sequence of GDF15 (or GDF15 mutein). Within the complex, the two heterodimers may be physically associated by a noncovalent interaction (e.g., hydrophobic effects), a covalent bond (e.g., a disulfide bond), or both. The first and second polypeptides in each of the heterodimers in the complex can be physically associated with each other by a noncovalent interaction (e.g., hydrophobic effects), a covalent bond (e.g., a disulfide bond), or both.

[068] The present disclosure also contemplates a complex that includes two heterodimers associated with each other, each heterodimer having a first polypeptide and a second polypeptide, wherein the first polypeptide is an Fc “button” and the second polypeptide is an Fc “hole ”, and wherein the first polypeptide or the second polypeptide is fused to an amino acid sequence of GDF15 (or GDF15 mutein). Within the complex, the two heterodimers can be physically associated by a non-covalent interaction (e.g., hydrophobic effects) or a covalent interaction (e.g., disulfide bond(s)) between the GDF15 polypeptides and the first and second polypeptides in Each of the heterodimers can be physically associated with each other by a noncovalent interaction (e.g., button-in-hole), a covalent bond (e.g., a disulfide bond), or both.

[069] The terms “patient” or “subject” are used interchangeably to refer to a human being or a non-human animal (e.g., a mammal).

[070] The terms “treat”, “treating”, treatment” and the like refer to a course of action (such as the administration of an agent, for example, a polypeptide, a complex or a pharmaceutical composition comprising a polypeptide, a complex) initiated after a disease, disorder or condition or a symptom thereof has been diagnosed, observed and the like, so as to eliminate, reduce, suppress, mitigate or improve, temporarily or permanently, at least one of the underlying causes of a disease, disorder or condition that afflicts a subject or at least one of the symptoms associated with a disease, disorder, condition that afflicts a subject. Thus, treatment includes inhibition (i.e., preventing the development or further development of the disease, disorder or condition or clinical symptoms in association therewith) of an active disease (e.g., so as to lower the insulin level and/or glucose in the bloodstream, to increase glucose tolerance in order to minimize fluctuation in glucose levels and/or in order to protect against diseases caused by disruption of glucose homeostasis, decrease body weight, prevent increase in body weight).

[071] The term “in need of treatment” as used herein refers to a judgment made by a doctor or other care provider that the subject needs or will benefit from treatment. This judgment is made based on a variety of factors that are within the domain of the doctor or caregiver's expertise.

[072] The terms “prevent”, “preventing”, “prevention” and the like refer to a course of action (such as the administration of an agent, for example, a polypeptide, a complex or a pharmaceutical composition comprising a polypeptide, a complex) initiated in a manner (e.g., before the onset of a disease, disorder, condition or symptom thereof) so as to prevent, suppress, inhibit or reduce, temporarily or permanently, a patient's risk of developing a disease, disorder, condition or the like (as determined through, for example, the absence of clinical symptoms) or delaying the appearance thereof, in general, in the context of a subject predisposed to having a particular disease, disorder or condition. In certain cases, the terms also refer to slowing the progression of the disease, disorder or condition or inhibiting its progression to a harmful or otherwise undesirable state.

[073] The term “in need of prevention”, as used herein, refers to a decision made by a doctor or other caregiver that a subject requires or will benefit from preventive care. This judgment is made based on a variety of factors that are within the domain of the doctor or caregiver's expertise.

[074] The phrase “therapeutically effective amount” refers to the administration of an agent to a subject, whether alone or as part of a pharmaceutical composition and, whether in a single dose or as part of a series of doses, in an amount that is capable of of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder, or condition when administered to a patient. The therapeutically effective amount can be determined by measuring the relevant physiological effects. For example, in the case of a hyperglycemic condition, a decrease or decrease in blood glucose or an improvement in the glucose tolerance test can be used to determine whether the amount of an agent is effective in treating the hyperglycemic condition. For example, a therapeutically effective amount is an amount sufficient to reduce or lower any level (e.g., a baseline level) of fasting plasma glucose (FPG), wherein, e.g., the amount is sufficient to reduce an FPG level greater than 200 mg/dl to less than 200 mg/dl, wherein the amount is sufficient to reduce an FPG level between 175 mg/dl and 200 mg/dL less than the starting level, in that the amount is sufficient to reduce an FPG level between 150 mg/dl and 175 mg/dL less than the starting level, wherein the amount is sufficient to reduce an FPG level between 125 mg/dl and 150 mg /dL less than the starting level, and so on (e.g., reducing FPG levels to less than 125 mg/dl, to less than 120 mg/dl, to less than 115 mg/dl, to less than 110 mg/dl, etc.). In the case of HbAIc levels, the effective amount is an amount sufficient to reduce or lower the levels by more than about 10% to 9%, by more than about 9% to 8%, by more than about from 8% to 7%, from more than about 7% to 6%, from more than about 6% to 5%, and so on. More particularly, a reduction or decrease in HbAIc levels by about 0.1%, 0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0. , 9%, 1%, 1, 5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more , is covered by this disclosure. The therapeutically effective amount can be adjusted in connection with the dosage regimen and diagnostic analysis of the subject's conditions and the like.

[075] The phrase “in an amount sufficient to effect a change” means that there is a detectable difference between a level of a measurement indicator before (e.g., a baseline level) and after administration of a particular therapy. Indicators include any objective parameter (e.g., glucose or insulin level, food intake, or body weight) or subjective parameter (e.g., a subject's feeling of well-being or appetite).

[076] The phrase “glucose tolerance”, as used herein, refers to a subject's ability to control the level of plasma glucose and/or plasma insulin when glucose intake fluctuates. For example, glucose tolerance encompasses the subject's ability to reduce, within about 120 minutes, the plasma glucose level back to a level determined before glucose ingestion.

[077] The terms “diabetes” and “diabetic” refer to a progressive disease of carbohydrate metabolism that involves inadequate production or utilization of insulin, often distinguished by hyperglycemia and glycosuria. The terms “prediabetes” and “prediabetic” refer to a state in which a subject does not have the characteristics, symptoms, and the like typically seen in patients with diabetes, but has characteristics, symptoms, and the like, which, if If left untreated, they can develop into diabetes. The presence of these conditions can be determined using, for example, either the fasting plasma glucose test (FPG) or the oral glucose tolerance test (OGTT). Both typically require a subject to fast for at least 8 hours before starting the test. In the FPG test, a subject's blood glucose is measured after fasting is complete; Generally, the subject fasts overnight and blood glucose is measured in the morning before the subject eats. A healthy subject will generally have an FPG concentration between about 90 and about 100 mg/dl, a subject with “pre-diabetes” will generally have an FPG concentration between about 100 and about 125 mg/dl. /dl and a subject with “diabetes” generally have an FPG level above about 126 mg/dl. In OGTT, a subject's blood glucose is measured after fasting and again two hours after drinking a high-glucose beverage. Two hours after consuming the high-glucose beverage, a healthy subject generally has a blood glucose concentration below about 140 mg/dl, a pre-diabetic subject generally has a blood glucose concentration of about 140 to about of 199 mg/dL and a diabetic subject generally has a blood glucose concentration of about 200 mg/dL or higher. While the aforementioned glycemia values refer to humans, normoglycemia, moderate hyperglycemia, and overt hyperglycemia are scaled differently in murine subjects. A healthy murine subject after a four-hour fast would generally have an FPG concentration between about 100 and about 150 mg/dl, a murine subject with “pre-diabetes” would generally have an FPG concentration between about 175 and about 250 mg/dl and a murine subject with “diabetes” would generally have an FPG concentration above about 250 mg/dl.

[078] The term “insulin resistance” as used herein refers to a condition in which a normal amount of insulin is unable to produce a normal physiological or molecular response. In some cases, a hyperphysiological amount of insulin, endogenously produced or exogenously administered, is capable of overcoming insulin resistance, in whole or in part, and producing a biological response.

[079] The term “metabolic syndrome” refers to an associated set of characteristics that includes, but is not limited to, hyperinsulinemia, abnormal glucose tolerance, obesity, redistribution of fat to the abdominal or upper compartment of the body, hypertension, dysfibrinolysis and dyslipidemia, distinguished by elevated triglycerides, low high-density lipoprotein (HDL) cholesterol, and high dense low-density lipoprotein (LDL) particles. Subjects having metabolic syndrome are at risk for developing type 2 diabetes and/or other disorders (e.g., atherosclerosis).

[080] The phrase “disorder of glucose metabolism” encompasses any disorder distinguished by a clinical symptom or a combination of clinical symptoms that is associated with an elevated glucose level and/or an elevated insulin level in a subject relative to a healthy individual. . Elevated glucose and/or insulin levels can manifest themselves in the following diseases, disorders and conditions: hyperglycemia, type II diabetes, gestational diabetes, type I diabetes, insulin resistance, reduced glucose tolerance, hyperinsulinemia, weakened glucose metabolism, pre -diabetes, other metabolic disorders (such as metabolic syndrome, which is also referred to as syndrome X) and obesity, among others. The polypeptides of the present disclosure, and compositions thereof, can be used, for example, to achieve and/or maintain glucose homeostasis, for example, to reduce the level of glucose in the bloodstream and/or to reduce the level of insulin to a band found in a healthy subject.

[081] The term “hyperglycemia”, as used herein, refers to a condition in which a high amount of glucose circulates in a subject's blood plasma compared to a healthy individual. Hyperglycemia can be diagnosed by methods known in the art, including measuring fasting blood glucose levels, as described herein.

[082] The term “hyperinsulinemia”, as used herein, refers to a condition in which there are high levels of circulating insulin, when, concomitantly, blood glucose levels are either high or normal. Hyperinsulinemia can be caused by insulin resistance, which is associated with dyslipidemia, such as high triglyceride levels, high cholesterol, high low-density lipoprotein (LDL) and low high-density lipoprotein (HDL); high levels of uric acids; polycystic ovary syndrome; type II diabetes and obesity. Hyperinsulinemia can be diagnosed as having a plasma insulin level greater than about 2 μU/ml.

[083] As used herein, the phrase “body weight disorder” refers to conditions associated with excess body weight and/or increased appetite. Several parameters are used to determine whether a subject is overweight compared to a healthy reference individual, including the subject's condition, age, height, sex, and health. For example, a subject may be considered overweight or obese by assessing the subject's body mass index (BMI), which is calculated by dividing a subject's weight in kilograms by the subject's height in square meters. An adult having a BMI in the range of ~18.5 to ~24.9 kg/m2 is considered to be of normal weight; an adult with a BMI between ~25 and ~29.9 kg/m2 can be considered overweight (pre-obese); and an adult with a BMI of ~30 kg/m2 or higher may be considered obese. Increased appetite often contributes to excess body weight. There are several conditions associated with improved appetite, including, for example, nocturnal eating syndrome, which is distinguished by morning anorexia, and nocturnal polyphagia often associated with insomnia, but which may be related to damage to the hypothalamus.

[084] The term “activators” refers to agents that, for example, stimulate, increase, activate, facilitate, intensify activation, sensitize or upregulate the function or activity of one or more agents, for example, polypeptides or complex used to treat or prevent a metabolic disorder. Furthermore, activators include agents that operate through the same mechanism of action as the polypeptides of the present invention (i.e., agents that modulate the same signaling pathway as the polypeptides in a manner analogous to that of the polypeptides) and that are capable of eliciting a response biological properties comparable to (or superior to) polypeptides. Examples of activators include agonists, such as small molecule compounds.

[085] The term “modulators” collectively refers to the polypeptides of the present invention and the activators.

[086] The terms “modulate”, “modulation” and the like refer to the ability of an agent (e.g., an activator) to increase the function or activity of one or more polypeptides (or the nucleic acid molecules that encode them ), either directly or indirectly; or the ability of an agent to produce an effect comparable to that of one or more polypeptides.

[087] It will be appreciated that throughout this reference amino acids are disclosed according to one or three letter codes. For the reader's convenience, the one- and three-letter amino acid codes are provided below:

[088] As used herein, the term “variant” encompasses naturally occurring variants (e.g., homologous and allelic variants) and non-naturally occurring variants (e.g., recombinantly modified). Naturally occurring variants include homologs, that is, nucleic acids and polypeptides that differ in nucleotide or amino acid sequence, respectively, from one species to another. Naturally occurring variants include allelic variants, that is, nucleic acids and polypeptides that differ in nucleotide or amino acid sequence, respectively, from one individual to another within a species. Non-naturally occurring variants include nucleic acids and polypeptides that comprise a change in nucleotide or amino acid sequence, respectively, where the change in sequence is introduced artificially, e.g., the change is generated in a laboratory or other facility by human intervention ( “hand of man”).

[089] The term “wild type” or “native”, in reference to GDF15, refers to naturally occurring, biologically active GDF15, including naturally occurring and biologically active GDF15 variants. The term includes the mature human GDF15 sequence of 112 amino acids (SEQ ID NO:1).

[090] The term “muteins”, for use herein, refers generically to recombinant proteins, that is, a polypeptide comprising an artificially introduced change in the amino acid sequence, for example, a change in the amino acid sequence generated in the laboratory or another installation by human intervention (“man’s hand”). These polypeptides typically carry single or multiple amino acid substitutions or deletions and are often derived from cloned genes that have undergone random or site-directed mutagenesis, or from completely synthetic genes. “GDF15 muteins” of the present disclosure thus encompass, for example, amino acid substitutions and/or amino acid deletions (e.g., N-terminal truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more amino acids) relative to a reference polypeptide, for example, relative to native/wild-type mature human GDF15 (SEQ ID NO:1).

[091] As used herein in reference to native human GDF15 or a GDF15 mutein, the terms “modified”, “modification” and the like refer to one or more changes that modify a property of human GDF15, a variant of Naturally occurring GDF15 or a GDF15 mutein, where the change does not affect the primary amino acid sequence of the GDF15 polypeptide itself (native or mutein). Such a property includes, for example, solubility, circulation half-life, stability, clearance, immunogenicity or allergenicity and manufacturability (e.g. cost and efficiency). “Modification” includes a covalent chemical modification that does not alter the primary amino acid sequence of the GDF15 polypeptide (native or mutein) itself. Changes to human GDF15, a naturally occurring GDF15 variant, or a GDF15 mutein that can be made include, but are not limited to, one or more of pegylation (covalent attachment of one or more polyethylene glycol (PEG) molecules) , or its derivatives); glycosylation (e.g. N-glycosylation), polysialylation and hesylation; maltose binding protein fusion; albumin fusion (e.g. HSA fusion); albumin binding via, for example, a conjugated fatty acid chain (acylation); Fc fusion; and fusion with a PEG mimetic. Some particular embodiments involve modifications involving fusion to an Fc, and still other particular modifications involve modifications involving glycosylation, or a combination thereof.

[092] The terms “DNA”, “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides or analogues thereof. same. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

[093] The term “probe” refers to a fragment of DNA or RNA corresponding to a gene or sequence of interest, in which the fragment has been radioactively labeled (for example, through the incorporation of 32P or 35S) or with some other molecule detectable, such as biotin, digoxigenin or fluorescein. As stretches of DNA or RNA with complementary sequences hybridize, a probe can be used, for example, to label viral plaques, bacterial colonies, or bands on a gel containing the gene of interest. A probe can be cloned DNA or it can be a strand of synthetic DNA; the latter can be used to obtain a cDNA or genomic clone from an isolated protein, for example, microsequencing a portion of the protein, deducing the nucleic acid sequence encoding the protein, synthesizing an oligonucleotide carrying that sequence, radiolabeling the sequence and using it as a probe to screen a cDNA library or a genomic library.

[094] The term “heterologous” refers to two components that are defined by structures derived from different sources. For example, in the context of a polypeptide, a “heterologous” polypeptide may include operably linked amino acid sequences that are derived from different polypeptides (e.g., a first component comprising a recombinant polypeptide and a second component derived from a native GDF15 polypeptide). ). Likewise, in the context of a polynucleotide encoding a chimeric polypeptide, a “heterologous” polynucleotide may include operably linked nucleic acid sequences that may be derived from different genes (e.g., a first component from a nucleic acid encoding a polypeptide according to an embodiment disclosed herein and a second component from a nucleic acid encoding a carrier polypeptide). Other exemplary "heterologous" nucleic acids include expression constructs in which a nucleic acid comprising a coding sequence is operably linked to a regulatory element (e.g., a promoter) that is of a different genetic origin than that of the coding sequence (e.g. , to provide expression in a host cell of interest, which may be of different genetic origin than the promoter, the coding sequence, or both). For example, a T7 promoter operably linked to a polynucleotide encoding a GDF15 polypeptide or domain thereof is said to be a heterologous nucleic acid. In the context of recombinant cells, “heterologous” may refer to the presence of a nucleic acid (or gene product, such as a polypeptide) that is of different genetic origin than the host cell in which it is present.

[095] The term “operationally linked” refers to the link between molecules to provide a desired function. For example, “operationally linked” in the context of nucleic acids refers to a functional link between nucleic acid sequences. By way of example, a nucleic acid expression control sequence (such as a promoter, signal sequence, or set of transcription factor binding sites) may be operably linked to a second polynucleotide, wherein the control sequence of expression affects transcription and/or translation of the second polynucleotide. In the context of a polypeptide, “operationally linked” refers to a functional link between amino acid sequences (e.g., different domains) to provide a described activity of the polypeptide.

[096] As used herein in the context of the structure of a polypeptide, “N-terminal” (or “amino-terminal”) and “C-terminal” (or “C-terminalarboxyl”) refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while the terms “N-terminus” and “C-terminus” refer to the relative positions in the amino acid sequence of the polypeptide toward the N-terminus and the C-terminus, respectively, and may include residues in the N-terminal and C-terminal, respectively. “Immediately N-terminal” or “immediately C-terminal” refers to a position of a first amino acid residue relative to a second amino acid residue, wherein the first and second amino acid residues are covalently linked to provide a sequence of contiguous amino acids.

[097] “Derived from”, in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence “derived from” a GDF15 polypeptide), is intended to mean that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid (e.g., a naturally occurring GDF15 polypeptide or a nucleic acid encoding GDF15), and is not intended to be limiting as to the source or method in which the protein or nucleic acid is produced. . By way of example, the term “derived from” includes homologs or variants of reference amino acids or DNA sequences.

[098] In the context of a polypeptide, the term “isolated” refers to a polypeptide of interest that, if naturally occurring, is in an environment different from that in which it can occur naturally. “Isolated” means that it includes polypeptides that are within samples that are substantially enriched for the polypeptide of interest and/or in which the polypeptide of interest is partially or substantially purified. When the polypeptide is not naturally occurring, “isolated” indicates that the polypeptide was separated from an environment in which it was produced either by synthetic or recombinant means.

[099] “Enriched” means that a sample is not naturally manipulated (e.g., in a laboratory, e.g., by a scientist or clinician) such that a polypeptide of interest is present in) a higher concentration (e.g., at least 3 times greater, at least 4 times greater, at least 8 times greater, at least 64 times greater or more) than the concentration of the polypeptide in the initial sample, such as a biological sample (e.g., a sample in which the polypeptide occurs naturally or in which it is present after administration), or b) a concentration greater than the environment in which the polypeptide was produced (e.g., as in a bacterial cell).

[0100] “Substantially pure” indicates that a component (e.g., a polypeptide, a dimer, a tetramide, a complex) becomes greater than about 50% of the total content of the composition, and typically greater than about 60%. % of total polypeptide content. More typically, "substantially pure" refers to compositions in which at least 75%, at least 85%, at least 90% or more of the total composition is the component of interest. In some cases, the polypeptide will become greater than about 90%, or greater than about 95% of the total content of the composition.

[0101] The terms “antibodies” (Abs) and “immunoglobulins” (Igs) refer to glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that do not have antigen specificity. Antibodies are described in detail hereinafter.

[0102] The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that constitute the population are identical except for possible naturally occurring mutations that may be present in smaller quantities . Monoclonal antibodies are highly specific, being directed against a single antigen. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each antibody is directed against a single determinant on an antigen.

[0103] In the context of an antibody, the term “isolated” refers to an antibody that has been separated and/or recovered from contaminating components of its natural environment; such contaminating components include materials that may interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes.

[0104] The phrase “conservative amino acid substitution” refers to the replacement of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E. Conservative amino acid substitutions can preserve protein activity by replacing an amino acid (or amino acids) in the protein with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or chain size side. Guidelines for substitutions, insertions, or deletions can be based on alignments of the amino acid sequences of different variant proteins or proteins from different species.

GROWTH DIFFERENTIATION FACTOR 15 (GDF15)

[0105] GDF15, also known as MIC-1 (macrophage inhibitory cytokines-1), PDF (prostate differentiation factor), PLAB (placental bone morphogenetic protein), NAG-1 (gene activated by non-steroidal anti-inflammatory drugs) steroids (NSAIDs), TGF-PL and PTGFB, is a member of the transforming growth factor β (TGF-β) superfamily.GDF15, which is synthesized as a 62 kDa intracellular precursor protein that is subsequently cleaved by a protease-like furin, is secreted as a 25 kDa disulfide-linked protein.(See, e.g., Fairlie et al., J. Leukoc. Biol 65:2 to 5 (1999)]. GDF15 mRNA is observed in several tissues, including liver, kidney, pancreas, colon, and placenta, and GDF15 expression in the liver can be significantly upregulated during injury to organs such as liver, kidneys, heart, and lungs.

[0106] The GDF15 precursor is a 308 amino acid polypeptide (NCBI Ref. Seq. NP_004855.2) containing a 29 amino acid signal peptide, a 167 amino acid prodomain and a 112 amino acid mature domain that is excised at from the prodomain by furin-like proteases. A GDF15 polypeptide of 308 amino acids is referred to as a “full-length” GDF15 polypeptide; a GDF15 polypeptide of 112 amino acids (amino acids 197 to 308 of “full-length” GDF15) is a “mature” GDF15 polypeptide (SEQ ID NO:1). Unless otherwise noted, the term “GDF15” refers to the mature human sequence of 112 amino acids. Furthermore, numerical references for particular GDF15 residues refer to the mature 112 amino acid sequence (i.e., residue 1 is Ala (A) and residue 112 is Ile (I); see SEQ ID NO:1). Of note, while the precursor amino acid sequence of GDF15 predicts three excision sites, resulting in three putative forms of “mature” human GDF15 (i.e., 110, 112, and 115 amino acids), the mature sequence of 112 amino acids is accepted as being correct.

[0107] The scope of the present disclosure includes orthologs of GDF15 and its modified forms, from other mammalian species, and their use, including mouse (NP_035949), chimpanzee (XP_524157), orangutan (XP_002828972), Rhesus monkey (EHH29815), giant panda (XP_002912774), gibbon (XP_003275874), guinea pig (XP_003465238), ferret (AER98997), cow (NP_001193227), pig (NP_001167527), dog (XP_541938), and platypus (Ornithorhynchus anatinus; AFV 61279). The mature form of human GDF15 has about 67% amino acid identity with the mouse ortholog.

[0108] For the sake of convenience, the modified human GDF15 molecules, GDF15 variants (e.g., muteins) and modified GDF15 muteins, described hereinafter, are collectively referred to hereinafter as the “Polypeptide(s)” . It should be noted that any reference to “human” in connection with the polypeptides and nucleic acid molecules of the present disclosure is not intended to be limiting with respect to the manner in which the polypeptide or nucleic acid is obtained or the source, but in rather it is only with reference to the sequence, as it may correspond to a sequence of a naturally occurring human polypeptide or nucleic acid molecule. In particular embodiments, the modified human GDF15 molecules are N-glycosylated dimers. In addition to human polypeptides and the nucleic acid molecules that encode them, the disclosure contemplates GDF15 related polypeptides and corresponding nucleic acid molecules from other species.

A. POLYPEPTIDES HAVING DESIRED PHYSICAL PROPERTIES

[0109] The present disclosure contemplates, in part, polypeptides that include a contiguous amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identical to the amino acid sequence of SEQ ID NO:1 (human GDF15 112 amino acids long). The polypeptides may include one or more amino acid substitutions and/or deletions with respect to the amino acid sequence of SEQ ID NO:1. In certain embodiments, in addition to amino acid substitutions, the polypeptides of the present disclosure may also include amino acid deletions relative to the amino acid sequence of SEQ ID NO:1. In some embodiments, the polypeptides of the present disclosure may include amino acid deletions relative to the amino acid sequence of SEQ ID NO:1.

[0110] For reasons of convenience and clarity, the amino acid sequence of SEQ ID NO:1 is used as a reference sequence for the polypeptides presented here. Therefore, amino acid residue positions are numbered herein with reference to SEQ ID NO:1. The sequence of SEQ ID NO:1 is shown below:

[0111] ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTM CIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSL QTYDDLLAKDCHCI

[0112] In some embodiments, the polypeptides of the present disclosure may include one, two, three or more amino acid substitutions, additions or deletions that introduce one or more consensus N-linked glycosylation site at a location where such site is not present. in SEQ ID NO:1. The consensus N-linked glycosylation site includes the sequence NXS/T, where N is Asn; X is an amino acid other than proline; followed by Ser (S) or Thr (T).

[0113] Examples of polypeptides of the present disclosure include polypeptides that have one, two, three, four or more consensus glycosylation sites (e.g., N-linked glycosylation consensus sites) at an amino acid site where such site is not present. present in the amino acid sequence of SEQ ID NO:1.

[0114] In certain embodiments, the polypeptide may include an amino acid substitution relative to SEQ ID NO:1 that provides an N-linked glycosylation consensus site at the position of the substitution (e.g., an NGD sequence in SEQ ID NO :1 can be changed to NGT/S by a substitution; substitution position underlined). In other cases, the polypeptide may include two amino acid substitutions relative to SEQ ID NO:1 that provide a consensus N-linked glycosylation site at the position of the substitutions (e.g., a KTD sequence in SEQ ID NO:1 may be changed to NTT/S by two substitutions; substitution positions underlined). In some embodiments, the polypeptide may include three amino acid substitutions relative to SEQ ID NO:1 that provide a consensus N-linked glycosylation site at the position of the substitution (e.g., a GPG sequence in SEQ ID NO:1 may be changed to NTT/S by three substitutions; position of substitutions underlined).

[0115] In certain embodiments, the polypeptide may include one or more amino acid deletions in relation to SEQ ID NO:1 that provide an N-linked glycosylation consensus site at the position of the deletion. For example, a sequence NGDHCPLGPGRCCRLHT (SEQ ID NO:119) in SEQ ID NO:1 can be modified by deleting amino acids D to H (underlined) thereby providing a consensus N:NGT-linked glycosylation site.

[0116] In certain embodiments, the polypeptide may include one or more amino acid additions relative to SEQ ID NO:1 that provide an N-linked glycosylation consensus site at the position (or positions) of the addition (or additions). An example of introducing a consensus N-linked glycosylation site through the addition of an amino acid includes the addition of an N to an LHT sequence in SEQ ID NO:1, thereby generating the sequence LNHT, where NHT is a N-linked glycosylation consensus site.

[0117] As noted above, the polypeptide may include one or more substitutions in relation to SEQ ID NO:1 and the substitutions may be numbered according to the position of the corresponding amino acid in SEQ ID NO:1.

[0118] In certain embodiments, the polypeptide may include a contiguous amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, wherein the contiguous amino acid sequence has the substitution D5T/S or R21N.

[0119] In certain embodiments, the polypeptide may include a contiguous amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, wherein the contiguous amino acid sequence has at least one of the following pairs of substitutions with respect to the corresponding amino acids in SEQ ID NO:1: i. R16N and H18T or R16N and H18S; ii. S23N and E25T or S23N and E25S; iii. L24N and D26T or L24N and D26S; iv. S50N and F52T or S50N and F52S; v. F52N and A54T or F52N and A54S; saw. Q51N and R53T or Q51N and R53S; viii. R53N and A55T or R53N and A55S; viii. S64N and H66T or S64N and H66S; ix. L65N and R67T or L65N and R67S; x. S82N and N84T or S82N and N84S; xi. K91N and D93T or K91N and D93S; xii. D93N and G95T or D93N and G95S; xiii. T94N and V96T or T94N and V96S; xiv. V96N and L98T or V96N and L98S; xv. S97N and Q99T or S97N and Q99S; and xvi. A106N and D108T or A106N and D108S.

[0120] For example, the substitutions in i) above indicate that the polypeptide has a threonine (T) or serine (S) at an amino acid position that corresponds to amino acid position 18 in SEQ ID NO: 1, wherein in SEQ ID NO:1 a histidine (H) is present at amino acid position 18. Similarly, a substitution of a D at position 5 by a T or S can be denoted by D5T/S. The position of the corresponding amino acid in a polypeptide relative to SEQ ID NO:1 can be determined by aligning the amino acid sequences.

[0121] In certain embodiments, the polypeptide may include two amino acid substitutions (a pair of substitutions) that provide a single N-glycosylation consensus sequence at a position in which an N-glycosylation consensus sequence is not present in SEQ ID NO: 1.1. Examples of such substitutions include R16N and H18T/S; K91N and D93T/S; T94N and V96T/S; and others listed above. R16N and H18T/S indicates that the polypeptide has an N in a position that corresponds to position 16 of SEQ ID NO:1, wherein in SEQ ID NO:1 an R is present and the polypeptide has a T or S in a position that corresponds to position 18 in SEQ ID NO:1, where H is present. Since the RXH sequence (at position 16-18) in SEQ ID NO:1 does not include any residue for the N-linked glycosylation consensus sequence, the pair of substitutions leads to the introduction of the N-linked glycosylation consensus sequence. .

[0122] In alternative embodiments, a single amino acid substitution may be sufficient to provide the N-linked glycosylation consensus sequence, for example, since the NGD sequence (at position 3-5) is present in SEQ ID NO: 1, a single substitution of D for T or S produces the sequence NGT or NGS, respectively, which are both N-glycosylation consensus sequences.

[0123] In certain cases, more than one N-glycosylation consensus sequence can be introduced into wild-type GDF15. For example, the amino acid sequence of wild-type GDF15 can be modified by substitutions and/or deletions to provide one, two, three, four or more N-glycosylation consensus sequences. In certain embodiments, the polypeptide may include 112 contiguous amino acids that have at least 90% sequence identity to the 112 amino acid sequence of SEQ ID NO:1, wherein the 112 contiguous amino acids include one, two, three, four or more N-glycosylation consensus sequences, such as, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3 or 1 to 2 N-glycosylation consensus sequences.

[0124] In certain embodiments, the polypeptide may include 112 contiguous amino acids that have a sequence identity of at least 90% to the 112 amino acid sequence of SEQ ID NO:1, wherein the 112 contiguous amino acids include one, two, three , four or more of the pairs of substitutions presented here.

[0125] The present disclosure also contemplates polypeptides that are active fragments (e.g., subsequences) of the above-described polypeptides. The length of the active fragments or subsequences can be from 40 amino acids to 111 amino acids, for example, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 106, 109 or even 111 amino acids.

[0126] Polypeptides have a defined sequence identity compared to a reference sequence over a defined length of contiguous amino acids (e.g., a “comparison window”). Sequence alignment methods for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), using the similarity search method of Pearson & Lipman, Proc. Nat'l. Academic. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Madison, Wisconsin) or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., Eds. 1995 supplement)).

[0127] As an example, a suitable polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , at least about 98% or at least about 99% amino acid sequence identity with a contiguous stretch of 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or up to 112 amino acids in SEQ ID NO:1.

[0128] Exemplary fragments of the polypeptides disclosed herein include polypeptides that have amino acid deletions in relation to SEQ ID NO:1. For example, polypeptides may have N-terminal truncations and/or C-terminal truncations relative to SEQ ID NO: 1.1. Truncations can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids relative to a reference polypeptide, e.g., SEQ ID NO: 1. In certain embodiments, a polypeptide of interest may include one or more substitutions that introduce an N-linked glycosylation consensus sequence, such as that disclosed herein, and N-terminal truncations and/or C-terminal truncations with respect to SEQ ID NO:1.

[0129] In certain embodiments, the polypeptide may be at least 98 amino acids in length and has an amino acid sequence identity of at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100 % with a corresponding 98 amino acid stretch of SEQ ID NO:1. This polypeptide may be devoid of the first two to first fourteen amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids) present at the N-terminus of SEQ ID NO:1, while retaining the amino acids present at the C-terminus of SEQ ID NO: 1. In other words, the deleted amino acid (or amino acids) corresponds to the N-terminal amino acids of SEQ ID NO:1.

[0130] In certain embodiments, the GDF15 mutein may be at least 106 amino acids in length and has an amino acid sequence identity of at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% with a corresponding lengthening of 106 amino acids of SEQ ID NO:1. The GDF15 mutein may be devoid of the first six amino acids present at the N-terminus of SEQ ID NO:1.

[0131] In certain embodiments, the polypeptide may be at least 109 amino acids in length and has an amino acid sequence identity of at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100 % with a corresponding stretch of 109 amino acids of SEQ ID NO:1. The GDF15 mutein may be devoid of the first three amino acids present at the N-terminus of SEQ ID NO:1.

[0132] Examples of polypeptides of the present disclosure may include a deletion of the two N-terminal amino acids (ΔN2) relative to WT hGDF15 and may fuse with an Fc sequence at the N-terminus. However, when referring to the position of amino acid substitutions, the residue number indicated is that which corresponds to the position in mature WT hGDF15 (WT; SEQ ID NO:1). Thus, amino acid N at the N-terminus of a polypeptide lacking the first two amino acids at the N-terminus may be referred to as residue 3, although it is the first amino acid in the GDF15 mutein polypeptide amino acid sequence and preceded by the heterologous amino acid sequences. (e.g. Fc).

[0133] As noted above, these polypeptide fragments may include one or more amino acid substitutions that introduce an N-glycosylation consensus sequence relative to the sequence of SEQ ID NO:1, such as one, two or more of the substitutions of amino acids disclosed herein.

[0134] As indicated above and as described in more detail below, the polypeptides of the present disclosure can be modified through, for example, pegylation (covalent attachment of one or more polyethylene glycol (PEG) molecules or derivatives thereof); glycosylation (e.g. N-glycosylation); polysialylation; albumin fusion molecules comprising serum albumin (e.g., human serum albumin (HSA), cyno serum albumin or bovine serum albumin (BSA)); albumin binding via, for example, a conjugated fatty acid chain (acylation); Fc fusion; and fusion with a PEG mimetic. In certain embodiments, the modifications are introduced in a location-specific manner. In other embodiments, the modifications include a linker. The linker can conjugate the modification moiety to the polypeptide.

[0135] In particular embodiments, the present disclosure contemplates the modification of mature human GDF15 and GDF15 muteins (such as the polypeptides described above) by conjugation with albumin. In other embodiments, the present disclosure contemplates modification of polypeptides through N-glycosylation or O-glycosylation. The characteristics of albumins and polypeptide conjugates thereof (e.g., fusion proteins), and glycosylated polypeptides are further described below.

FC-GDF15 FUSION POLYPEPTIDES AND COMPLEXES THEREOF

[0136] In exemplary embodiments, the GDF15 polypeptides disclosed herein may be present as a fusion polypeptide comprising an Fc polypeptide or fragment thereof fused to the amino acid sequence of one or more of the polypeptides described herein (e.g., human GDF15 molecules, modified human GDF15 molecules, GDF15 muteins and modified GDF15 muteins). As provided herein, the GDF15 polypeptide can be a wild-type polypeptide or a mutein, e.g., a glycosylation mutein. As used herein, a "glycosylation mutein" or "glycomutein" or "glycosylation variant" or "glycovariant", in the context of a polypeptide, e.g., a GDF15 polypeptide, refers to a polypeptide that includes one or more glycosylation consensus sites at a position in the amino acid sequence where the position of the reference polypeptide (wild type) does not include the glycosylation consensus site. In certain cases, the fusion polypeptide may include an Fc sequence fused to the N-terminus of a GDF15 glycomutein disclosed herein.

[0137] Any Fc polypeptide sequence described herein or known in the art may be a component of the fusion proteins of the present disclosure. The components of the fusion proteins may optionally be covalently linked through a linker, such as the linkers described herein. In some embodiments of the present disclosure, the fusion proteins comprise the Fc polypeptide sequence as an N-terminal moiety and the polypeptides described herein as a C-terminal moiety.

[0138] In certain cases, the Fc partner of the Fc-GDF15 fusion polypeptides disclosed herein may be an Fc having the human IgG Fc sequence (e.g., IgG1, IgG2, IgG3 or IgG4) or a variant of the same. The amino acid sequence of human IgG1 Fc is provided as SEQ ID NO:2:

[0139] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2)

[0140] The hinge region is in italics, the CH2 domain is underlined and the CH3 domain is double underlined. The numbering of the amino acid position in the Fc sequence is according to the EU numbering (Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78 to 85 (1969)). Thus, the glutamic acid residue “E” at position 1 in SEQ ID NO:2 is numbered 216; the CH2 domain starts with alanine (A) which is numbered 231; The CH3 domain starts at glycine (G) which is numbered 341, according to EU numbering.

[0141] The Fc partner of the Fc-GDF15 fusion polypeptides disclosed herein may be an Fc that has a contiguous amino acid sequence that is at least 90% identical to SEQ ID NO:2, for example, at least 93%, at least at least 95%, at least 97%, at least 98% or more identical to SEQ ID NO:2. In certain embodiments, the Fc partner may be an Fc fragment comprising a CH3 domain or a contiguous amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CH3 domain in SEQ ID NO:2. In certain embodiments, the Fc partner may be an Fc fragment comprising a CH2 domain and a CH3 domain or a contiguous amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CH2 and CH3 domains in SEQ ID NO:2. In certain embodiments, the Fc partner may be an Fc fragment comprising a partial hinge region, a CH2 domain, and a CH3 domain, or an amino acid sequence contiguous that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the hinge region, CH2 domain and CH3 domain in SEQ ID NO:2. In certain embodiments, the Fc partner may have an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: two.

[0142] In certain cases, the Fc partner of the Fc-GDF15 fusion polypeptides may include an engineered protrusion, such a protrusion may associate with another Fc polypeptide that includes an engineered cavity. In other cases, the Fc partner of the Fc-GDF15 fusion polypeptides may include an engineered cavity, such a cavity may associate with another Fc polypeptide that includes an engineered protrusion. Examples of Fc sequences with engineered bulge and/or cavity are described in US 8,216,805. In certain cases, the bulge and cavity can be engineered into the CH3 domain of the Fc polypeptide. In certain cases, the Fc partner that associates with the Fc-GDF15 fusion polypeptides of the present disclosure is not conjugated to a GDF15 polypeptide. Thus, the Fc partner dimerizes with the Fc-GDF15 fusion polypeptide forming a heterodimer, with one GDF15 molecule per heterodimer.

[0143] “Protuberances” or “buttons” can be manipulated by replacing small amino acid side chains in a CH3 domain of a first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” or “holes” of identical or similar size to the bulges are optionally created in the CH3 domain of a second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).

[0144] The “first polypeptide” can be any polypeptide that must be associated with a second polypeptide. The first and second polypeptides meet at an “interface” (defined below). In addition to the interface, the first polypeptide may comprise one or more additional domains, such as a CH2 domain or a hinge region. In certain cases, the first polypeptide includes a CH3 domain that may form the interface of the first polypeptide.

[0145] The “second polypeptide” can be any polypeptide that must be associated with the first polypeptide through an “interface”. In addition to the interface, the second polypeptide may comprise one or more additional domains, such as a CH2 domain or a hinge region. In certain cases, the second polypeptide includes a CH3 domain that may form the interface of the second polypeptide.

[0146] The “interface” comprises those “contact” amino acid residues (or other non-amino acid groups, such as carbohydrate groups, NADH, biotin, FAD or heme group) in the first polypeptide that interact with one or more amino acid residues of “contact” (or other non-amino acid groups) at the interface of the second polypeptide. In certain cases, the interface may be a domain of an immunoglobulin, such as a constant domain (or fragments thereof). In certain cases, the interface comprises the CH3 domain of an immunoglobulin that is derived from an IgG antibody, for example, a human IgG1, IgG2, IgG3 or IgG4 antibody.

[0147] A “bulge” refers to at least one amino acid side chain that projects from the interface of the first polypeptide and is therefore positionable in a compensatory cavity at the adjacent interface (i.e., the interface of the second polypeptide) of in order to stabilize the heterodimer, and thus favor the formation of heterodimer over the formation of homodimer, for example. The cavity may exist in the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). The protrusion can be introduced synthetically (e.g., by altering the nucleic acid encoding the interface), for example, by recombinant means.

[0148] A “cavity” refers to at least one amino acid side chain that is recessed from the interface of the second polypeptide and therefore accommodates a corresponding protuberance at the adjacent interface of the first polypeptide. The cavity may exist in the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). For example, the nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity.

[0149] A protuberance is also referred to as a “button” and a cavity is also referred to as a “hole”. Examples of bulges and cavities are disclosed in US 8,216,805 and include substitutions at the following amino acid positions: 347, 366, 368, 394, 405 and 407. Amino acid position numbering is in accordance with EU numbering. The engineered protrusion may include at least one corresponding amino acid substitution in a human IgG1 Fc sequence, wherein the substitution is at a position selected from the group consisting of amino acid residues 347, 366 and 394. For example, at least one replacement is selected from the group consisting of Q347W/Y, T366W/Y and T394W/Y. In certain cases, the engineered cavity comprises at least one corresponding amino acid substitution in a human IgG1 Fc sequence, wherein the substitution is at a position selected from the group consisting of amino acid residues 366, 368, 394, 405 and 407. For example, at least one replacement is selected from the group consisting of T366S, L368A, T394S, F405T/V/A, and Y407T/V/A.

[0150] In certain cases, the bulge may include the T366W/Y substitution and the cavity may include the T366S, L368A and Y407T/V/A substitutions.

[0151] For example, the bulge may include the T366W/Y substitution and the cavity may include the Y407T/V/A substitution. In other cases, the bulge may include the T366Y substitution and the cavity may include the Y407T substitution. In other examples, the bulge may include the T366W substitution and the cavity may include the Y407A substitution. In additional examples, the bulge may include the T394Y substitution and the cavity may include the Y407T substitution.

[0152] In certain embodiments, the GDF15 polypeptide Fc partner in the fusion polypeptide may include additional mutations that improve a property of the fusion polypeptide. As such, the FC sequences in the first and second polypeptides described herein may include additional mutations. For example, the Fc partner sequence may include a mutation (or mutations) that abrogates (e.g., decreases or eliminates) IgG effector function that otherwise may be a characteristic of the Fc partner. In certain cases, the Fc partner sequence may include mutation (mutations) that abrogate effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0153] The four human IgG isotypes bind the activating Fcy receptors (FcyRI, FcYRIIa, FcYRIIIa), the inhibitory FcYRIIb receptor, and the first complement component (C1q) with different affinities, producing very different effector functions. Thus, mutations in linker regions can have a significant impact on effector function.

[0154] IgG binding to FCYRS or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for the binding of FCYRS and C1q and have unique sequences in IgG2 and IgG4. Substitutions in human IgG1 of residues in IgG2 at positions 233 to 236 and IgG4 residues in positions 327, 330 and 331 have been shown to greatly reduce ADCC and CDC activity (Armour KL. et al., 1999. Eur J Immunol. 29 (8): 2,613 to 2,624; Shields RL et al., 2001, J. Biol. 276 (9): 6,591 to 6,604). Furthermore, Idusogie et al. demonstrated that alanine substitution at different positions, including K322, significantly reduced complement activation (Idusogie EE. et al., 2000. J Immunol. 164 (8): 4,178 to 4,184). Likewise, mutations in the CH2 domain of murine IgG2A have been shown to reduce binding to FcyRI, and C1q (Steurer W. et al., 1995. J Immunol. 155 (3): 1165 to 1174). In certain embodiments, the Fc polypeptide may include a mutation in the CH2 domain that abolishes effector function (or effector functions) of IgG. Exemplary mutations in the CH2 regions include: APELLGGP (SEQ ID NO:96) ^ APALLGGP (SEQ ID NO:98); APELLGGP (SEQ ID NO:96) ^ APELAGGP (SEQ ID NO:99); and APELLGGP (SEQ ID NO:96) ^ APALAGGP (SEQ ID NO:97)

[0155] In some embodiments, an Fc polypeptide conjugated to GDF15 glycomuteins comprises part or all of a wild-type hinge sequence (generally at its N-terminus). In some embodiments, an Fc polypeptide does not comprise a wild-type or functional hinge sequence. In certain cases, the Fc sequence may include one of the following hinge sequences: EPKSCDKTHTCPPCP (SEQ ID NO:100); KSCDKTHTCPPCP (SEQ ID NO:101); SCDKTHTCPPCP (SEQ ID NO:102); CDKTHTCPPCP (SEQ ID NO:103); DKTHTCPPCP (SEQ ID NO:104); KTHTCPPCP (SEQ ID NO:105); THTCPPCP (SEQ ID NO:106); or CPPCP (SEQ ID NO:107); or a variant thereof having one or more substitutions (e.g., 1 to 6 substitutions, e.g., 1 to 5, 1 to 4, 1, 2, 3, 4, 5 or 6 substitutions). In certain cases, the Fc sequence may include the hinge region that forms a covalent bond (e.g., one or more disulfide bonds) with the hinge region of another Fc. Thus, in certain embodiments, the first and second polypeptides in the complexes disclosed herein may be associated through a covalent interaction between the hinge regions of the first and second polypeptides. The covalent interaction may include one or two intermolecular disulfide bonds.

[0156] Described in detail herein, a first polypeptide comprising an Fc button or hole sequence conjugated to a GDF15 glycomutein is contemplated. Such a polypeptide may be a complex with a second Fc polypeptide to which the first polypeptide may physically associate by placing the button in the hole of the Fc sequence.

[0157] In certain cases, a complex of a first Fc polypeptide and a second Fc polypeptide is disclosed. One of the first or second polypeptide may be a fusion polypeptide of Fc and GDF15. As noted herein, the GDF15 polypeptide may include a glycosylation mutation (or mutations) that leads to glycosylation of the GDF15 polypeptide. A glycosylated GDF15 polypeptide may also be referred to as GDF15-glycan or GDF15-glycomutein. The GDF15-glycan or GDF15-glycomutein may be as disclosed herein. In certain cases, the GDF15-glycan or GDF15-glycomutein fused to the Fc-button or Fc-hole polypeptide provided herein may be a polypeptide that includes an amino acid sequence that is at least 80%, 85%, 90% contiguous. , 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, wherein the contiguous amino acid sequence has the substitution D5T, D5S or R21N in relation to the sequence of amino acids of SEQ ID NO:1. In certain embodiments, the GDF15-glycan or GDF15-glycomutein fused to the Fc-button or Fc-hole polypeptide may be a polypeptide that has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96 %, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence includes one or more of the following pairs of substitutions with respect to the amino acid sequence of SEQ ID NO: :1: xvii. R16N and H18T or R16N and H18S; xviii. S23N and E25T or S23N and E25S; xix. S50N and F52T or S50N and F52S; xx. F52N and A54T or F52N and A54S; xxi. R53N and A55T or R53N and A55S; xxii. S64N and H66T or S64N and H66S; xxiii. K91N and D93T or K91N and D93S; xxiv. D93N and G95T or D93N and G95S; xxv. T94N and V96T or T94N and V96S; xxvi. V96N and L98T or V96N and L98S; xxvii. S97N and Q99T or S97N and Q99S; and xxviii. A106N and D108T or A106N and D108S.

[0158] In certain cases, the complex may include a first and a second polypeptide. The first polypeptide may include an IgG Fc sequence, wherein the IgG Fc sequence may include a CH3 sequence that includes at least one engineered protuberance; and a second polypeptide comprising an IgG Fc sequence, wherein the IgG Fc sequence comprises a CH3 sequence comprising at least one engineered cavity, wherein the first polypeptide dimerizes with the second polypeptide through the positioning of the protuberance of the first polypeptide in the cavity of the second polypeptide, and wherein the C-terminus of the first polypeptide or the C-terminus of the second polypeptide is conjugated to the N-terminus of a GDF15 mutein comprising at least one N-linked glycosylation consensus site. , the complex comprises a heterodimer of the first polypeptide and the second polypeptide. Once the first or second polypeptide is fused to the GDF15 muteins disclosed herein, one GDF15 molecule is present per heterodimer. In certain cases, the GDF15 mutein may be a GDF15 mutein described herein.

[0159] As discussed herein, the first and second polypeptides can interact to form a heterodimer through covalent and/or non-covalent interactions, such as hydrophobic interaction, disulfide bonds, or both.

[0160] In certain embodiments, a complex is disclosed that comprises a first heterodimer and a second heterodimer. Each of the first heterodimer and the second heterodimer may include a first polypeptide and a second polypeptide, wherein the first polypeptide may include an IgG Fc sequence, the IgG Fc sequence may include a CH3 sequence comprising at least one manipulated bulge; the second polypeptide may include an IgG Fc sequence, the IgG Fc sequence may include a CH3 sequence comprising at least one engineered cavity; wherein the first polypeptide dimerizes with the second polypeptide by positioning the protuberance of the first polypeptide in the cavity of the second polypeptide, wherein the C-terminus of the first polypeptide or the C-terminus of the second polypeptide is conjugated to the N-terminus of a GDF15 mutein comprising at least one N-linked glycosylation consensus site, wherein the GDF15 mutein in the first heterodimer dimerizes with the GDF15 mutein in the second heterodimer thus forming the complex comprising the first heterodimer and the second heterodimer. In a complex of the present disclosure, such complex includes the first heterodimer physically associated with a second heterodimer, two GDF15 molecules are present per heterodimer-heterodimer complex.

[0161] As noted herein, the first and second polypeptides may interact to form a heterodimer through covalent and/or non-covalent interactions, such as hydrophobic interaction, disulfide bonds, or both, and the first and second dimers of heterodimers can interact to form a dimer-dimer complex through covalent and/or non-covalent interactions, such as hydrophobic interaction, disulfide bonds, or both.

[0162] In certain cases, the GDF15 muteins present in each of the heterodimers described here, for example, in a complex of two heterodimers, may be identical or different in SEQUENCE. In certain cases, the GDF15 mutein in a complex of two heterodimers may be identical in SEQUENCE.

[0163] By way of example, Fc sequences for fusion to GDF15 muteins and as binding partners to FC-GDF15 fusion proteins are disclosed herein. In certain embodiments, the Fc sequences present in the complexes of the present disclosure may be similar or identical in sequence other than the engineered “button” and “hole” sequences.

[0164] In certain cases, the first and second polypeptides that can interact to form the complexes disclosed herein can be as shown below as Pair I to VIII. In the sequences presented below, the human immunoglobin G1 (hIgG1) Fc sequence is followed by a linker sequence (underlined), followed by the GDF15 mutein sequence (bold).

PAIR I: FIRST POLYPEPTIDE: hIgG1-Fc(AA)(T366W)-(G4S)2-ΔN2-GDF15 (N3-I112) (D5T)

[0165] DKTHTCPPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSNGTHCPLGPGRCCRLHTVRASL EDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHHRLKPDTVPAP CVCPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:3)

SECOND POLYPEPTIDE: hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0166] DKTHTCPPPCAPALAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:4)

PAR II: FIRST POLYPEPTIDE: hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN2-GDF15 (N3-I112) (D5T)

[ 0167 ] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHN HYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSNGTHC PLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHA QIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:5)

SECOND POLYPEPTIDE: hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0167] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:6)

PAR III: FIRST POLYPEPTIDE: hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15 (G4-I112)(R21N)

[0169] DKTHTCPPCPAPALAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGDHCP LGPGRCCRLHTVNASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQ IKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:7)

SECOND POLYPEPTIDE: hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[ 0170 ] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:8)

PAR IV: FIRST POLYPEPTIDE: hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15 (G4-I112)(S23N/E25T)

[0171] DKTHTCPPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGDHCP LGPGRCCRLHTVRANLTDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQ IKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:9)

SECOND POLYPEPTIDE: hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0171] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:10)

PAR V: FIRST POLYPEPTIDE: hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15 (G4-I112)(F52N/A54T)

[ 0173 ] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGDHCP LGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQNRTANMHAQ IKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:11)

SECOND POLYPEPTIDE: hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[ 0174 ] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:12)

PAIR VI: FIRST POLYPEPTIDE: hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15 (G4-I112)(R53N/A55T)

[0175] DKTHTCPPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGDHCP LGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFNATNMHAQ IKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:13)

SECOND POLYPEPTIDE: hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[ 0176 ] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:14)

PAR VII: FIRST POLYPEPTIDE: hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15 (G4-I112) (K91N/D93T)

[ 0177 ] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGDHCP LGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQ IKTSLHRLKPDTVPAPCCVPASYNPMVLIQNTTTGVSLQTYDDLLAKDCHCI (SEQ ID NO:15)

SECOND POLYPEPTIDE: hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[ 0178 ] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:16)

PAIR VIII: FIRST POLYPEPTIDE: hIgG1-Fc(AA)(T366W)- (G4S)5-ΔN3-GDF15 (G4-I112)(D93N/G95T)

[ 0179 ] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGDHCP LGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQ IKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTNTTVSLQTYDDLLAKDCHCI (SEQ ID NO:17)

SECOND POLYPEPTIDE: hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[ 0180 ] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:18)

[0181] In certain cases, the first and second polypeptides that can interact to form the complexes disclosed herein can have an amino acid sequence at least 80% identical to the amino acid sequence of the first and second polypeptide, respectively, as disclosed in Pares I to VIII. For example, the sequence identity may be at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more.

[0182] In certain embodiments, the complex may include a first polypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3; and a second polypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4, wherein the first and the second polypeptides are covalently linked through at least one intermolecular disulfide bond. Also provided herein is a complex comprising a first heterodimer and a second heterodimer, each of the first heterodimer and the second heterodimer comprising a first polypeptide having an amino acid sequence of at least 90%, 92%, 95%, 96% , 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3; and a second polypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4.

[0183] In certain embodiments, the complex may include a first polypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:5; and a second polypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 6, wherein the first and the second polypeptides are covalently linked through at least one intermolecular disulfide bond. Also provided herein is a complex comprising a first heterodimer and a second heterodimer, each of the first heterodimer and the second heterodimer comprising a first polypeptide having an amino acid sequence of at least 90%, 92%, 95%, 96% , 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:5; and a second polypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 6.

[0184] In certain embodiments, the complex may include a first polypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7; and a second polypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8, wherein the first and the second polypeptides are covalently linked through at least one intermolecular disulfide bond. Also provided herein is a complex comprising a first heterodimer and a second heterodimer, each of the first heterodimer and the second heterodimer comprising a first polypeptide having an amino acid sequence of at least 90%, 92%, 95%, 96% , 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7; and a second polypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0185] In particular embodiments, the complexes disclosed herein may include two heterodimers, each heterodimer comprising:

[0186] (a) a hIgG1-Fc polypeptide comprising a button (Fc-button) and having the sequence:

[0187] DKTHTCPPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:127); It is

[0188] (b) a hIgG1-Fc polypeptide comprising a hole (Fc-hole) and having the sequence:

[ 0189 ] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:4),

[0190] in which the Fc-button (a) or the Fc-hole (b) is fused at the C-terminus to the N-terminus of a GDF15 glycomutein. The sequence of GDF15 glycomutein can be as shown below:

[0191] ARNGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMC IGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ TYDDLLAKDCHCI (SEQ ID NO:128; GDF15 (A1-I112) D5T); or

[0192] NGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIG ACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI (SEQ ID NO:129; ΔN2-GDF15 (N3-I112) D5T); or

[0193] GTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGA CPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY DDLLAKDCHCI (SEQ ID NO:130; ΔN3-GDF15 (G4-I112) D5T); or

[0194] GDHCPLGPGRCCRLHTVNASLEDLGWADWVLSPREVQVTMCIGA CPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY DDLLAKDCHCI (SEQ ID NO:131; ΔN3-GDF15 (G4-I112) R21N); or

[0195] GDHCPLGPGRCCRLHTVRANLTDLGWADWVLSPREVQVTMCIGA CPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY DDLLAKDCHCI (SEQ ID NO:132; ΔN3-GDF15 (G4-I112) (S23N/E25T)); or

[0196] GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGA CPSQNRTANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY DDLLAKDCHCI (SEQ ID NO:133; ΔN3-GDF15 (G4-I112)(F52N/A54T)); or

[0197] GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGA CPSQFNATNMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY DDLLAKDCHCI (SEQ ID NO:134; ΔN3-GDF15 (G4-I112)(R53N/A55T)); or

[0198] GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGA CPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQNTTTGVSLQTY DDLLAKDCHCI (SEQ ID NO:135; ΔN3-GDF15 (G4-I112) (K91N/D93T)); or

[0199] GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGA CPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTNTTVSLQTYD DLLAKDCHCI (SEQ ID NO:136; ΔN3-GDF15 (G4-I112)(D93N/G95T)).

[0200] In certain examples, the amino acid sequence of the Fc-button may be at least 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:127. In certain examples, the amino acid sequence of Fc-hole may be at least 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:4. In certain examples, the amino acid sequence of the GDF15 mutein may be at least 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of any of SEQ ID NOs:128 to 136.

[0201] The Fc-button or Fc-hole can be joined to the GDF15 glycomutein through a (G4S)n linker sequence, where n = 1 to 10, such as 2, 3, 4 or 5.

[0202] In certain examples, the complexes of the present disclosure may have a recovery of at least 50 mg/l, for example, at least more than 55 mg/l, 60 mg/l, 65 mg/l, 70 mg /l, 75 mg/l, 80 mg/l, 85 mg/l, 90 mg/l, 95 mg/l, 100 mg/l, 110 mg/l, 120 mg/l, 130 mg/l, 140 mg /l, 150 mg/l, 160 mg/l, 170 mg/l, 180 mg/l, 190 mg/l, 200 mg/l or more. In certain cases, the complexes of the present disclosure may have a recovery of at least 50 mg/l to 300 mg/l, such as 60 mg/l to 300 mg/l, 75 mg/l to 300 mg/l, 75 mg/l to 250 mg/l, 75 mg/l to 200 mg/l, 75 mg/l to 175 mg/l, 75 mg/l to 150 mg/l, 100 mg/l to 300 mg/l, 100 mg/l to 250 mg/l, 100 mg/l to 200 mg/l, 100 mg/l to 150 mg/l, 100 mg/l to 125 mg/l, 110 mg/l to 300 mg/l or 150 mg/l to 300 mg/l. Complex recovery refers to the amount of fully assembled dimer-dimer complex obtained from culture media in which a host cell expressing the first and second polypeptides that form the two dimers present in each fully assembled complex is grown. .

[0203] The present disclosure also contemplates Fc polypeptide fusion partners, and fusion proteins comprising such, where the Fc polypeptide fusion partner is modified to be a partner of a charged Fc pair. A “charged Fc pair partner” refers to either (i) a “negatively charged” Fc sequence (optionally without the hinge region) and comprising a charged pair mutation or (ii) a “negatively charged” Fc sequence (optionally without the hinge region) and comprising a charged pair mutation or (ii) a “charged Fc pair partner” positive” (optionally without the hinge region) and comprising a charged pair mutation. The terms “positive charge” and “negative charge” are used herein by reference to describe the nature of the charge of pairwise mutations in Fc sequences and not to indicate that the overall sequence or construct necessarily has a positive or negative charge. Charged Fc amino acid sequences suitable for use in polypeptide constructs (e.g., GDF15 glycomutein, modified GDF15 glycomuteins) of the present disclosure are described, for example, in WO 2013/113008.

[0204] Examples of a positively charged Fc (“Fc(+)”) include an Fc comprising an aspartic acid to lysine (E356K) mutation and a glutamic acid to lysine (D399K) mutation of an Fc sequence lacking the region of hinge. Examples of a negatively charged Fc (“Fc(-)”) include an Fc comprising two lysine to aspartate mutations (K392D, K409D) in an Fc sequence lacking the hinge region. The C-terminal lysine (K477) can also be optionally deleted. When an Fc(+)polypeptide fusion protein (e.g., Fc(+)GDF15 mutein fusion protein) and an Fc(-)polypeptide fusion protein (e.g., Fc(-) mutein fusion protein) GDF15) are incubated together, the aspartate residues associate with the lysine residues through electrostatic force, which facilitates the formation of Fc heterodimers between the Fc(+) and Fc(-) sequences of the GDF15 polypeptide fusion proteins.

[0205] The present disclosure also contemplates constructs designated “hemi” or “hemiFc” constructs, which comprise two Fc sequences joined together by a linker that connects the N-terminus of a first Fc sequence to the C-terminus of a second Fc sequence. . In some embodiments, a monomer comprises a polypeptide sequence (e.g., a GDF15 glycomutein or mature modified GDF15) 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 sequence. Fc, 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 are also associated by the Fc hinge regions. Two such monomers associate to form a dimer to which the monomers are linked via an interchain disulfide bond between the two polypeptide sequences. For examples of hemiFc polypeptides suitable for use with the GDF15 muteins of the present disclosure see document under WO 2013/113008.

[0206] The present disclosure also contemplates fusion proteins having a multimer of Fc polypeptides, or fragments thereof, including a partner of a charged Fc pair (e.g., an Fc multimer).

[0207] The complexes of the present disclosure have improved properties, such as increased solubility, decreased aggregation and/or increased serum half-life. In certain cases, the solubility of the complexes is generally improved relative to unconjugated recombinant human GDF15 and Fc-conjugated wild-type GDF15 (button or hole). In certain embodiments, the complex has a solubility of at least 1 mg/ml in phosphate buffered saline (PBS) at pH 7.0. In other embodiments, the complex has a solubility of at least 2 mg/ml, at least , 3 mg/ml, at least 4 mg/ml or at least 5 mg/ml. In other embodiments, the complex has a solubility of at least 6 mg/ml in phosphate buffered saline (PBS) at pH 7.0, at least 7 mg/ml, at least 8 mg/ml, at least 9 mg/ml. ml or at least 10 mg/ml. In particular embodiments, the complex has a solubility greater than 10 mg/ml.

[0208] Glycosylation: For purposes of the present disclosure, “glycosylation” is intended to refer generically to enzymatic processes that attach glycans to proteins, lipids or other organic molecules. Use of the term “glycosylation” in conjunction with the present disclosure is generally intended to mean the addition or deletion of one or more carbohydrate chemical moieties (by removing the underlying glycosylation site or deleting the glycosylation by chemical and/or enzymatic means). ) and/or the addition of one or more glycosylation sites that may or may not be present in the native sequence. Furthermore, the phrase includes qualitative changes in the glycosylation of native proteins involving a change in the nature and proportions of the various carbohydrate moieties present.

[0209] Glycosylation can dramatically affect the physical properties of proteins and may also be important in protein stability, secretion and subcellular localization. Indeed, the glycosylation of GDF15 mutein polypeptides described here confers beneficial improvements to their physical properties. By way of example, but not limitation, the solubility of GDF15 muteins can be improved by glycosylation, and such improvement can be substantial (see Examples). The improved solubility exhibited by such modified GDF15 muteins may, for example, allow the generation of formulations more suitable for pharmaceutical delivery of non-glycosylated GDF15/GDF15 muteins. Glycosylated GDF15/GDF15 mutein polypeptides may also exhibit increased stability. Furthermore, polypeptides can improve one or more pharmacokinetic properties, such as half-life.

[0210] The addition of a glycosylation site can be carried out by changing the amino acid sequence as described above. The change to the polypeptide can be made, for example, by the addition of, or substitution for, one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites). . The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type can be different. One type of sugar that is commonly found in both is N-acetylneuraminic acid (hereafter referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, can impart acidic properties to the glycoprotein. A particular embodiment of the present invention comprises the generation and use of N-glycosylation variants as described above.

[0211] Another means of increasing the number of carbohydrate moieties in the polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide.

[0212] Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) are a commonly used host cell for the production of recombinant glycoproteins. These cells do not express the enzyme beta-galactoside alpha-2,6-sialyltransferase and therefore do not add sialic acid at the alpha-2,6 linkage to N-linked oligosaccharides of glycoproteins produced in these cells.

[0213] In particular embodiments, GDF15 muteins comprising at least one N-linked glycosylation consensus site are glycosylated. Thus, in particular embodiments, the GDF15 muteins in the complexes disclosed herein can be glycosylated at the N-linked glycosylation consensus site introduced into the GDF15 mutein. In certain cases, while a complex disclosed herein may include glycosylated GDF15, such as glycosylated GDF15 produced during expression from a cell line, the complex may be post-production treated to remove the carbohydrate moiety. Post-production removal of the carbohydrate moiety can result in the removal of substantially all carbohydrate groups attached to the GDF15 mutein (and the Fc sequence) during expression in a eukaryotic host cell.

[0214] Thus, the present disclosure contemplates the conjugation of one or more additional components or molecules at the N-terminus and/or C-terminus of a polypeptide sequence, such as another protein (for example, a protein having a sequence of amino acids heterologous to the present protein) or a carrier molecule. Thus, an exemplary polypeptide sequence may be provided as a conjugate with another component or molecule.

[0215] A polypeptide can also be conjugated to large, slowly metabolized macromolecules, such as proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads; polymeric amino acids, such as polyglutamic acid, polylysine; amino acid copolymers; inactivated virus particles; inactivated bacterial toxins, such as diphtheria toxoid, tetanus, cholera, leukotoxin molecules; inactivated bacteria; and dendritic cells. Such conjugated forms, if desired, can be used to produce antibodies against a polypeptide of the present disclosure. In certain cases, the GDF15 in the complexes described here may be a polypeptide conjugated to a large, slowly metabolized macromolecule.

[0216] Additional candidate components and molecules for conjugation include those that are suitable for isolation or purification. Particular non-limiting examples include binding molecules such as biotin (biotin-avidin specific binding pair), an antibody, a receptor, a ligand, a lectin, or molecules comprising a solid support, including, for example, plastic beads. or polystyrene, plates or beads, magnetic beads, test strips and membranes.

[0217] Purification methods such as cation exchange chromatography can be used to separate conjugates by charge difference, which effectively separates conjugates into their various molecular weights. For example, the cation exchange column can be loaded and then washed with ~20 mM sodium acetate, pH ~4, and then eluted with a linear NaCl gradient (0 to 0.5 M) buffered to a pH of about from 3 to 5.5, for example, at pH ~ 4.5. The content of fractions obtained by cation exchange chromatography can be identified by molecular weight using conventional methods, for example, mass spectroscopy, SDS-PAGE, or others Known methods for separating molecular entities by molecular weight.

[0218] Linkers: Any of the previously mentioned components and molecules used to modify the polypeptide sequences of the present disclosure can be optionally conjugated through a linker. Suitable linkers include “flexible linkers” that are generally of sufficient length to allow some movement between the modified polypeptide sequences and the attached components and molecules. Ligand molecules can be about 6 to 50 atoms long. Ligand molecules can also be, for example, aryl acetylene, ethylene glycol oligomers containing 2 to 10 monomeric units, diamines, diacids, amino acids or combinations thereof. Suitable linkers can be easily selected and can be of any suitable size, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 30, 30 to 50 amino acids.

[0219] Exemplary flexible linkers include glycine (G)n polymers, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., (GmSo)n, (GSGGS)n (SEQ ID NO :120), (GmSoGm)n, (GmSoGmSoGm)n (SEQ ID NO:121), (GSGGSm)n (SEQ ID NO:122), (GSGSmG)n (SEQ ID NO:123) and (GGGSm)n ( SEQ ID NO:124), and combinations thereof, wherein m, n and o are each independently selected from an integer of at least 1 to 20, e.g., 1 to 18, 2 to 16, 3 to 14, 4 to 12, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), and other flexible binders. Glycine and glycine-serine polymers are relatively unstructured and therefore can serve as a neutral link between components. Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO: 21), GGSGG (SEQ ID NO: 22), GSGSG (SEQ ID NO: 23), GSGGG (SEQ ID NO: 24), GGGSG ( SEQ ID NO: 25), and GSSSG (SEQ ID NO: 26).

[0220] Additional flexible linkers include glycine polymers (G)n or glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO:120), (GGGS)n (SEQ ID NO :125) and (GGGGS)n (SEQ ID NO:126), where n = 1 to 50, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20 , 20 to 30, 30 to 50). Exemplary flexible linkers include, but are not limited to, GGGS (SEQ ID NO:19), GGGGS (SEQ ID NO:20), GGSG (SEQ ID NO:21), GGSGG (SEQ ID NO:22), GSGSG ( SEQ ID NO:23), GSGGG (SEQ ID NO:24), GGGSG (SEQ ID NO:25), and GSSSG (SEQ ID NO:26). A multimer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 30, or 30 to 50) of these linker sequences can be linked together to provide linkers flexible forms that can be used to conjugate a heterologous amino acid sequence to the polypeptides disclosed herein. As described herein, the heterologous amino acid sequence may be a signal sequence and/or a fusion partner, such as albumin, Fc sequence and the like.

[0221] Examples of linkers include, for example, (GGGGS)n (SEQ ID NO:126), wherein n is an integer from 1 to about 10 (e.g., n = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); GGGSGGGSIEGR (SEQ ID NO:48); GGGGG (SEQ ID NO:27); EGGGS (SEQ ID NO:28).

[0222] In some cases, the linker may be a cleavable linker, for example, an enzymatically cleavable linker. In other cases, the linker may be a non-cleavable linker, for example, a linker that is not enzymatically cleaved under normal physiological conditions in vivo.

[0223] For example, a proteolytically cleavable linker may include a matrix metalloproteinase (MMP) cleavage site, e.g., a cleavage site for an MMP selected from collagenase-1, -2 and -3 (MMP-1, -8 and -13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2 and 3 (MMP-3, -10 and -11), matrilysin (MMP-7) and membrane metalloproteinases (MT1 -MMP and MT2 -MMP). The cleavage sequence of MMP-9 is Pro-X-X-Hy (where, X represents an arbitrary residue; Hy, a hydrophobic residue) (SEQ ID NO:29), for example, Pro-X-X-Hy- (Ser/ Thr) (SEQ ID NO:30), for example, Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ ID NO:31) or Pro-Leu/Gln-Gly-Met-Thr (SEQ ID NO: 32). Another example of a protease cleavage site is a plasminogen activator cleavage site, for example, a uPA or a tissue plasminogen activator (tPA) cleavage site. Specific examples of uPA and tPA cleavage sequences include sequences comprising Val-Gly-Arg. Another example is a thrombin cleavage site, for example, CGLVPAGSGP (SEQ ID NO:33). Additional suitable linkers comprising protease cleavage sites include linkers comprising one or more of the following amino acid sequences: 1) SLLKSRMVPNFN (SEQ ID NO:34) or SLLIARRMPNFN (SEQ ID NO:35), cleaved by cathepsin B; SKLVQASASGVN (SEQ ID NO:36) or SSYLKASDAPDN (SEQ ID NO:37), cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO:38) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO:39) cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ ID NO:40) cleaved by MMP-9; DVDERDVRGFASFL (SEQ ID NO:41) cleaved by an MMP similar to thermolysin; SLPLGLWAPNFN (SEQ ID NO:42) cleaved by matrix metalloproteinase 2 (MMP-2); SLLIFRSWANFN (SEQ ID NO:43) cleaved by catespin L; SGVVIATVIVIT (SEQ ID NO:44) cleaved by cathepsin D; SLGPQGIWGQFN (SEQ ID NO:45) cleaved by matrix metalloproteinase 1 (MMP-1); KKSPGRVVGGSV (SEQ ID NO:46) cleaved by urokinase-type plasminogen activator; PQGLLGAPGILG (SEQ ID NO:47) cleaved by membrane matrix metalloproteinase type 1 (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQ ID NO:94) cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase and stromelysin-1; GPQGLAGQRGIV (SEQ ID NO:49) cleaved by matrix metalloproteinase 13 (collagenase-3); GGSGQRGRKALE (SEQ ID NO:50) cleaved by tissue-type plasminogen activator (tPA); SLSALLSSDIFN (SEQ ID NO:51) cleaved by human prostate specific antigen; SLPRFKIIGGFN (SEQ ID NO:52) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ ID NO:53) cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO:54) cleaved by calpain (calcium-activated neutral protease).

[0224] In addition to the specific amino acid sequences and nucleic acid sequences provided herein, the disclosure also contemplates polypeptides and nucleic acids that have sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical in sequence to amino acids and nucleic acids. The terms “identical” or percentage “identity”, in the context of two or more polynucleotide sequences or two or more amino acid sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of residues. amino acids or nucleotides that are the same (e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical over a specified region) when compared and aligned for maximum correspondence over a designated region. The disclosure specifically contemplates the first and second polypeptides present in a complex, the first polypeptide and the second polypeptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical in sequence to the amino acid sequence of the first and second polypeptide, respectively, of the first and second pairs of polypeptides provided herein.

POLYPEPTIDE PRODUCTION METHODS

[0225] A polypeptide of the present disclosure can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis).

A. CHEMICAL SYNTHESIS

[0226] Where a polypeptide is chemically synthesized, the synthesis can proceed through the liquid phase or solid phase. Solid-phase peptide synthesis (SPPS) allows the incorporation of unnatural amino acids and/or modification of the peptide/protein backbone. Various forms of SPPS, such as Fmoc and Boc, are available for the synthesis of polypeptides of the present disclosure. The details of the chemical synthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. Med. Chem. 6: 3 to 10; and Camarero JA et al., 2005 Protein Pept Lett. 12: 723 to 728).

B. RECOMBINANT PRODUCTION

[0227] When a polypeptide is produced using recombinant techniques, the polypeptide can be produced as an intracellular protein or as a secreted protein, with the use of any suitable construct and any suitable host cell, which may be a prokaryotic cell or eukaryotic, such as a bacterial cell (e.g., E. coli) or a yeast host cell, respectively. Other examples of eukaryotic cells that can be used as host cells include insect cells, mammalian cells and/or plant cells. When mammalian host cells are used, they may include human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells (e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g. Cos 1, Cos 7 and CV1) and hamster cells (e.g. Chinese hamster ovary (CHO) cells). In specific embodiments, the polypeptide and complexes comprising the polypeptide are produced in CHO cells. In other embodiments, the polypeptide and complexes comprising the polypeptide are produced in a yeast cell and in particular embodiments may be a yeast cell genetically modified to produce glycoproteins with mammalian-type N-glycans.

[0228] A variety of host vector systems suitable for expressing a polypeptide may be employed according to conventional procedures known in the art. See, for example, Sambrook et al., 1989, Current Protocols in Molecular Biology Cold Spring Harbor Press, New York. ; and Ausubel et al. 1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for introducing genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The transfer method can be selected to provide stable expression of the nucleic acid encoding the introduced polypeptide. The nucleic acid encoding the polypeptide may be provided as an inherited episomal element (e.g., a plasmid) or may be genomically integrated. A variety of vectors suitable for use in producing a polypeptide of interest are commercially available.

[0229] Vectors can provide extrachromosomal maintenance in a host cell or can provide integration into the host cell genome. The expression vector provides transcription and translation regulatory sequences, and can provide inducible or constitutive expression, where the coding region is operatively linked, under the transcriptional control of the transcription initiation region and a transcriptional and translational termination region. In general, transcription and translation regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding site, transcriptional start and stop sequences, translation initiation and stop sequences, and enhancer or activator sequences. Promoters may be constitutive or inducible, and may be a strong constitutive promoter (e.g., T7).

[0230] Expression constructs generally have convenient restriction sites located close to the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest. A selectable marker operational in the expression host may be present to facilitate the selection of cells containing the vector. Furthermore, the expression construct may include additional elements. For example, the expression vector may have one or two replication systems, thereby allowing it to be maintained in organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Furthermore, the expression construct may contain a selectable marker gene to allow selection of transformed host cells. Selectable genes are well known in the art and will vary with the host cell used.

[0231] Isolation and purification of a protein can be achieved according to methods known in the art. For example, a protein can be isolated from a lysate of cells genetically modified to express the protein constitutively and/or at the time of induction; from the culture medium in which the host cell is grown; or from a synthetic reaction mixture, by affinity purification, which may involve contacting the sample (cell lysate, culture medium or reaction mixture) with a label that specifically binds to the protein, washing to remove material specifically unbound, and elution of the specifically bound protein. The isolated protein can be further purified by dialysis and other methods normally employed in protein purification methods. In one embodiment, the protein can be isolated using metal chelate chromatography methods. Proteins may contain modifications to facilitate isolation. In certain embodiments, the complexes of the present disclosure can be separated based on size.

[0232] In certain embodiments, a complex comprising a first polypeptide and a second polypeptide, the first polypeptide comprising an IgG Fc sequence, wherein the IgG Fc sequence comprises a CH3 sequence comprising at least one engineered protuberance; the second polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered pocket; wherein the first polypeptide dimerizes with the second polypeptide by positioning the protuberance of the first polypeptide in the cavity of the second polypeptide, wherein the C-terminus of the first polypeptide or the C-terminus of the second polypeptide is conjugated to the N-terminus of a GDF15 mutein comprising at least one N-linked glycosylation consensus site can be isolated from the medium in which a host cell expressing the first and second polypeptides is cultivated.

[0233] In certain embodiments, a complex comprising a first heterodimer and a second heterodimer, each of the first heterodimer and the second heterodimer comprising a first polypeptide and a second polypeptide, the first polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered protuberance; and the second polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered pocket; wherein the first polypeptide dimerizes with the second polypeptide by positioning the protuberance of the first polypeptide in the cavity of the second polypeptide, wherein the C-terminus of the first polypeptide or the C-terminus of the second polypeptide is conjugated to the N-terminus of a GDF15 mutein comprising at least one N-linked glycosylation consensus site, wherein the GDF15 mutein in the first heterodimer dimerizes with the GDF15 mutein in the second heterodimer thus forming the complex comprising the first heterodimer and the second heterodimer, may be isolated from the medium in which a host cell expressing the first and second polypeptides is cultivated.

[0234] As noted herein, a first and a second nucleic acid may be present in a single vector or separate vectors in a single host cell or two different host cells. In certain cases, the first and second polypeptides of the present disclosure may be encoded by a first and a second nucleic acid, respectively, which may be present expressed in the same cell. In embodiments in which the first and second nucleic acids are present in different cells, the cells may be fused, such as some time during the production process.

[0235] The complexes can be prepared in substantially pure or isolated form (e.g., free from other polypeptides). The complexes may be present in a composition that is enriched for the complexes relative to other components that may be present (e.g., other polypeptides or other complexes (e.g., homodimers, homotetramers) or other host cell components). For example, the purified complex (e.g., a heterodimer-heterodimer complex) may be provided such that the complex is present in a composition that is substantially free of other expressed proteins, e.g., less than 90%, less than 60%. %, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% of the composition consists of other proteins expressed.

ANTIBODIES

[0236] The present disclosure provides antibodies, including isolated antibodies that specifically bind to a polypeptide or fusion protein of the present disclosure. The term “antibody” encompasses intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody binding fragments, including Fab and F(ab)'2 fragments, as long as they exhibit the desired biological activity. The basic total antibody structural unit comprises a tetramer and each tetramer is composed of two identical pairs of polypeptide chains, each pair having a “light” chain (about 25 kDa) and a “heavy” chain (about 50-70 kDa). kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. In contrast, the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda, while human heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. . Linker fragments are produced by recombinant DNA techniques or by chemical or enzymatic cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single chain antibodies.

[0237] Each heavy chain has at one end a variable domain (VH) followed by several constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Within the light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. All antibody chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. The CDRs of the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From the N-terminus to the C-terminus, both the light and heavy chains comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains.

[0238] An intact antibody has two binding sites and, except in bifunctional or bispecific antibodies, the two binding sites are the same. A bispecific or bifunctional antibody is an artificial hybrid antibody with two pairs of different heavy/light chains and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or ligation of Fab' fragments.

[0239] As presented above, binding fragments can be produced by enzymatic or chemical cleavage of intact antibodies. Digestion of antibodies with the enzyme papain results in two identical antigen-binding fragments, also known as “Fab” fragments, and one “Fc” fragment, which has no antigen-binding activity. Digestion of antibodies with the enzyme pepsin results in an F(ab')2 fragment in which the two arms of the antibody molecule remain linked and comprise two antigen-binding sites. The F(ab')2 fragment has the ability to cross-link the antigen

[0240] As used herein, the term “Fab” refers to a fragment of an antibody that comprises VH and VL regions, as well as the constant domain of the light chain and the CH1 domain of the heavy chain.

[0241] When used here, the term “Fv” refers to the minimal fragment of an antibody that retains both the recognition antigen and the antigen-binding sites. In a double-chain Fv species, this region includes a dimer of a heavy chain variable domain and a light chain variable domain in a noncovalent association. In a single-chain Fv species, a heavy-chain variable domain and a light-chain variable domain can be covalently linked by a flexible peptide linker, such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a type of double-chain Fv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the dimeric VH-VL. While the six CDRs collectively confer antigen-binding specificity to the antibody, even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind antigen.

[0242] When used here, the term “complementarity determining regions” or “CDRs” refers to parts of immune receptors that contact a specific ligand and determine its specificity.

[0243] The term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for binding to the antigen. The hypervariable region generally comprises amino acid residues of a CDR and/or residues of a “hypervariable loop”.

[0244] As used herein, the term “epitope” refers to binding sites for antibodies on protein antigens. Epitopic determinants typically comprise chemically active surface clusters of molecules, such as amino acids or sugar side chains, as well as specific structural and three-dimensional charge features. An antibody is said to bind an antigen when the dissociation constant is < 1 μM, < 100 nM, or < 10 nM. An increase in the equilibrium constant (“KD”) means there is less affinity between the epitope and the antibody, whereas a decrease in the equilibrium constant means there is more affinity between the epitope and the antibody. An antibody with a KD of “no more than” a certain amount means that the antibody will bind to the epitope with the given KD or more strongly. While KD describes the binding characteristics of an epitope and an antibody, “potency” describes the effectiveness of the antibody itself for an antibody function. There is not necessarily a correlation between a balance constant and power; so, for example, a relatively low KD does not automatically mean high power.

[0245] The term “selectively binds”, in reference to an antibody, does not mean that the antibody binds only to a single substance, but rather that the KD of the antibody to a first substance is lower than the KD of the antibody to a second substance. An antibody that binds exclusively to one epitope only binds to that single epitope.

[0246] When administered to humans, antibodies that contain rodent (i.e., murine or rat) variable and/or constant regions are sometimes associated with, for example, rapid clearance from the body or generation of an immune response by the body against the antibody. In order to avoid the use of rodent-derived antibodies, fully human antibodies can be generated by introducing human antibody function into a rodent, so that the rodent produces fully human antibodies. Unless specifically identified herein, “human” and “fully human” antibodies may be used interchangeably. The term “completely human” can be useful in distinguishing antibodies that are only partially human from those that are completely, or wholly, human. One skilled in the art is aware of several methods of generating fully human antibodies.

[0247] In order to address possible human anti-mouse antibody responses, chimeric or otherwise humanized antibodies may be used. Chimeric antibodies have a human constant region and a murine variable region, and as such, anti-human chimeric antibody responses may be observed in some patients. Therefore, it is advantageous to provide fully human antibodies against multimeric enzymes in order to avoid possible human anti-mouse antibodies or anti-human chimeric antibodies.

[0248] Fully human monoclonal antibodies can be prepared, for example, by generating hybridoma cell lines, by techniques known to those skilled in the art. Other preparation methods involve the use of sequences encoding specific antibodies for transformation of a suitable mammalian host cell, such as a CHO cell. Transformation may be carried out by any known method for introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide into a virus (or viral vector) and transducing a host cell with the virus (or vector), or by procedures known in the art. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into cores. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, CHO cells, HeLa cells, and human hepatocellular carcinoma cells.

[0249] Antibodies can be used to detect a polypeptide of the present disclosure. For example, antibodies can be used as a diagnosis by detecting the level of one or more polypeptides of the present invention in a subject and, or comparing the detected level to a normal control level or to a baseline level in a subject. previously determined (for example, before any illness).

THERAPEUTIC AND PROPHYLACTIC USES

[0250] The present disclosure provides methods for treating or preventing metabolic and metabolism-associated diseases, such as obesity and other body weight disorders, hyperglycemia, hyperinsulinemia, glucose intolerance, and glucose metabolism disorders, by means of administration of the complex of the present disclosure, or compositions thereof, as described herein. Such methods may also have an advantageous effect on one or more symptoms associated with a disease, disorder or condition, for example, decreasing the severity or frequency of a symptom.

[0251] In order to determine whether a subject may be a candidate for the treatment or prevention of a body weight disorder (e.g., obesity) by the methods provided herein, parameters such as, but not limited to, etiology and extent of the The subject's condition (e.g., how overweight the subject is compared to the healthy reference individual) should be assessed. For example, an adult having a BMI between ~25 and ~29.9 kg/m2 may be considered overweight (pre-obese), whereas an adult having a BMI of ~30 kg/m2 or greater may be considered obese. . As discussed herein, the polypeptides of the present invention can effect appetite suppression, for example, a decrease in appetite that leads to a reduction in body weight.

[0252] In order to determine whether a subject may be a candidate for the treatment or prevention of hyperglycemia, hyperinsulinemia, glucose intolerance and/or glucose disorders by the methods provided herein, various diagnostic methods known in the art may be utilized. Such methods include those described elsewhere herein (e.g., fasting plasma glucose (FPG) assessment and oral glucose tolerance test (oGTT)).

[0253] The complexes provided herein, when administered to a subject for the treatment or prevention of metabolic and metabolism-associated diseases, such as obesity and other body weight disorders, hyperglycemia, hyperinsulinemia, glucose intolerance, metabolism disorders of glucose can lead to a reduction in blood glucose level, a reduction in body weight and/or a reduction in food intake.

[0254] In certain embodiments, the complexes contemplated herein can decrease blood glucose level, body weight and/or food intake by at least 5% compared to those in the absence of administration of the complexes. For example, the complexes contemplated herein may decrease blood glucose level, body weight and/or food intake by at least 10%, 20%, 30%, 50%, 60%, 70%, 80% or 90% compared to before starting treatment or prevention.

[0255] In certain embodiments, a complex of the present disclosure used to treat a metabolic disorder may be a complex that includes two heterodimer molecules per complex, wherein each heterodimer is the same and includes a first polypeptide and a second polypeptide, wherein the first polypeptide includes an IgG Fc sequence, the IgG Fc sequence may include a CH3 sequence comprising at least one engineered protuberance; the second polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered pocket; wherein the first polypeptide dimerizes with the second polypeptide by positioning the protrusion of the first polypeptide into the cavity of the second polypeptide to form a heterodimer, wherein the C-terminus of the first polypeptide or the C-terminus of the second polypeptide in each heterodimer is conjugated to the N-terminus of a GDF15 mutein comprising at least one N-linked glycosylation consensus site, wherein the GDF15 mutein in the heterodimer dimerizes with the GDF15 mutein in another heterodimer, thus forming the complex comprising two heterodimers.

[0256] In still other embodiments, a complex of the present disclosure used to treat a metabolic disorder may be a complex that includes two heterodimer molecules (heterodimer associated with heterodimer) per complex, wherein each heterodimer is equal and each heterodimer includes a first polypeptide having an IgG Fc sequence, the IgG Fc sequence may include a CH3 sequence comprising at least one engineered protuberance, wherein the C-terminus of the first polypeptide is fused to the N-terminus of the GDF15 glycomutein; and the second polypeptide comprising an IgG Fc sequence, the IgG Fc sequence comprising a CH3 sequence comprising at least one engineered pocket; wherein the first polypeptide dimerizes with the second polypeptide by positioning the protrusion of the first polypeptide in the cavity of the second polypeptide to form the heterodimer, wherein the GDF15 mutein in the heterodimer dimerizes with the GDF15 mutein in another heterodimer thus forming the complex comprising the two heterodimers.

PHARMACEUTICAL COMPOSITIONS

[0257] The complexes of the present disclosure may be in the form of compositions suitable for administration to a subject. In general, such compositions are “pharmaceutical compositions” comprising one or more complexes and one or more pharmaceutically or physiologically acceptable diluents, carriers or excipients. In certain embodiments, the complexes are present in a therapeutically effective amount in the pharmaceutical composition. Pharmaceutical compositions can be used in the methods of the present disclosure; thus, for example, the pharmaceutical compositions may be administered ex vivo or in vivo to a subject in order to practice the therapeutic and prophylactic methods and uses described herein. As noted herein, the complexes may or may not be glycosylated. For example, the complexes may be glycosylated as produced in a eukaryotic host cell and may be subjected to a process to remove the carbohydrate moiety prior to formulation into a pharmaceutical composition. Removal of carbohydrate moieties may result in a significant reduction in glycosylation of the polypeptides in the complexes or a complete absence of glycosylation of the polypeptides in the complexes.

[0258] In specific embodiments, the present disclosure provides methods for treating a disorder in glucose metabolism or body weight through administration of the complexes, N-glycosylated complexes or compositions thereof. In the particular embodiment, the present disclosure provides methods for reducing food intake or decreasing body weight through administration of the complexes, N-glycosylated complexes or compositions thereof. The present disclosure further provides a use of the aforementioned sequences, complexes, N-glycosylated complexes or compositions thereof in the manufacture of a medicament for use in the treatment of a condition selected from metabolic and metabolism-associated diseases, such as , obesity and other body weight disorders, hyperglycemia, hyperinsulinemia, glucose intolerance and glucose metabolism disorders. The present disclosure further provides a use of the aforementioned sequences, complexes, N-glycosylated complexes or compositions thereof in the manufacture of a medicament for use in treating a disorder in glucose metabolism or body weight. The present disclosure further provides a use of the aforementioned sequences, complexes, N-glycosylated complexes or compositions thereof in the manufacture of a medicament for use in reducing food intake or body weight.

[0259] Also provided herein are compositions, for example, pharmaceutical compositions of the sequences, complexes and N-glycosylated complexes disclosed herein for the treatment or prevention of a condition selected from metabolic and metabolism-associated diseases, such as obesity. and other body weight disorders, hyperglycemia, hyperinsulinemia, glucose intolerance and glucose metabolism disorders. The present disclosure further provides a composition (e.g., pharmaceutical composition) of the aforementioned N-glycosylated sequences, complexes, or complexes for treating a disorder in glucose metabolism or body weight. The present disclosure further provides a composition (e.g., pharmaceutical composition) of the aforementioned N-glycosylated sequences, complexes, or complexes for reducing food intake or body weight.

[0260] The pharmaceutical compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration; Exemplary routes of administration are presented here. Furthermore, the pharmaceutical compositions may be used in combination with other therapeutically active agents or compounds (e.g., glucose-lowering agents) as described herein in order to treat or prevent diseases, disorders and conditions as contemplated. by this disclosure.

[0261] Pharmaceutical compositions typically comprise a therapeutically effective amount of at least one of the complexes contemplated by the present disclosure and one or more pharmaceutically and physiologically acceptable formulating agents. Pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methylparabens, ethyl or n-propyl, p- hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, fillers, bulking agents, detergents, buffers, vehicles, diluents and/or adjuvants. For example, a suitable carrier may be physiological saline or citrate-buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are the most exemplary vehicles. Those skilled in the art will readily recognize a variety of buffers that can be used in pharmaceutical compositions and dosage forms. Typical buffers include, but are not limited to, weak pharmaceutically acceptable acids, weak bases or mixtures thereof. As an example, the components of the buffer may be water-soluble materials such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid and salts thereof. Acceptable buffering agents include, for example, a Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino) ethanesulfonic acid (MES), sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino) propanesulfonic acid (MOPS) and N-Tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0262] After a pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form, in a lyophilized form that requires reconstitution before use, in a liquid form that requires dilution before use, or in another acceptable form. In some embodiments, the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, ampoule, syringe, or auto-injector (similar to, for example, an EpiPen®)), whereas a multi-use container ( e.g., a multipurpose bottle) is provided in other embodiments. Any drug delivery apparatus can be used to deliver the complexes, including implants (e.g., implantable pumps) and catheter systems, both of which are well known to those skilled in the art. Depot injections, which are generally administered subcutaneously or intramuscularly, can also be used to release the complexes disclosed herein over a defined period of time. Depot injections are generally either solid or oil based and generally comprise at least one of the formulation components set forth herein. One skilled in the art is familiar with possible formulations and uses of depot injections.

[0263] Pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated in accordance with the known art using the dispersing or wetting agents and suspending agents mentioned herein. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable diluents, solvents and dispersion media that may be employed include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS), ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), and suitable mixtures thereof. Furthermore, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Furthermore, fatty acids, such as oleic acid, find use in the preparation of injectables. Prolonged absorption of certain injectable formulations may be achieved by the inclusion of an agent that delays absorption (e.g., aluminum monostearate or gelatin).

[0264] Pharmaceutical compositions containing the active ingredient (e.g., the complexes of the present disclosure) may be in a form suitable for oral use, for example, as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microgranules or elixirs. Pharmaceutical compositions intended for oral use may be prepared according to any method known in the art for manufacturing pharmaceutical compositions, and such compositions may contain one or more agents, such as, for example, sweetening agents, flavoring agents, coloring agents and preservative agents in order to provide pharmaceutically elegant and tasty preparations. Tablets, capsules and the like contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for tablet manufacturing. These excipients may be, for example, diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.

[0265] Tablets, capsules and the like suitable for oral administration may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide sustained action. For example, a delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by techniques known in the art to form osmotic therapeutic tablets for controlled release. Additional agents include biodegradable or biocompatible particles or a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide/glycolide copolymers, polylactide copolymers /glycolide, or ethylene vinyl acetate copolymers, in order to control the distribution of an administered composition. For example, the oral agent may be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, through the use of hydroxymethyl cellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively, or in a colloid drug delivery system. Colloidal dispersion systems include complexes of macromolecules, nanocapsules, microspheres, microgranules and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. Methods of preparing liposomes are described, for example, in US Patent 4,235,871, 4,501,728 and 4,837,028. Methods for preparing the above-mentioned formulations will be apparent to those skilled in the art.

[0266] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose, or as capsules of soft gelatin in which the active ingredient is mixed with water or an oily medium, for example, peanut oil, liquid paraffin, or olive oil.

[0267] Aqueous suspensions contain active materials in mixture with suitable excipients for their manufacture. Such excipients may be suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, dispersing agents or wetting agents, for example, a phosphatide of naturally occurring (e.g., lecithin) or condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate) or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., monooleate polyethylene sorbitan). Aqueous suspensions may also contain one or more preservatives.

[0268] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, for example, peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil, such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those presented above, and flavoring agents can be added to provide a palatable oral preparation.

[0269] Dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide the active ingredient in mixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

[0270] The pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures thereof. Suitable emulsifying agents may be naturally occurring gums, e.g. acacia gum or gum tragacanth, naturally occurring phosphatides, e.g. soybean, lecithin and esters or partial esters derived from fatty acids and hexitol anhydrides, e.g. , sorbitan monooleate; and condensation products of partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.

[0271] Formulations may also include carriers to protect the composition against rapid degradation or elimination from the body, such as a controlled-release formulation, including implants, liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, a delay material, such as glyceryl monostearate or glyceryl stearate alone or in combination with a wax, may be employed.

[0272] The present disclosure contemplates the administration of the complexes in the form of suppositories for rectal administration of the drug. Suppositories can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at normal temperatures but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycols.

[0273] The complexes contemplated by the present disclosure may be in the form of any other suitable pharmaceutical composition (e.g., sprays for nasal or inhalation use) currently known or developed in the future.

[0274] The concentration of a polypeptide complex in a formulation can vary widely (e.g., from less than about 0.1%, typically to or at least about 2% to as much as 20% to 50% or more, in weight) and will normally be selected primarily based on fluid volumes, viscosities and subject-based factors, in accordance with, for example, the particular mode of administration selected.

[0275] Contemplated here is the use of Nano Precision Medical depot dispensing technology (Nano Precision Medical, Emeryville, CA). The technology utilizes a titanium oxide nanotube membrane, which produces zero-order release rates of macromolecules such as therapeutic proteins and peptides. The biocompatible membrane is housed in a small subcutaneous implant that provides long-term (e.g., up to one year) delivery of therapeutic macromolecules. The technology is currently being evaluated for the delivery of GLP-1 agonists for the treatment of Type II diabetes. In certain embodiments, the complex (or complexes) disclosed herein may be a formulation with a membrane. For example, the complex may be embedded in the membrane or surrounded by the membrane. The membrane may be in the form of a disc, tube or sphere. In certain embodiments, the tube may be a nanotube or the sphere may be a nanosphere.

ROUTES OF ADMINISTRATION

[0276] The present disclosure contemplates the administration of the disclosed complexes, and compositions thereof, in any appropriate manner. Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intra-articular, intraperitoneal, intracerebral (intraparenchymal), and intracerebroventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), sublingual, and inhalation.

[0277] Depot injections, which are generally administered subcutaneously or intramuscularly, can also be used to release the complexes disclosed herein over a defined period of time. Depot injections are generally either solid or oil based and generally comprise at least one of the formulation components set forth herein. One skilled in the art is familiar with possible formulations and uses of depot injections.

[0278] With respect to antibodies, in an exemplary embodiment an antibody or antibody fragment of the present disclosure is stored at 10 mg/ml in sterile isotonic aqueous saline for injection at 4°C and is diluted in 100 ml or 200 ml of chloride of 0.9% sodium for injection prior to administration to the subject. The antibody is administered by intravenous infusion over 1 hour at a dose of between 0.2 and 10 mg/kg. In other embodiments, the antibody is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via subcutaneous bolus injection.

[0279] The present disclosure contemplates methods in which the complexes of the present disclosure are administered to a subject at least twice a day, at least once a day, at least once every 48 hours, at least once a every 72 hours, at least once a week, at least once every 2 weeks, or once a month.

COMBINATION THERAPY

[0280] The present disclosure contemplates the use of a complex provided herein in combination with one or more active therapeutic agents or other therapeutic or prophylactic modalities. In such combination therapy, the different active agents often have different mechanisms of action. Such combination therapy may be particularly advantageous in allowing a reduction in the dose of one or more of the agents, reducing or eliminating adverse effects associated with one or more of the agents; Furthermore, such combination therapy may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder or condition.

[0281] As used herein, “combination” is intended to include therapies that can be administered separately, e.g., separately formulated for separate administration (e.g., as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a “co-formulation”).

[0282] In certain embodiments, a complex is administered or applied sequentially, for example, in which one agent is administered before one or more other agents. In other embodiments, the complex is administered simultaneously, for example, in which two or more agents are administered at or about the same time; the two or more agents may be present in two or more separate formulations or combined in a single formulation (i.e., a co-formulation). Regardless of whether the two or more agents are administered sequentially or simultaneously, they are intended to be administered in combination for the purposes of the present disclosure.

[0283] The complexes of the present disclosure may be used in combination with other agents useful in the treatment, prevention, suppression or amelioration of the diseases, disorders or conditions disclosed herein, including those that are normally administered to subjects suffering from obesity, disorders food, hyperglycemia, hyperinsulinemia, glucose intolerance, and other glucose metabolism disorders.

[0284] The present disclosure contemplates combination therapy with various agents (and classes thereof), including: 1) insulin, insulin mimetics, and agents that involve the stimulation of insulin secretion, including sulfonylureas (e.g., chlorpropamide, tolazamide , acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) and meglitinides (e.g., repaglinide (PRANDIN) and nateglinide (STARLIX)); 2) biguanides (e.g., metformin (GLUCOPHAGE)) and pharmaceutically acceptable salts thereof, in particular, metformin hydrochloride, and sustained-release formulations thereof, such as Glumetza™, Fortamet™ and GlucophageXR™) and other agents that act to promote the use of glucose, reducing hepatic glucose production and/or decreasing intestinal glucose production; 3) alpha-glucosidase inhibitors (e.g., acarbose, voglibose and miglitol) and other agents that delay carbohydrate digestion and, consequently, absorption from the intestine and reduce postprandial hyperglycemia; 4) thiazolidinediones (e.g. rosiglitazone (AVANDIA), troglitazone (REZULIN), pioglitazone (ASTOS), glipizide, balaglitazone, rivoglitazone, netoglitazone, AMG 131, MBX2044, mitoglitazone, lobeglitazone, IDR-105, troglitazone, englitazone, ciglitazone, a daglitazone , darglitazone that enhance insulin action (e.g., by insulin sensitization), including insulin, insulin and mimetics (e.g., insulin degludec, insulin glargine, insulin lispro, insulin detemir, insulin glulisine, and inhalable formulations of each), thereby promoting glucose utilization in peripheral tissues; 5) glucagon-like peptides including DPP-IV inhibitors (e.g., alogliptin, omarigliptin, linagliptin, vildagliptin (GALVUS), and sitagliptin (JANUVIA)) and glucagon-like peptide 1 (GLP-1) and GLP-1 analogs and antagonists (e.g., exenatide (BYETTA and ITCA 650 (a subcutaneously inserted osmotic pump that delivers an exenatide analog over a 12-month period; Intarcia, Boston, MA)) and GLP-1 receptor agonists (e.g., dulaglutide, semaglutide, albiglutide, exenatide, liraglutide, lixisenatide, taspoglutide, CJC-1131, and BIM-51077, including intranasal, transdermal, and one-time formulations per week); 6) and DPP-IV resistant analogues (incretin mimetics), PPAR gamma agonists, PPAR alpha agonists such as fenofibric acid derivatives (e.g. gemfibrozil, clofibrate, ciprofibrate, fenofibrate, bezafibrate), PPAR agonists of dual-acting (e.g., ZYH2, ZYH1, GFT505, chiglitazar, muraglitazar, aleglitazar, sodelglitazar, and naveglitazar), pan-acting PPAR agonists, PTP1B inhibitors (e.g., ISIS-113715 and TTP814), SGLT inhibitors (e.g., , ASP1941, SGLT-3, empagliflozin, dapagliflozin, canaglifozin, BI-10773, PF-04971729, remogloflozin, TS-071, tofogliflozin, ipragliflozin and LX-4211), insulin secretion, angiotensin-converting enzyme inhibitors (e.g. alacepril, benazepril, captopril, ceronapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril, temocapril or trandolapril), angiotensin II receptor antagonists (e.g. losartan, i.e. is, COZAAR®, valsartan, candesartan, olmesartan, telmisartan and any of these drugs used in combination with hydrochlorothiazide, such as HYZAAR®) or other antihypertensive drugs, such as LCZ 696, RXR agonists, glycogen synthase kinase inhibitors -3, immune modulators, sympatholytics, beta-adrenergic blocking drugs (e.g., propranolol, atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartate), alpha-adrenergic blocking drugs (e.g., doxazocin, Prazocin, or alpha methyldopa) , central alpha adrenergic agonists, peripheral vasodilators (eg, hydralazine); beta-3 adrenergic receptor agonists, 11beta-HSD1 inhibitors, neutral endopeptidase inhibitors (e.g., thiorphan and phosphoramidon), aldosterone antagonists, aldosterone synthase inhibitors, renin inhibitors (e.g., urea derivatives of di- and tripeptides (see US Patent No. 5,116,835), amino acids and derivatives (US Patent No. 5,095,119 and 5,104,869), amino acid chains linked by non-peptide bonds (US Patent No. 5,114,937), di- and tri-peptide (US Patent 5,106,835), peptidyl amino diols (US Patents 5,063,208 and 4,845,079) and peptidyl beta-aminoacyl aminodiol carbamates (US Patent 5,089,471); in addition, a variety of other analogues of peptides, as disclosed in the following US Patents 5,071,837; 5,064,965; 5,063,207; 5,036,054; 5,036,053; 5,034,512 and 4,894,437, and small molecule renin inhibitors (including diol sulfonamides and sulfinyls (Patent US5,098,924), N-morpholino derivatives (Patent US5,055,466), N-heterocyclic alcohols (Patent US4,885,292) and pyrolimidazolones (Patent US. 5,075,451); in addition, pepstatin derivatives (US Patent 4,980,283) and fluoro- and chloro-derivatives of statone-containing peptides (US Patent 5,066,643), enalcrein, RO 42-5892, A 65317, CP 80794, ES 1005, 8891 ES , SQ 34017, aliskiren (2(S), 4(S), 5(S), 7(S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2, 7- diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)-phenyl]-octanamide hemifumarate) SPP600, SPP630 and SPP635), endothelin receptor antagonists, phosphodiesterase-5 inhibitors (e.g. sildenafil, tadalfil and vardenafil ), vasodilators, calcium channel blockers (e.g., amlodipine, nifedipine, veraparmil, diltiazem, galopamil, niludipine, nimodipines, nicardipine), potassium channel activators (e.g., nicorandil, pinacidil, cromacalim, minoxidil, aprilcalim, loprazolam ), lipid-lowering agents, for example, HMG-CoA reductase inhibitors such as simvastatin and lovastatin, which are marketed as ZOCOR® and MEVACOR® in lactone prodrug form and function as inhibitors upon administration, and pharmaceutically acceptable salts of open-ring dihydroxy acid HMG-CoA reductase inhibitors, such as atorvastatin (particularly the calcium salt sold in LIPITOR®), rosuvastatin (particularly the calcium salt sold in CRESTOR®), pravastatin (particularly the sodium salt sold in PRAVACHOL®), cerivastatin and fluvastatin (particularly the sodium salt sold in LESCOL®); a cholesterol absorption inhibitor such as ezetimibe (ZETIA®) and ezetimibe in combination with any other lipid-lowering agent such as the HMG-CoA reductase inhibitors noted above and particularly with simvastatin (VYTORIN®) or with calcium from atorvastatin; drugs to raise HDL, (e.g., niacin and nicotinic acid receptor agonists and controlled or sustained release versions thereof, and/or with an HMG-CoA reductase inhibitor; niacin receptor agonists, such as acipimox and acifran, as well as niacin receptor partial agonists, glucagon receptor antagonists (e.g. MK-3577, MK-0893, LY-2409021 and KT6-971); bile acid sequestering agents (e.g. colestilan, colestimide, colesevalam hydrochloride, colestipol, cholestyramine, and dialkylaminoalkyl derivatives of a cross-linked dextran), acyl-CoA:cholesterol acyltransferase inhibitors, (e.g., avasimib); agents intended for use in inflammatory conditions, such as aspirin, not anti-inflammatory drugs. nonsteroidal inflammatory drugs or NSAIDs, glucocorticoids, and selective cyclooxygenase-2 or COX-2 inhibitors; glucokinase activators (GKAs) (e.g., AZD6370); 11β-hydroxysteroid dehydrogenase type 1 inhibitors, (e.g., such as those disclosed in U.S. Patent No. 6,730,690, and LY-2523199); CETP inhibitors (e.g. anacetrapib, evacetrapib and torcetrapib); fructose-1,6-bisphosphatase inhibitors (e.g., such as those disclosed in patent Nos. US6,054,587; 6,110,903; 6,284,748; 6,399,782; and 6,489,476); acetyl-CoA carboxylase-1 or 2 (ACC2 or ACC1) inhibitors; PCSK9 inhibitors; GPR-40 partial agonists; SCD modulators; fatty acid synthase inhibitors; amylin and amylin analogues (e.g. pramlintide); including pharmaceutically acceptable salt forms of the above active agents, where chemically possible.

[0285] Furthermore, the present disclosure contemplates combination therapy with agents and methods for promoting weight loss, such as agents that stimulate metabolism or decrease appetite, and modified diets and/or exercise regimens to promote weight loss. weight loss.

[0286] The complexes of the present disclosure may be used in combination with one or more other agents in any manner appropriate under the circumstances. In one embodiment, treatment with the at least one active agent and at least one complex of the present disclosure is maintained for a period of time. In another embodiment, treatment with the at least one active agent is reduced or interrupted (e.g., when the subject is stable), while treatment with a complex of the present disclosure is maintained at a constant dosage regimen. In another embodiment, treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable), while treatment with the complex (or complexes) of the present disclosure is reduced (e.g., higher dose). low, less frequent dosing or shorter treatment regimen). In yet another embodiment, treatment with the at least one active agent is reduced or discontinued (e.g., when the object is stable), and treatment with the complex of the present disclosure is increased (e.g., higher dose, more frequent dosing or longer treatment regimen). In yet another embodiment, treatment with the at least one active agent is continued and treatment with a complex of the present disclosure is reduced or discontinued (e.g., lower dose, less frequent dosing, or shorter treatment regimen). In yet another embodiment, treatment with the at least one active agent and treatment with the complex (or complexes) of the present disclosure are reduced or discontinued (e.g., lower dose, less frequent dosing, or shorter treatment regimen).

DOSAGE

[0287] The complexes of the present disclosure may be administered to a subject in an amount that depends, for example, on the objective of administration (e.g., the degree of resolution desired); the age, weight, sex, health and physical condition of the subject to be treated; the nature of the polypeptide and/or formulation being administered; the route of administration; and the nature of the disease, disorder, condition or symptom thereof (for example, the severity of the glucose/insulin dysregulation and the stage of the disorder). The dosage regimen may also take into account the existence, nature and extent of any adverse effects associated with the agent(s) being administered. Effective dosage amounts and dosage regimens can be readily determined from, for example, safety and dose-escalation studies, in vivo studies (e.g., animal models), and other methods known to those skilled in the art.

[0288] In general, dosage parameters dictate that the dosage amount be less than an amount that would be irreversibly toxic to the subject (i.e., the maximum tolerated dose, “MTD”) and not less than an amount necessary to produce a measurable effect on the subject. Such quantities are determined, for example, by pharmacokinetic and pharmacodynamic parameters associated with absorption, distribution, metabolism and excretion (“ADME”), taking into account the route of administration and other factors.

[0289] An effective dose (ED) is the dose or amount of an agent that produces a therapeutic response or desired effect, in some fraction of the subjects taking it. The “median effective dose” or ED50 of an agent is the dose or amount of an agent that produces a therapeutic response or desired effect in 50% of the population to which it is administered. Although the ED50 is commonly used as a measure of an agent's reasonable expectation of effect, it is not necessarily the dose that a physician would consider appropriate, taking into account all relevant factors. Thus, in some situations the effective amount is greater than the calculated ED50, in other situations the effective amount is less than the calculated ED50, and in other situations the effective amount is the same as the calculated ED50.

[0290] Furthermore, an effective dose of the complex (or complexes) of the present disclosure may be an amount that, when administered in one or more doses to a subject, produces a desired result with respect to a healthy subject. For example, an effective dose may be one that, when administered to a subject having elevated plasma glucose and/or plasma insulin, achieves a desired reduction relative to that of a healthy subject by at least about 10%, at least. at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more than 80%.

[0291] An appropriate dosage level will generally be about 0.001 to 100 mg/kg of patient body weight per day, which can be administered in single or multiple doses. In some embodiments, the dosage level will be from about 0.01 to about 25 mg/kg per day, and in other embodiments from about 0.05 to about 10 mg/kg per day. A suitable dosage level may be about 0.01 to 25 mg/kg per day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day. Within this range, the dosage can be 0.005 to 0.05, 0.05 to 0.5 or 0.5 to 5.0 mg/kg per day.

[0292] For administering an agent orally, compositions may be provided in the form of tablets, capsules and the like, containing from 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 3.0, 5 ,0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250, 0.300, 0.400, 0.500, 0.600, 0.750 , 0.800, 0.900.0, and 1000.0 milligrams of the active ingredient. The complex can be administered on a regimen of, for example, 1 to 4 times a day, and often once or twice a day.

[0293] Dosage of the complex (or complexes) of the present disclosure may be repeated with an appropriate frequency, which may be in the range of once per day to once per month, depending on the pharmacokinetics of the complex (e.g., half- lifetime) and pharmacodynamic response (for example, the duration of the therapeutic effect of the complex). In some embodiments, the dosage is frequently repeated between once a week, once every two weeks, once a month. In other embodiments, the complex may be administered approximately once a month.

[0294] In certain embodiments, the dosage of the disclosed complex is contained in a “unit dosage form”. The phrase “unit dosage form” refers to physically discrete units, each unit containing a predetermined amount of a complex of the present disclosure, alone or in combination with one or more additional agents, sufficient to produce the desired effect. It will be appreciated that the parameters of a unit dosage form will depend on the particular agent and the effect to be achieved.

KITS

[0295] The present disclosure also contemplates kits comprising the disclosed complex (or complexes) and pharmaceutical compositions thereof. Kits are generally in the form of a physical structure housing various components, as described below, and may be used, for example, in the practice of the methods described above (e.g., administering a complex to a subject in need of weight reduction). ).

[0296] A kit may include one or more of the complex (or complexes) disclosed herein (provided, for example, in a sterile container), which may be in the form of a pharmaceutical composition suitable for administration to a subject. The complex(es) may be supplied in a form that is ready for use or in a form that requires, for example, reconstitution or dilution prior to administration. When the complex (or complexes) is in a form that must be reconstituted by a user, the kit may also include buffers, pharmaceutically acceptable excipients and the like, packaged with or separately from the complex (or complexes). When combined therapy is contemplated, the kit may contain the various agents separately or they may already be combined in the kit. Each component of the kit can be enclosed within an individual container and all of the various containers can be contained within a single package. A kit of the present disclosure may be designed for the conditions necessary to adequately maintain the components housed therein (e.g., refrigeration or freezing).

[0297] A kit may contain a label or leaflet, including identifying information for the components contained therein and instructions for use (e.g., dosage parameters, clinical pharmacology of the active ingredient(s), including mechanism of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.). Labels or leaflets may include manufacturer information, such as batch numbers and expiration dates. The label or leaflet may be, for example, integrated into the physical structure that houses the components, contained separately within the physical structure or attached to a component of the kit (for example, an ampoule, tube or vial). Exemplary instructions include those for lowering or lowering blood glucose, treating hyperglycemia, treating diabetes, etc. with the disclosed modulators and pharmaceutical compositions thereof.

[0298] Labels or leaflets may additionally include or be embedded in a computer-readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk, such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape or an electrical storage medium such as RAM and ROM or hybrids thereof such as magnetic/optical storage media, FLASH media or memory-type cards. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the Internet, are provided.

EXPERIMENTAL

[0299] The following examples are presented in order to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors consider their invention nor are they intended to represent that the experiments Below are all or the only experiments performed. Efforts have been made to ensure accuracy regarding the numbers used (e.g. quantities, temperature, etc.), but some experimental errors and deviations must be considered.

[0300] Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C) and pressure is at or near atmospheric. Standard abbreviations are used, including the following: bp = base pair(s); KB = kilobase(s); PL = picoliter(s); s = second(s); min. = minute(s); h = hour(s); AA = amino acid(s); KB = kilobase(s); NT = nucleotide(s); ng = nanogram; μg = microgram; mg = milligram; g = grass; kg = kilogram; dl = deciliter; μl = microliter; ml = milliliter =; l = liter; μM = micromolar; mM = millimolar; M = molar; kDa = kilodalton; i. m. = intramuscular(mind); i. P. = intraperitoneal(mind); s. w. = subcutaneous(mind); bid = twice a day; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = statistically not significant; PG = fasting plasma glucose; FPI = fasting plasma insulin; ITT = insulin tolerance test; PTT = pyruvate tolerance test; oGTT = oral glucose tolerance test; GSIS = glucose-stimulated insulin secretion; PBS = phosphate buffered saline; PCR = polymerase chain reaction; NHS = N-hydroxysuccinimide; DMEM = Dulbeco's Modification of Eagle's Medium; GC = genome copy; EDTA = ethylenediaminetetraacetic acid.

MATERIALS AND METHODS

[0301] The following methods and materials were used in the Examples below:

[0302] Animals. Diet-induced obese (DIO) male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were maintained on a high-fat diet (D12492, Research Diets, Inc., New Brunswick, NJ) containing 60 kcal per % fat, 20 kcal per % protein and 20 kcal per % carbohydrate for 12 to 20 weeks. All animal studies were approved by the NGM Institutional Animal Care and Use Committee. DIO C57BL/6J mice provide a human model of obesity, where obesity is based on excessive calorie intake. C57BL/6J mice are prone to obesity in which pronounced weight gain is observed, as well as hyperinsulinemia and sometimes hyperglycemia. This strain is the most commonly used mouse strain to model diet-induced obesity. (Nilsson C., et al., Acta Pharmacologica Sinica (2012) 33:173 to 181).

[0303] Nucleic Acid and Amino Acid Sequences. Genbank Accession No. BC000529.2 shows the ORF cDNA encoding the human GDF15 variants and GenBank Accession No. NP_004855.2 shows the amino acid sequence encoded by the cDNA. The cDNA for the Fc fusion partner was purchased from Invivogen (pFUSE-Chig-HG1, GenBank:AY623427.1, Protein ID = AAT49050) and modified as indicated. The amino acid sequence of the Fc fusion partner encoded by the pFUSE-Chig-HG1 vector is:

[0304] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO:55)

[0305] Construction of expression constructs. The mammalian expression vector pTT5 (National Research Council of Canada) was modified by inserting a Kozak element and human IgK signal peptide sequence:(CACCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCT ACTCTGGCTCCGAGGTGCCAGATGT) (SEQ ID NO:56) between the EcoRI site and PmeI. Although both restriction sites were eliminated, an AgeI site was created for additional in-frame cloning of secreted factors. For single fragment insertion (e.g., Fc portion of human IgG1), fusion technology (Clontech) was used. For the insertion of two or more fragments generated by PCR (i.e., hIgG1-Fc + GDF15), the Gibson Master Mix (NEB) assembly was used according to factory protocols. All PCR fragments were amplified by Sapphire PCR mix and gel purified using the Qiagen gel extraction kit. Electrocompetent TOP10 cells (Life Technologies) were transformed with the cloning reactions, distributed on LB-agar plates containing carbenicillin and incubated overnight at 37°C. Isolated colonies were harvested and analyzed by sequencing. DNA from positive colonies was amplified (DNA-Maxi-prep, Qiagen), completely sequence confirmed, and used to transfect mammalian cells for recombinant protein expression.

[0306] To create specific muteins, site-directed mutagenesis was performed with QuikChange Lightning or QuikChange Lightning Multi site-directed mutagenesis kits (Agilent) and the appropriate primers, following factory protocols.

[0307] Expression of fusion molecule (Fc/Fc)-GDF15, wild-type GDF15 and GDF15 glycomutein. All molecules were recovered from 293F cells transiently transfected in Expi (Invitrogen Corporation, Carlsbad, CA). Cells were routinely subcultured in Expi expression medium (Invitrogen) and maintained as suspension cultures in shake flasks of varying sizes. Typically, cells were subcultured to a cell density of 5.5 viable cells/ml and cultured for 3 days prior to subculture. Vials were maintained in a humidified CO2 incubator (37°C and 5% CO2) on New Brunswick shaker platforms (New Brunswick Scientific Company, Edison, NJ) at a shaking rate of 110 RPM.

[0308] Transfections were performed when the cell density of the culture reached 2.5e6 viable cells/ml, at more than 95% viability. Typically, for 50 ml transfection, 2.5e6 cells/ml x 50 ml cells were inoculated into a 250 ml shake flask into 42.5 ml culture volume. Fifty micrograms (50 μg) of plasmid DNA constituting the expression vector containing the gene of interest was first diluted in 2.5 ml of serum medium reduced with OPTI-MEM (Invitrogen). Simultaneously, Expifectamine transfection reagent (Invitrogen), 2.67 times the volume (of the amount of plasmid DNA) was also diluted in 2.5 ml of serum medium reduced with OPTI-MEM. After a 5-min incubation at room temperature, the diluted transfection reagent was slowly added to the diluted plasmid DNA to form competent transfection complexes. After an incubation period of 20 minutes at room temperature, 5 ml of the transfection complex was added to the 42.5 ml cell culture. The transfected cells were then placed in the humidified CO2 incubator on an orbital shaker maintained at 110 RPM. Twenty-four hours after transfection, the transfected culture was fed with 250 μl of enhancer solution 1 (Invitrogen) and 2.5 ml of enhancer solution 2 (Invitrogen). The culture was then returned to the humidified CO2 incubator on an orbital shaker. Six to seven days after transfection, cultures were harvested by centrifugation at 3,000 RPM for 30 min. before being filtered through a 0.2 μm filter (Nalgene). Samples were then analyzed on a coomassie staining gel for expression.

[0309] Purification of recombinant protein. The (Fc/Fc)- GDF15 molecules expressed in conditioned media (CM) were evaluated for recovery and activity after purification. CM was passed through a SelectSuRe (GE) mAb column at a loading capacity of no more than 20 mg/ml resin. CM volumes ranged from 50 ml to 1,000 ml to evaluate recoveries. After loading CM SelectSuRe mAb, the column was washed with 5 to 10 column volumes of 1X PBS (Corning Cellgro), followed by the elution step with low pH glycine buffer (Polysciences Inc.). After elution, the (Fc/Fc)-GDF15 clusters were pH neutralized with 1 µM of Tris pH 8.0 (Teknova) and then injected onto a Superdex200 (GE) column pre-equilibrated in 1X PBS (Corning Cellgro ). Fractions of intact, fully assembled (Fc/Fc)-GDF15 molecules were pooled and analyzed for purity and quantified using A280 methods using appropriate extinction coefficients and molecular weights to determine recovery based on CM volumes. of departure. The fully assembled molecules were dimer-dimer complex of two heterodimers. Each heterodimer having one Fc associated with glycomutein Fc-GDF15 through the button-in-hole interaction, and two heterodimers associated through the GDF15 - GDF15 interaction.

[0310] Purification of WT GDF15 and GDF15 glycomuteins. Wild-type GDF15 and non-Fc-conjugated GDF15 glycomuteins were purified from cultured media using ion exchange capture. GDF15 and WT GDF15 glycomuteins were eluted using an appropriate salt/pH gradient favorable for optimal elution and separation of impurities from host cell proteins. All GDF15 molecules were then further purified using HiTrap Phenyl HP GE at pH 8.0, using a linear decreasing gradient of ammonium sulfate. Fractions were evaluated and pooled based on purity and glycosylation properties via gel shift on non-reduced SDS-PAGE gels. Similar to the (Fc/Fc)-GDF15 molecules, the glycomuteins GDF15 and wild-type GDF15 were expressed using the IgK signal peptide.

EXAMPLE 1: DESIGN OF HETERODIMERIC BUTTON-IN-HOLE (Fc/Fc)-GDF15 FUSION MOLECULES

[0311] The Fc-GDF15 designs are described in Figure 1 and the primary sequences are represented below (Constructs B1a/b to B19a/b). To achieve productive assembly of Fc-GDF15 molecules, an effective system was designed to enable Fc/Fc dimerization while simultaneously enabling GDF15/GDF15 dimerization. To avoid the abnormal folding and aggregation potential of single-chain Fc-GDF15, a heterodimeric fusion partner was designed for the Fc/Fc interaction to enable high-fidelity GDF15/GDF15 homodimerization. Knob-in-hole Fc/Fc heterodimers were designed to address GDF15 assembly and secretion from Expi 293F transient systems. Fc/Fc heterodimeric button-in-hole systems were evaluated using a [T366Y (button) // Y407T (hole)] or [T366W (button) // T366S-L368A- Y407V (hole)] system coupled to a ligand (G4S)n (n = 2, 3, 4 or 5) and GDF15. It is to be noted that the numbering of the amino acid position in the Fc CH3 domain is based on the EU numbering system (Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78 to 85 (1969)). In all cases, the Fc fusion partner (knob/hole) was coupled to the N-terminus of mature GDF15 comprising amino acid residues A1-I112, R2-I112, N3-I112, G4-I112, D5-I112, H6 -I112 or C7-I112. GDF15 N-terminal truncations (Δ1= R2-I112, Δ2= N3-I112, Δ3= G4-I112, Δ4= D5-I112, Δ5= H6-I112 or Δ6= C7-I112) were incorporated to reinforce the stability as the N-terminal sequence (ARNGDH, SEQ ID NO:95) has previously been demonstrated as a site of proteolytic susceptibility and N-terminal truncations provide superior stability vs. GDF15 which does not include these N-terminal truncations.

[0312] Figures 1A to 1D depict the placement of the knob versus hole in Fc-GDF15 (chain A), coupled to the corresponding hole versus knob in the Fc heterodimeric partner (chain B), coupled to a wild-type IgG hinge containing two intermolecular disulfide bonds or without the hinge domain (Hinge). For heterodimeric Fc button or hole A/B chains, an AA mutation (APELLGGP (SEQ ID NO: 96) ^APALAGGP (SEQ ID NO: 97)) was introduced to remove IgG1 effector functionality. Heterodimeric button-in-hole designs of (Fc/Fc)-GDF15 were profiled in expression for assembly and are reported in Figure 2A. In all cases, transient button-in-hole (Fc/Fc)-GDF15 expression resulted in recoveries, after purification, between 0 mg/l and 74.9 mg/l of correctly assembled product (0 = aggregates/no expression, <25 mg/l, 25 mg/l to 49.9 mg/l, 50 mg/l to 74.9 mg/l, 75 mg/l to 99.0 mg/l, > 100 mg/l). In all cases, the assembly and secretion of the button-in-hole heterodimeric Fc/Fc-GDF15 molecules was accompanied with varying levels of contamination from abnormally folded homodimeric species, such as Fc(hole):Fc(hole), Fc (button):Fc(button), Fc(button)-GDF15:Fc(button)-GDF15 and Fc(hole)-GDF15:Fc(hole)-GDF15. Based on the expression profile, it was found that T366W (button) placed on the Fc-GDF15 chain, coupled to T366S-L368A-Y407V (hole) on the heterodimeric Fc partner chain (Figure 1D) produces a product with stable maximum and minimized abnormal pairing of Fc/Fc homodimeric products (Figure 2A - B5a/B5b variant). This project was the focus of additional expression engineering and optimization.

[0313] Recovery of the B5a/B5b variant from the transiently expressed Expi 293F source provided recoveries in the range of 0.0 mg/l to 24.9 mg/l. To increase expression, assembly and recoveries, N-glycosylation sites were introduced into the mature sequence of GDF15 (Figure 1F). In the designed constructs, the presence of a single N-linked glycan consensus site on GDF15 significantly improved the expression, assembly, and recovery of the fully mature (Fc/Fc)-GDF15 button-in-hole B5a/B5b heterodimer (Figure 2A - variants B9a/B9b to B19a/B19b). Linker length was considered optimal when n = 5 for (G4S)n for receptor binding and activity through an in vitro assay. The presence of a glycan at the D5T position completely removes a primary deamidation site at the N3 position of mature GDF15 and appears to further increase the stability of the molecule as evidenced in Example 2.

[0314] The presence of N-linked glycans in the GDF15 sequence is proposed to aid expression and minimize abnormally folded products against accumulation due to increased residence time in the endoplasmic reticulum and Golgi apparatus during the secretion process. This additional residence time is proposed to have a beneficial effect on folding kinetics and allows for significantly improved heterodimeric button-in-hole (Fc/Fc) recoveries and pairing from mammalian tissue culture.

[0315] The sequences of the variants (B1a/b to B19a/b) are provided below. In the sequences illustrated below, the human IgK signal peptide is in lower case followed by the Fc sequence. In sequences that also include linker sequence and GDF15, the Fc sequence is followed by the linker sequence (underlined) which is followed by the GDF15 sequence (bold). The numbering of the position of amino acid substitutions in the Fc sequence is based on the EU numbering, the substitutions with reference to the amino acid present in the corresponding position in human IgG1Fc (SEQ ID NO:2). Numbering of N-terminal deletion in the GDF15 sequence and amino acid substitutions is with reference to wild-type human mature GDF15 (SEQ ID NO:1).

B1 a:hIgK-hIgG1 -Fc(AA)(T366Y)-(G4S)5-ΔN3-GDF15 (G4-I112)

[0316] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLYC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGG GGSGGGGSGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIG ACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ TYD DLLAKDCHCI (SEQ ID NO:57)

B1 b:hIgK-hIgG1 -Fc(AA)(Y407T)

[0317] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:58)

B2a:hIgK-hIgG1-Fc(AA)(Y407T)- (G4S)5-ΔN3-GDF15 (G4-I112)

[0318] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIG ACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ TYDDL LAKDCHCI (SEQ ID NO:59)

B2b:hIgK-hIgG1 -Fc(AA)(T366Y)

[0319] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLYC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:60)

BSai^gK-hJgGI-FciΔdobradigaj-AAllTSββYl-iGiSJδ-Δ^^^Flδ-JG^z I112)

[0320] mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLV KGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSC SVM HEALHN HYTQ KSLSLSPG KGGGGSGGGGSGGGGSGGGGSGGGGSG DHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAAN MHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCH CI (SEQ ID NO:61)

B3b:hIgK-hIgG1 -Fc(.Hinge, AA)(Y407T)

[0321] mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:62)

B4a^IgK2hlgG^Fc(Adobrad^aJ AA)(Y407T)2(G4S)^\^32GDF1^(G4; I112)

[0322] mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNV FSC SVM HEALHN HYTQ KSLSLSPG KGGGGSGGGGSGGGGSGGGGSGGGGSG DHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAAN MHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCH CI (SEQ ID NO:63)

B4b:hIgK-hIgG1 -FcUhinge, AA)(T366Y)

[0323] mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLV KGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:64)

B5a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15 (G4-I112)

[0324] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIG ACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ TYDDL LAKDCHCI (SEQ ID NO:65)

B5b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0325] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:66)

B6a:hIgK- hlgGI-Fc(AA) (T366S)(L368A)(Y407V)-(G4S)5-ΔN6-GDF15 (C7I112)

[0326] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYD DL LAKDCHCI (SEQ ID NO:67)

B6b:hIgK- hIgG1-Fc(AA)(T366W)

[0327] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:68)

B7a:hIgK-hIgG1-FcUhinge, AA)(T366W)-(G4S)5-ΔN3-GDF15 (G4- I112)

[0328] mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCL VKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVM HEALHN HYTQ KSLSLSPGKGGGGSGGGGSGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAA NMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDC H CI (SEQ ID NO:69)

B7b:hIgK-hIgG1 -Fcf.Hingge AA)(T366S)(L368A)(Y407V)

[0329] mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAV KGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:70)

B8a:hIgK-hIgG1-Fc(Δh, AA) (T366S)(L368A)(Y407V)-(G4S)5-ΔN6-GDF15 (C7-I112)

[0330] mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAV KGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSC SVM HEALHN HYTQ KSLSLSPG KGGGGSGGGGSGGGGSGGGGSGGGGSC PLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHA QIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI ( SEQ ID NO:71)

B8b:hIgK-hIgG1-Fc(Δh, AA)(T366W)

[0331] mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCL VKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:72)

B9a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)3-GDF15 (A1-I112) (D5T)

[0332] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSAR NGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRA ANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKD CHCI (SEQ ID NO:73)

B9b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0333] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:74)

B10a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)4-GDF15 (A1-I112) (D5T)

[0334] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSARNGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYD DL LAKDCHCI (SEQ ID NO:75)

B10b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0335] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:76)

B11a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)5-GDF15 (A1-I112) (D5T)

[0336] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV LQTY DDLLAKDCHCI (SEQ ID NO:77)

B11 b:hIgK-hIgG1 -Fc(AA)(T366S)(L368A)(Y407V)

[0337] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:76)

B12a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)2-ΔN2-GDF15 (N3-I112) (D5T)

[0338] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV (SEQ) ID NO:78)

B12b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0339] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:79)

B13a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN2-GDF15 (N3-I112) (D5T)

[0340] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSNGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSL QTYD DLLAKDCHCI (SEQ ID NO:80)

B13b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0341] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:81)

B14a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15 (G4-I112)(R21N)

[0342] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSGDHCPLGPGRCCRLHTVNASLEDLGWADWVLSPREVQVTMCIG ACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ TYDDL LAKDCHCI (SEQ ID NO:82)

B14b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0343] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:83)

B15a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15(G4- I112)(S23N/E25T)

[0344] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV TYD DLLAKDCHCI (SEQ ID NO:84)

B15b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0345] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:85)

B16a:hIgK^(G4- I112)(F52N/A54T)

[0346] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIG ACPSQNRTANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ TYDDLLA KDCHCI (SEQ ID NO:86)

B16b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0347] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:87)

B17a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15(G4- I112)(R53N/A55T)

[0348] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIG ACPSQFNATNMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDL LAKDCHCI (SEQ ID NO:88)

B17b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0349] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:89)

B18a:hIgK- hIgG1-Fc(AA)(T366W)-(G4S)5-ΔN3-GDF15 (G4-I112) (K91N/D93T)

[0350] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIG ACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQNTTTGVSLQT YDDL LAKDCHCI (SEQ ID NO:90)

B18b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0351] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:91)

B19a:hIgK- hIgG1-Fc(AA)(T366W)- (G4S)5-ΔN3-GDF15 (G4- I112)(D93N/G95T)

[0352] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSGG GGSGGGGSGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIG ACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTNTTVSLQT YDDL LAKDCHCI (SEQ ID NO:92)

B19b:hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)

[0353] mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:93)

[0354] Sequences of wild-type human mature GDF15 (SEQ ID NO: 1) and GDF15 glycomuteins listed in Figure 2B are as follows:

Wild-type human mature IgK-GDF15

[0355] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:108)

IgK-GDF15-glucomutein R21N

[0356] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVNASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:109)

IgK-GDF15-glucomutein R53N/A55T

[0357] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFNATNMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:110)

IgK-GDF15-glucomutein S64N/H66T

[0358] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTNLTRLKPDTVPAPCCVPASY NPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:111)

IgK-GDF15-glucomutein P70N

[0359] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKNDTVPAPCCVPASY NPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:112)

IgK-GDF15-glucomutein Q90N

[0360] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLINKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO:113)

IgK-GDF15-glucomutein K91N/D93T

[0361] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQNTTTGVSLQTYDDLLAKDCHCI (SEQ ID NO:114)

IgK-GDF15-glucomutein D93N/G95T

[0362] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTNTTVSLQTYDDLLAKDCHCI (SEQ ID NO:115)

IgK-GDF15-glucomutein G95N

[0363] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTDTNVSLQTYDDLLAKDCHCI (SEQ ID NO:116)

IgK-GDF15-glucomutein S97N/Q99T

[0364] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTDTGVNLTTYDDLLAKDCHCI (SEQ ID NO:117)

IgK-GDF15-glucomutein L98N

[0365] mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTDTGVSNQTYDDLLAKDCHCI (SEQ ID NO:118)

[0366] GDF15 molecules were expressed using the IgK signal peptide, which is cleaved from the secreted polypeptide by a signal peptidase expressed by 293 cells. Recovery of wild-type human mature GDF15 (SEQ ID NO: 1) and glycomuteins GDF15 is listed in Figure 2B.

[0367] Exemplary GDF15 glycomuteins that can be expressed as Fc-Fc(button/hole)GDF15 glycomuteins are described in the document under USSN14/811,578 filed on July 28, 2015.

EXAMPLE 2: EFFECTS OF (Fc/Fc)-GDF15 FUSION MOLECULES ON BODY WEIGHT AND FOOD INTAKE IN A DIO MOUSE MODEL

[0368] The effects of a subcutaneously administered fusion molecule having recombinant Fc heterodimer fused to recombinant human GDF15 (i.e., a complex of two heterodimers, each heterodimer having an Fc polypeptide dimerized with an Fc-GDF15 glycomutein polypeptide) on the body weight were assessed over a 35-day period. Briefly, B9a/B9b, B11a/B11b, and B13a/B13b fusion molecules were administered weekly for 21 days at doses of 0.4 nmol/kg and 4 nmol/kg as a single subcutaneous bolus injection (10 ml/kg). with DIO mice weighing approximately 35 to 40 g. Following administration of vehicle control or the fusion molecules, body weight reduction was monitored at multiple time points over a 35-day time period, which was comprised of 21 days of protein dosing followed by a washout. 14 days (post-dose), to monitor effectiveness.

[0369] As depicted in Figures 3 to 6, administration of the Fc fusion molecules (heterodimer-heterodimer complexes) at a dose of 0.4 nmol/kg and 4 nmol/kg resulted in significant reduction in body weight. In each group of mice, n = 6 and p values (*, p < 0.05; **, p < 0.01; ***, p < 0.001, ns = not significant) were determined by T test unpaired Student's score comparing to the vehicle control group at each specified time point. As depicted in Figure 7, total body weight with SEM analysis is shown at each sampling time point for all groups. As depicted in Figure 8, changes in body weight (g) with SEM analysis and p-values are shown at each sampling time point for all groups. As depicted in Figure 9, percentages of body weight changes (%) with SEM analysis and p-values are shown at each sampling time point for all groups.

[0370] As depicted in Figures 3 and 5, there is an increased efficacy observed in reducing body weight for B13a/B13b compared to B9a/B9b and B11a/B11b in the 0.4 nmol/kg dose study. The increased in vivo efficacy for B13a/B13b is attributed to increased stability due to truncation of the 2 N-terminal residues of GDF15(ΔAR).

[0371] Particular embodiments of the present invention are described herein, including the best way known to the inventors to carry out the invention. Upon reading the foregoing description, variations of the disclosed embodiments may become apparent to individuals working in the art, and it is expected that those skilled in the art may employ such variations as appropriate. Therefore, it is intended that the invention be practiced otherwise than as specifically described herein, and that the invention includes all modifications and equivalents of the subject matter recited in the appended claims, as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention, unless otherwise indicated in this document or otherwise clearly contradicted by the context.

[0372] All publications, patent applications, accession numbers and other references cited in this specification are incorporated herein by reference, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims (16)

1. Complex, characterized by comprising a first heterodimer and a second heterodimer, each of the first and second heterodimers comprising: (i) a first polypeptide comprising from the N-terminus to the C-terminus: a first human IgG1 Fc comprising a first hinge region and a first CH3 sequence comprising at least one engineered protuberance comprising at least one corresponding amino acid substitution in an IgG1 Fc sequence, wherein the substitution is selected from the group consisting of Q347W/Y, T366W/Y, and T394W/Y, according to EU numbering, a linker, and an N-glycosylated GDF15 mutein comprising the amino acid sequence of SEQ ID NO: 129, and (ii) a second polypeptide comprising a second human IgG1 Fc comprising a second hinge region and a second CH3 sequence comprising at least one engineered pocket comprising at least one corresponding amino acid substitution in an IgG1 Fc sequence, wherein the substitution is selected from the group consisting of T366S, L368A, T394S, F405T /V/A, and Y407T/V/A, according to EU numbering; wherein the first polypeptide dimerizes with the second polypeptide by positioning the protuberance of the first polypeptide in the cavity of the second polypeptide; and wherein the GDF15 mutein in the first heterodimer dimerizes with the GDF15 mutein in the second heterodimer, thereby forming the complex comprising the first heterodimer and the second heterodimer. 2. Complex, according to claim 1, characterized by the fact that the ligand comprises the sequence Glycine-Glycine-Glycine-Glycine-Ser (G4S)n, where n=2-5. 3. Complex according to claim 1, characterized by the fact that the ligand comprises the sequence (G4S)3. 4. Complex according to claim 1, characterized by the fact that the ligand comprises the sequence (G4S)5. 5. Complex according to claim 1, characterized in that the first polypeptide comprises the T366W/Y substitution and the second polypeptide comprises the Y407T/V/A substitution; wherein the first polypeptide comprises the T366Y substitution and the second polypeptide comprises the Y407T substitution; or wherein the first polypeptide comprises the T366W substitution and the second polypeptide comprises the Y407A substitution. 6. Complex according to claim 1, characterized by the fact that the N-glycosylated GDF15 mutein comprises the amino acid sequence of SEQ ID NO: 128. 7. Complex according to claim 1, characterized in that the first polypeptide comprises the amino acid sequence established in SEQ ID NO: 3 and the second polypeptide comprises the amino acid sequence established in SEQ ID NO: 4. 8. Complex according to claim 1, characterized in that the first polypeptide comprises the amino acid sequence established in SEQ ID NO: 5 and the second polypeptide comprises the amino acid sequence established in SEQ ID NO: 6. 9. Complex according to any one of claims 1 to 8 characterized by the fact that the first polypeptide is glycosylated. 10. Complex characterized by comprising a first heterodimer and a second heterodimer, each of the first and second heterodimers comprising: a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, wherein the first polypeptide is N-glycosylated, and wherein the first polypeptide is dimerized with the second polypeptide via one or two disulfide bonds formed between regions of hinge present in the Ig Fc sequences in the first and second polypeptides. 11. Pharmaceutical composition characterized by comprising the complex, as defined in any one of claims 1 to 10, and a pharmaceutically acceptable diluent, carrier or excipient. 12. Use of the complex, as defined in any one of claims 1 to 10, characterized in that it is in the preparation of a pharmaceutical composition or medicine for treating obesity or hyperglycemia in a mammalian subject. 13. Use according to claim 12, characterized in that the subject is a human and in which the treatment results in the reduction of the subject's body weight. 14. Use according to claim 12, characterized in that the subject is a human and in which the treatment results in a reduction in the subject's blood glucose. 15. Use according to claim 12, characterized by the fact that the subject is human and has diabetes mellitus. 16. Use according to claim 12, characterized in that the subject is human and obese.