GIP AND GLP-1 RECEPTOR DUAL-AGONISTS FOR THE TREATMENT

OF DIABETES

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The content of the electronically submitted sequence listing in ASCII text file

(Name: 46736-134883_SL.txt; Size: 281,150 bytes; and Date of Creation: August 11, 2014) filed with the application is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Diabetes mellitus type 2 (type-2 diabetes) is characterized by high blood glucose and insulin resistance. Type-2 diabetes makes up about 90% of cases of diabetes. Type-2 diabetes is frequently associated with obesity.

[0003] Incretin hormones, e.g., glucagon and glucagon-like peptide- 1 are hormones that provide glycemic control during digestion. Incretin mimetics are a class of pharmacological agents currently available or in clinical trials for treatment of type-2 diabetes. Incretin mimetics have multiple antihyperglycemic actions that mimic several of the actions of incretin hormones originating in the gut, such as glucagon-like peptide (GLP)-l.

[0004] Glucagon-like peptide-1 (GLP-1) derives from pre-proglucagon, a 158 amino acid precursor polypeptide that is processed in different tissues to form a number of different proglucagon-derived peptides, including glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM), that are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the regulation of food intake. GLP-1 is produced as a 37-amino acid peptide that corresponds to amino acids 72 through 108 of proglucagon (92 to 128 of preproglucagon). GLP-l(7-36) amide or GLP-l(7-37) acid are biologically active forms of GLP-1, that demonstrate essentially equivalent activity at the GLP-1 receptor. [0005] GLP-1 is secreted from gut L cells and binds to the GLP-1 receptor. Its activities include stimulation of insulin synthesis and secretion, inhibition of glucagon secretion, and inhibition of food intake.

[0006] GLP-1 and GLP-1 analogs, acting as agonists at the GLP-1 receptor, have been shown to be effective hypoglycemic control, e.g., type-2 diabetes. Certain GLP-1 analogs are being sold or are in development for treatment of type-2 diabetes including, e.g., liraglutide (Victoza® from Novo Nordisk), dulaglutide (Eli Lilly), Bydureon (AZ/BMS), Aliblutide (GSK) and Exenatide (Byetta® from Eli Lilly/Amylin).

[0007] Gastric inhibitory peptide, also known as glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid peptide hormone secreted from K cells in the intestinal epithelium. GIP secretion is regulated by food intake. GIP is also expressed in pancreatic islet a-cells and promotes insulin secretion. GIP acts at the GIP receptor, and its activities include, without limitation, stimulation of glucose-dependent insulin secretion, an increase in β-cell mass, and a decrease in gastric acid secretion.

[0008] A recent study shows that GLP-1 and GIP, alone or in combination, have shown increased expression of PDX-1, Bcl-2 and insulin in pancreatic islet cells, in both normal subjects and subjects with type-2 diabetes. Lupi et al., (2010) Regulatory peptides 165: 129-132 (2010).

[0009] There remains a need for more agents for effective treatment, including improved incretin mimetics of hypoglycemic conditions such as type-2 diabetes.

BRIEF SUMMARY

[0010] This disclosure provides an isolated polypeptide comprising or consisting of the amino acid sequence:

Y S Xi G T F T S D X ₂ S X ₃ X ₄ X ₅ E X ₆ X ₇ X ₈ X9 X10 X11 Xi ₂ F X ₁₃ X ₁₄ W L X ₁₅ X ₁₆ X ₁₇

wherein X ₁ is E or Q; X ₂ is Y, V, or L; X is K, S, or I; X ₄ is L, Y, or A; X5 is L or M; X ₆ is E or D; X ₇ is E, G, or K; X ₈ is E, Q, or I; X ₉ is A or H; X ₁₀ is V, A, or Q; Xn is R, K, or Q; X ₁₂ is L, E, or D; X ₁₃ is I or V; X ₁₄ is E, A, or N; X ₁₅ is L, K, or V; X ₁₆ is A, K, or N; X ₁₇ is G or Q; and X ₁₈ is no amino acid, G, N, P, K, or T.

[0011] In some embodiments of the polypeptide of SEQ ID NO: l, X ₄ is L. In other embodiments, X ₁ is E; X ₂ is Y; X is K; X5 is L; X ₆ is E; X ₇ is E; X ₈ is E; X ₉ is A; X ₁₀ is V; Xn is R, X ₁₂ is L; X ₁₃ is I; X ₁₄ is E; and X ₁₇ is G. In other embodiments, X15 is L and X ₁₆ is A; i5 is K and X ₁₆ is N; or 15 is V and X ₁₆ is K.

[0012] In specific embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-32.

[0013] The invention also provides an isolated polypeptide comprising the amino acid sequence: Xi X ₂ E G T F T S D X ₃ S X ₄ X ₅ X ₆ X ₇ X _{8 9} A I D E F X ₁₀ Xn X _l2 L L X ₁₃ Xi ₄ Xi ₅ (SEQ ID NO: 190); wherein Χ is H or Y; X ₂ is G or S; X ₃ is V or Y; X ₄ is S, K, or I; X5 is Y, L, or A; X ₆ is M or L; X ₇ is E or D; X ₈ is R or E; X9 is Q or E; X^ is I or V; Xn is A or N; X ₁₂ is W or D; X ₁ is A, K, or G; X ₁₄ is G or Q; and X^ is no amino acid, G, or K.

[0014] In some embodiments, the polypeptide of SEQ ID NO:33, Xi is Y and X ₂ is S. In other embodiments, X ₁₀ is I, Xn is A, and X ₁₂ is W. In other embodiments, X ₁₄ is G and X ₁₅ is no amino acid or G. In still other embodiments, X ₇ is E. In other embodiments, X ₈ is R and X ₉ is Q. In other embodiments, X ₁ is K. In other embodiments, X is V. In still further embodiments X ₄ is S, X5 is Y, and X ₆ is M.

[0015] In specific embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:33-43.

[0016] The invention also provides an isolated polypeptide comprising the amino acid sequence of IP171, IP176, IP206, IP214, IP215, IP175. IP208, IP204, IP199, IP173, IP150, IP174, IP205, IP127, or IP200.

[0017] This disclosure also provides an isolated polypeptide comprising or consisting of the amino acid sequence:

Y S X3 G T F T S D Xio S X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ Xn Xi8 X19 X20 X21 F X ₂₃ X ₂₄ W L X ₂₇ X ₂ 8

X ₂₉ X ₃₀ (SEQ ID NO: 461);

[0018] wherein X is E or Q; X ₁₀ is Y, V, or L; X ₁₂ is K, S, or I; X ₁ is L, Y, or A; X ₁₄ is

L or M; X^ is E or D; X ₁₆ is E, G, or K; X ₁₇ is E, Q, or I; X ₁₈ is A or H; X ₁₉ is V, A, or Q;

X ₂₀ is R, K, or Q; X ₂₁ is L, E, or D; X ₂₃ is I or V; X ₂₄ is E, A, or N; X ₂₇ is L, K, or V; X ₂₈ is

A, K, or N; X29 is G or Q; and X ₃ o is no amino acid, G, N, P, K, or T.

[0019] In some embodiments of the polypeptide of SEQ ID NO:461, X ₁₃ is L. In other embodiments, X is E; X ₁₀ is Y; X ₁₂ is K; X ₁₄ is L; X^ is E; X ₁₆ is E; X ₁₇ is E; X ₁₈ is A;

X ₁₉ is V; X ₂ o is R, X ₂₁ is L; X ₂₃ is I; X ₂₄ is E; and X29 is G. In other embodiments, X ₂₇ is L and X ₂₈ is A; X ₂₇ is K and X ₂₈ is N; or X ₂₇ is V and X ₂₈ is K. In specific embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-32.

[0020] This disclosure also provides an isolated polypeptide comprising or consisting of the amino acid sequence:

Xi X ₂ X ₃ G X ₅ X ₆ X ₇ S X9 Xio X11 X12 Xi ₃ Xi ₄ X15 X½ X17 Xis X19 X20 X21 F X ₂₃ X ₂₄ X25 L X ₂₇ X ₂₈ X ₂₉ X ₃₀ (SEQ ID NO: 462);

[0021] wherein Xi is Y or H; X ₂ is S or G; X ₃ is E or Q; X ₅ is T or M; X ₆ if F or H; X ₇ is

T or I; X ₉ is D or L; X ₁₀ is Y, V, F, or L; X _n is S, R, or M; X ₁₂ is K, S, M, or I; X ₁₃ is L, Y, Q, H, I or A; X ₁₄ is L, K, or M; X ₁₅ is E or D; X ₁₆ is E, G, or K; X ₁₇ is E, Q, or I; X ₁₈ is A, C, or H; X ₁₉ is V, A, E, I, N, S, T, or Q; X ₂₀ is R, K, or Q; X _2i is L, E, C, or D; X ₂₃ is I or V; X ₂₄ is E, C, A, or N; X ₂₅ is W, C, F, H, Y, or K; X ₂₇ is L, K, or V; X ₂₈ is A, K, C, or N; X ₂ 9 is G or Q; and X ₃ o is no amino acid, G, N, P, K, or T. In specific embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-32 and 191-256.

[0022] The disclosure also provides an isolated polypeptide comprising the amino acid sequence:

Xi X ₂ E G X5 X ₆ X ₇ S X9 Xio Xn X ₁₂ X ₁₃ X ₁₄ X15 X½ Xn A I X ₂ o X ₂₁ F X ₂₃ X ₂₄ X25 L X ₂₇ X ₂₈ X ₂₉ X30 (SEQ ID NO: 463);

[0023] wherein Xi is H or Y; X ₂ is G or S; X ₅ is T or M; X ₆ is F or H; X ₇ is T or I; X ₉ is

D or L; X ₁₀ is V, F, L, or Y; Xn is S, A, or R; X ₁₂ is S, K, A, M, Q or I; X ₁₃ is Y, L, H, or A; X ₁₄ is M, A, K, I, or L; X ₁₅ is E or D; X ₁₆ is R, K, or E; Xn is Q or E; X ₂₀ is D or E; X ₂₁ is E or A; X ₂₃ is I, A, or V; X ₂₄ is A or N; X ₂₅ is W, E, L, F, I , S, N, G, M, H, K or D; X ₂₇ is L or A; X ₂₈ is A, K, or G; 29 is G, A, or Q; and X ₃ o is no amino acid, G, A, E, or K. In specific embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:33-43 and 257-294.

[0024] The polypeptides of the invention may further a heterologous polypeptide fused thereto. In some embodiments, the heterologous polypeptide comprises a linker, a hinge, an Fc domain, or a combination thereof. In some embodiments, the linker comprises (GGGGS)n, wherein n 1, 2, 3, or 4. In certain specific embodiments, the linker comprises the amino acid sequence: G GGGGS GGGGS GGGGS GGGGS A (SEQ ID NO:66), A PPGGS GGGGS GGGGS GGGGS A (SEQ ID NO:67), GT GGGGS GGGGS GGGGS GGGGS A (SEQ ID NO:68), G GGGGS GGGGS GGGGS GGGGS A (SEQ ID NO:69), G GGGGS A (SEQ ID NO:70), G GGGGS GGGGS A (SEQ ID NO:71), G GGGGS GGGGS GGGGS A (SEQ ID NO:72), G KGGGS GGGGS GGGGS GGGGS A (SEQ ID NO:73), G GGGGS GGGGS GGGGS GGGGS A (SEQ ID NO: 179), G GGGG GGGG GGGG GGGG A (SEQ ID NO: 180) or any combination, fragment or variant thereof.

[0025] In some embodiments, the linker comprises GG SGSTA SSGSG SATGG GGAA

(SEQ ID NO:74), any variant thereof, or any fragment thereof. In other embodiments, the linker comprises AAAGG SGSTA SSGSG SATGG GGAA (SEQ ID NO:75), APPGG SGSTA SSGSG SATGG GGAA (SEQ ID NO:76), or any combination, fragment or variant thereof.

[0026] In some embodiments, the hinge comprises an amino acid sequence of an IgGl hinge, an IgG4 hinge, a fragment thereof, a variant thereof, or any combination thereof. In certain specific embodiments, the hinge comprises ESKYGPPCPPCPAPEAA (SEQ ID NO:77), THTCPPCPAPEF (SEQ ID NO:78), THTCPPC (SEQ ID NO:79), CPPCPAPEF (SEQ ID NO:80), TYTCPPCPAPEF (SEQ ID NO:81), TSTCPPCPAPEF (SEQ ID NO:82), PPCPPCPAPEF (SEQ ID NO:83), ESKYGPPCPPCPAPEF (SEQ ID NO:84), APEF (SEQ ID NO:85), ESKYGPPCPPC (SEQ ID NO:86), THTCPPCPAPELL (SEQ ID NO: 87), any variant thereof, any fragment thereof, or any combination thereof.

[0027] In some embodiments, the Fc region comprises an IgGl Fc region, an IgGl-TM

Fc region, an IgGl-FQQ Fc region, an IgG4 Fc region, an IgGl-YTE Fc region, any fragment thereof, any variant thereof, or any combination thereof

[0028] In certain specific embodiments, the polypeptide and heterologous polypeptide comprises the amino acid sequence selected from the group consisting of: SEQ ID NOS:88-175. In a specific embodiment, the polypeptide and heterologous polypeptide comprises IP088 (SEQ ID NO: 105).

[0029] The polypeptides of the invention (with or without the heterologous polypeptide) may further comprise a heterologous moiety attached thereto.

[0030] In some embodiments, the heterologous moiety is a polypeptide, an organic polymer, an inorganic polymer, a polyethylene glycol (PEG), biotin, an albumin, a human serum albumin (HSA), a HSA FcRn binding portion, an antibody, a domain of an antibody, an antibody fragment, a single chain antibody, a domain antibody, an albumin binding domain, an enzyme, a ligand, a receptor, a binding peptide, a non-FnIII scaffold, an epitope tag, a recombinant polypeptide polymer, a cytokine, or a combination of two or more of the recited moieties.

[0031] In some embodiments of the invention, the polypeptide binds to a GIP receptor, a

GLP-1 receptor, or to both a GIP and a GLP-1 receptor. In certain embodiments, the polypeptide binds to a GIP receptor. For example, but not by way of limitation, the GIP receptor may be a mouse GIP receptor, a rat GIP receptor, or a human GIP receptor.

[0032] In some embodiments, the polypeptide binds to a human GIP receptor with an

EC50 in the cAMP assay 1 of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.

[0033] In some embodiments, the polypeptide binds to a human glucagon receptor with at least 1000-fold lower affinity than its binding affinity for a human GIP receptor, as measured in the cAMP assay.

[0034] In some embodiments, the polypeptide of the invention binds to a GLP-1 receptor.

For example, but not by way of limitation, the GLP- 1 receptor is a mouse GLP- 1 receptor, a rat GLP-1 receptor, or a human GLP-1 receptor.

[0035] In some embodiments, the polypeptide binds to a human GLP-1 receptor with an

EC50 in the cAMP assay 1 of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.

[0036] In some embodiments, the polypeptide binds to a human glucagon receptor with at least 1000-fold lower affinity than its binding affinity for a human GLP-1 receptor, as measured in the cAMP assay.

[0037] In some embodiments, the polypeptide of the invention is an agonist of GLP-1 activity, an agonist of GIP activity, or an agonist of both GLP-1 and GIP activity.

[0038] The invention also provides an isolated polynucleotide encoding the polypeptide of any one of the polypeptides of the invention. The invention also provides a vector comprising the polynucleotides of the invention and host cells comprising the polynucleotides of the invention, optionally operably connected to a vector.

[0039] The invention also provides a method of making a polypeptide of the invention comprising culturing a host cell containing a polynucleotide of the invention under conditions allowing expression of the polypeptide, and recovering the polypeptide. In some embodiments, the polynucleotide is operably linked to a vector.

[0040] The invention further provides a pharmaceutical composition comprising a polypeptide of the invention and a carrier and kits comprising such a pharmaceutical composition.

[0041] The invention further provides a method of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, comprising administering to a subject in need of treatment an effective amount of at least one polypeptide of the invention or a composition of the invention.

[0042] In some embodiments, the disease or condition is diabetes, such as, for example,

Type-2 diabetes.

[0043] In some embodiments of the method of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, the administration further improves glycemic control, provides body weight control, improves β-cell function and mass, reduces the rate of gastric acid secretion and gastric emptying, or any combination thereof.

[0044] In some embodiments of the method of treating or preventing a disease or condition caused or characterized by hypoglycemia or impaired insulin release, the peptide is administered orally or by injection (e.g. , subcutaneously or intravenously). In some embodiments, the polypeptide is administered once per day. In some embodiments, the method further comprises administering one or more additional therapies. For example, but not by way of limitation, the additional therapy comprises blood sugar monitoring, diet modifications, exercise, insulin, a thiazolidinedione, a sulfonylurea, an incretin, metformin, a glyburide, a dipeptidyl peptidase 4 inhibitor, a bile acid sequestrant, or any combination thereof. In some embodiments, the subject is human. BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0045] FIGURE 1 shows the structure of an exemplary GIP/GLP-1 agonist polypeptide as provided herein.

[0046] FIGURE 2A shows an alignment of exendin-4 (1-30) (SEQ ID NO: 187), human

GLP-1 (SEQ ID NO: 176), human GIP(l-30) (SEQ ID NO: 188), GIP/GLP-1 agonist polypeptide portion of IP088 (SEQ ID NO: 15), and the GLP-1 analog Fc control (GLPlaFc) (SEQ ID NO: 189).

[0047] FIGURES 2B and 2C show potency curves for IP088 on hGLP-lr- and GlPr- expressing cells. The curve is compared with hGLP-1 in FIG. 2B and with hGIP in FIG. 2C. Both panels show a comparison to a GLP-1 analogue-Fc fusion control (GLP-laFc).

[0048] FIGURE 2D is a curve showing pharmacokinetics of IP088 (circles) in mouse out to 48 hours, compared to GLP-laFc (inverted triangles).

[0049] FIGURE 3A-C shows the pharmacokinetic profile of IP088 (panel A) and an

GLP-1 analogue-Fc (gamma4) fusion control (GLP-laFc) (panel B) in mouse over 168 hours as measured by the presence of human IgGl Fc by ELISA. Circles: IV injection of 1 mg/kg, squares: subcutaneous injection of 1 mg/kg. Pharmacokinetic parameters C _max , bioavailability (F ) and half-life Tm are compared in panel C.

[0050] FIGURE 3D-F shows the pharmacokinetic activity of IP088 (panel D) and

GLP-laFc (panel E) in mouse over 168 hours, as measured by potency in a cAMP reporter activity assay on cells expressing human GLP-lr. Circles: Γ injection of 1 mg/kg, squares: subcutaneous injection of 1 mg/kg. Pharmacokinetic parameters C _max , bioavailability (F ) and half life T _1/2 are compared in panel F.

[0051] FIGURE 4A shows a schematic of the oral glucose tolerance tests carried out in

C57 mice.

[0052] FIGURE 4B shows the results of the OGTT on day 0. □ = IP040 (vehicle only),

• = liraglutide, * = GLP-laFc (Peptide SEQ ID NO:62-Fc), and 0 = IP088.

[0053] FIGURE 4C shows the results of the OGTT on day 2. □ = IP040 (vehicle only),

• = liraglutide, * = GLP-laFc (Peptide SEQ ID NO:62-Fc), and 0 = IP088. DETAILED DESCRIPTION

Definitions

[0054] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a polynucleotide," is understood to represent one or more polynucleotides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0055] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0056] It is understood that wherever aspects are described herein with the language

"comprising," otherwise analogous aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided.

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to whom this disclosure is directed. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0058] Units, prefixes, and symbols are denoted in their Systeme International de Unites

(SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. [0059] As used herein, the term "polypeptide" is intended to encompass a singular

"polypeptide" as well as plural "polypeptides," and comprises any chain or chains of two or more amino acids. Thus, as used herein, a "peptide," a "peptide subunit," a "protein," an "amino acid chain," an "amino acid sequence," or any other term used to refer to a chain or chains of two or more amino acids, are included in the definition of a "polypeptide," even though each of these terms can have a more specific meaning. The term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term further includes polypeptides that have undergone post-translational or post-synthesis modifications, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.

[0060] More specifically, the term "polypeptide" as used herein encompasses full length peptides and fragments, variants or derivatives thereof, e.g., a GIP/GLP-1 agonist polypeptide (e.g., 29, 30, or 31 amino acids in length). A "polypeptide" as disclosed herein, e.g., a GIP/GLP-1 agonist polypeptide, can comprise a fusion polypeptide comprising one or more additional components such as, e.g., a linker, a hinge, an Fc domain or an albumin domain, to increase half-life, impart flexibility, allow for dimerization or other desired properties. A polypeptide as described herein can also be derivatized in a number of different ways.

[0061] A polypeptide as provided herein can be multimeric. As used herein, the terms

"multimer," "multimeric" and "multivalent" refer to a molecule, e.g., a GIP/GLP-1 agonist polypeptide, that comprises at least GIP/GLP-1 agonist polypeptides in association. The multimer, e.g., a dimer, trimer, tetramer, or larger polypeptide, can be linked through a disulfide bonds, hydrogen bonds, or other covalent or non-covalant linkages.

[0062] The terms "fragment," "analog," "derivative," or "variant" when referring to a

GIP/GLP-1 agonist polypeptide includes any polypeptide that retains at least some desirable activity, e.g., binding to GIP and/or GLP-1 receptors. Fragments of GIP/GLP-1 agonist polypeptides provided herein include proteolytic fragments, deletion fragments that exhibit desirable properties during expression, purification, and or administration to a subject.

[0063] The term "variant," as used herein, refers to a polypeptide that differs from the recited polypeptide due to amino acid substitutions, deletions, insertions, and/or modifications. Variants can be produced using art-known mutagenesis techniques. Variants can also, or alternatively, contain other modifications-for example a polypeptide can be conjugated or coupled, e.g., fused to a heterologous amino acid sequence or other moiety, e.g., for increasing half-life, solubility, or stability. Examples of moieties to be conjugated or coupled to a polypeptide provided herein include, but are not limited to a linker, a hinge, albumin, an immunoglobulin Fc region, polyethylene glycol (PEG), and the like. The polypeptide can also be conjugated or produced coupled to an element for ease of synthesis, purification or identification of the polypeptide (e.g., 6-His), or to enhance binding of the polypeptide to a solid support.

] The term "sequence identity" as used herein refers to a relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid in the corresponding position of the comparator sequence, the sequences are said to be "identical" at that position. The percentage "sequence identity" is calculated by determining the number of positions at which the identical nucleic acid base or amino acid occurs in both sequences to yield the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of "sequence identity." Percentage of "sequence identity" is determined by comparing two optimally aligned sequences over a comparison window. In order to optimally align sequences for comparison, the portion of a polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant. An optimal alignment is that alignment that, even with gaps, produces the greatest possible number of "identical" positions between the reference and comparator sequences. Percentage "sequence identity" between two sequences can be determined using the version of the program "BLAST 2 Sequences" that was available from the National Center for Biotechnology Information as of September 1, 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing "BLAST 2 Sequences," parameters that were default parameters as of September 1, 2004, can be used for word size (3), open gap penalty (11), extension gap penalty (1), gap drop-off (50), expect value (10), and any other required parameter including but not limited to matrix option.

[0065] The terms "polynucleotide" or "nucleotide" as used herein are intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). In certain aspects, a polynucleotide comprises a conventional phosphodiester bond or a non- conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).

[0066] The term "nucleic acid" refers to any one or more nucleic acid segments, e.g.,

DNA or RNA fragments, present in a polynucleotide. When applied to a nucleic acid or polynucleotide, the term "isolated" refers to a nucleic acid molecule, DNA or RNA that has been removed from its native environment, for example, a recombinant polynucleotide encoding an polypeptide comprising a variant Fc domain contained in a vector is considered isolated for the purposes of the present disclosure. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0067] The term "vector" means a construct that is capable of delivering, and in some aspects, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0068] As used herein, the term "host cell" refers to a cell or a population of cells harboring or capable of harboring a recombinant nucleic acid. Host cells can be a prokaryotic cells (e.g., E. coli), or alternatively, the host cells can be eukaryotic, for example, fungal cells (e.g., yeast cells such as Saccharomyces cerivisiae, Pichia pastoris, or Schizosaccharomyces pombe), and various animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS-7, NIH-3T3). [0069] The terms "composition" or "pharmaceutical composition" refer to compositions containing a polypeptide comprising a GIP/GLP-1 agonist polypeptide provided herein, along with e.g., pharmaceutically acceptable carriers, excipients, or diluents for administration to a subject in need of treatment, e.g., a human subject being treated for a hypoglycemic condition, e.g., type-2 diabetes.

[0070] The term "pharmaceutically acceptable" refers to compositions that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity or other complications commensurate with a reasonable benefit/risk ratio.

[0071] An "effective amount" is that amount of a polypeptide comprising a GIP/GLP-1 agonist polypeptide provided herein, the administration of which to a subject, either in a single dose or as part of a series, is effective for treatment, e.g., treatment of type-2 diabetes. An amount is effective, for example, when its administration results in one or more of prevention or modulation of hyperglycemia, promotion of insulin synthesis, an increase in β-cell mass, weight loss or weight maintenance (e.g., prevention of weight gain), reduction in food intake, modulation of gastric acid secretion, or modulation of gastric emptying. This amount can be a fixed dose for all subjects being treated, or can vary depending upon the weight, health, and physical condition of the subject to be treated, the extent of glycemic control desired, the formulation of polypeptide, a professional assessment of the medical situation, and other relevant factors.

[0072] The term "subject" is meant any subject, particularly a mammalian subject, in need of treatment with a GIP/GLP-1 agonist polypeptide provided herein. Mammalian subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows, apes, monkeys, orangutans, and chimpanzees, and so on. In one aspect, the subject is a human subject.

[0073] As used herein, an "subject in need thereof" refers to an individual for whom it is desirable to treat, e.g., a subject diagnosed with a hypoglycemic condition, e.g., type-2 diabetes, or a subject prone to contract a hypoglycemic condition, e.g., type-2 diabetes.

[0074] As used herein a "GIP/GLP-1 agonist polypeptide" is a chimeric polypeptide that exhibits activity at the GIP receptor of at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more (up to 100%) relative to native GIP and also exhibits activity at the GLP-1 receptor of about at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more (up to 100%) relative to native GLP-1, under conditions provided elsewhere herein, e.g., the cAMP assay performed in Hanks Balanced Salt Solution supplemented with bovine serum albumin ("BSA-cAMP assay"), described in Example 2.

[0075] As used herein the term "native GIP" refers to naturally-occurring GIP, e.g., human GIP (i.e., dPi-42), comprising the sequence of SEQ ID NO: 178, or an active fragment thereof. The term "native GLP-1 " refers to naturally- occurring GLP-1, e.g., human GLP-1, and is a generic term that encompasses, e.g., GLP- 1(7-36) amide (SEQ ID NO: 176), GLP- 1(7-37) acid (SEQ ID NO: 177) or active fragments thereof, or a mixture of those two compounds. As used herein, a general reference to "GIP" or "GLP-1 " in the absence of any further designation is intended to mean native human GIP or native human GLP-1, respectively. Unless otherwise indicated, "GIP" refers to human GIP, and "GLP-1" refers to human GLP- 1.

GIP/GLP-1 agonist polypeptides

[0076] Provided herein are polypeptides that bind both to a GIP receptor and to a GLP-1 receptor. In certain aspects, the peptides provided herein are dual-agonists of GIP and GLP-1 activity. Such peptides are referred to herein as GIP/GLP-1 agonist polypeptides. GIP/GLP-1 agonist polypeptides as provided herein possess GLP-1 and GIP activities with favorable ratios to promote enhanced control of a hypoglycemic condition, e.g., type-2 diabetes. Polypeptides as provide herein can promote one or more of glycemic control, increased insulin production, decreased glucagon production, increased β-cell mass, or decreased body fat. Polypeptides provided herein can further possess optimized solubility, formulatability, and stability. In certain aspects, GIP/GLP-1 agonist polypeptides as provided herein are active at the human GLP-1 and human GIP receptors. In certain aspects, GIP/GLP-1 agonist polypeptides as provided herein are also active at rodent GLP- 1 and rodent GIP receptors, e.g., rat or mouse GLP-1 receptor or rat or mouse GIP receptor. In certain aspects, GIP/GLP-1 agonist polypeptides as provided herein are also active at non-human primate GLP-1 and GIP receptors, e.g., cynomolgus monkey GLP-1 and GIP receptors.

[0077] Certain GIP/GLP-1 agonist peptides provided herein are fusion proteins comprising a GIP/GLP-1 -like peptide domain, and one or more additional domains including, but not limited to one or more of a linker, a hinge, or an Fc domain. Suitable linkers, hinges, and Fc domains are described elsewhere in this disclosure. Additional linkers, hinges, and Fc domains are well-known to those of ordinary skill in the art and can be incorporated into GIP-GLP-1 agonist polypeptides as described herein, and tested for potency, activity, and efficacy in treating hypoglycemic conditions, e.g., type-2 diabetes without undue experimentation according to the methods provided herein.

[0078] In one aspect, this disclosure provides a GIP/GLP-1 agonist polypeptide comprising a peptide sequence derived from G89. The G89 peptide (SEQ ID NO:2) was derived by introducing amino acid substitutions into a truncated version of exendin-4 (SEQ ID NO: 187), a GLP-l-like peptide derived from Heloderma suspectum venom (commercially available under the generic name exenatide, see U.S. Pat. No. 5,424,286, incorporated herein by reference in its entirety). G89 was identified by cAMP potency assays on cells expressing GIPr and cells expressing GLP-lr and has 7 amino acid substitutions relative to amino acids 1 to 29 of exendin-4.

[0079] According to this aspect, this disclosure provides an isolated polypeptide comprising the amino acid sequence:

Y S Xi G T F T S D X ₂ S X ₃ X ₄ X ₅ E X ₆ X ₇ X ₈ X ₉ Xio Xii X12 F Xi3 Xi4 W L X ₁₅ Xi6 Xi7 Xi8 (SEQ ID NO: 1);

where Xi is E or Q; X ₂ is Y, V, or L; X ₃ is K, S, or I; X ₄ is L, Y, or A; X5 is L or M; X ₆ is E or D; X ₇ is E, G, or K; X ₈ is E, Q, or I; X ₉ is A or H; X ₁₀ is V, A, or Q; Xn is R, K, or Q; X ₁₂ is L, E, or D; X ₁ is I or V; X ₁₄ is E, A, or N; X ₁₅ is L, K, or V; X ₁₆ is A, K, or N; X ₁₇ is G or Q; and X ₁₈ is no amino acid, G, N, P, K, or T. In certain aspects, X ₄ is L. In certain aspects, Xi is E; X ₂ is Y; X ₃ is K; X ₄ is L; X5 is L; X ₆ is E; X ₇ is E; X ₈ is E; X9 is A; Xio is V; Xn is R, X ₁₂ is L; X ₁₃ is I; X ₁₄ is E; and X ₁₇ is G. In further aspects, related to any of the aspects noted above, X15 is L and X ₁₆ is A; X^ is K and X ₁₆ is N; or X^ is V and X ₁₆ is K.

[0080] This aspect of the disclosure also provides an isolated polypeptide comprising or consisting of the amino acid sequence:

Y S X3 G T F T S D Xio S X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ Xn X ₁₈ X19 X20 X21 F X ₂₃ X ₂₄ W L X ₂₇ X ₂₈ X ₂₉ X ₃₀ (SEQ ID NO: 461); wherein X ₃ is E or Q; X^ is Y, V, or L; X ₁₂ is K, S, or I; X ₁₃ is L, Y, or A; X ₁₄ is L or M; X ₁₅ is E or D; X ₁₆ is E, G, or K; X ₁₇ is E, Q, or I; X ₁₈ is A or H; X ₁₉ is V, A, or Q; X ₂₀ is R, K, or Q; X ₂₁ is L, E, or D; X ₂ is I or V; X ₂₄ is E, A, or N; X ₂₇ is L, K, or V; X ₂₈ is A, K, or N; X29 is G or Q; and X ₃₀ is no amino acid, G, N, P, K, or T. In some embodiments of the polypeptide of SEQ ID NO:461, X ₁₃ is L. In other embodiments, X is E; X ₁₀ is Y; X ₁₂ is K; X ₁₄ is L; X ₁₅ is E; X ₁₆ is E; X ₁₇ is E; Xig is A; X ₁₉ is V; X ₂ o is R, X21 is L; X ₂₃ is I; X ₂₄ is E; and X29 is G. In other embodiments, X ₂₇ is L and X ₂ 8 is A; X ₂₇ is K and X ₂ 8 is N; or

[0081] This aspect of the disclosure also provides an isolated polypeptide comprising or consisting of the amino acid sequence:

Xi X ₂ X ₃ G X5 X ₆ X ₇ S X9 Xio Xu X12 Xl ₃ Xl ₄ Xl5 Xl6 Xl7 Xl8 X19

X ₂₀ X21 F X ₂₃ X ₂₄ X ₂₅ L X ₂₇ X ₂₈ X ₂₉ X ₃₀ (SEQ ID NO: 462); wherein Xi is Y or H; X ₂ is S or G; X ₃ is E or Q; X5 is T or M; X ₆ if F or H; X ₇ is T or I; X ₉ is D or L; X ₁₀ is Y, V, F, or L; X _n is S, R, or M; X ₁₂ is K, S, M, or I; X ₁₃ is L, Y, Q, H, I or A; X ₁₄ is L, K, or M; X ₁₅ is E or D; X ₁₆ is E, G, or K; X ₁₇ is E, Q, or I; X ₁₈ is A, C, or H; X ₁₉ is V, A, E, I, N, S, T, or Q; X ₂₀ is R, K, or Q; X ₂₁ is L, E, C, or D; X ₂₃ is I or V; X ₂₄ is E, C, A, or N; X ₂₅ is W, C, F, H, Y, or K; X ₂₇ is L, K, or V; X ₂₈ is A, K, C, or N; X ₂₉ is G or Q; and X ₃ o is no amino acid, G, N, P, K, or T.

[0082] In certain specific aspects, this disclosure provides a G89-related GIP/GLP-1 agonist polypeptide comprising any one or more of the amino acid sequences listed in Table 1.

Table 1: GIP/GLP-1 Agonist Peptides based on G89

Peptide SEQ ID NO

YSEGT FTSDY SKLLE EEAVR LFIEW LLAG (G89) 2

YSEGT FTSDV SKLLE EEAVR LFIEW LLAG 3

YSEGT FTSDY SKLLD EEAVR LFIEW LLAG 4

YSEGT FTSDY SKLLE EEAVR LFIEW LVKG 5

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGN 6

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGG HNITQ 7

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGG 8

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGP 9

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGP SSGAP PPS 10

YSEGT FTSDY SKLLE EEAVR LFIEW LLAQK 11

YSEGT FTSDY SKLLE EEAVR LFIEW LKNGG 12

YSEGT FTSDY SKLLE EEAVR LFIEW LKNGG PSSGA PPPS 13

YSEGT FTSDY SKLLE EEAVR LFIEW LKNGT 14 YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 15

YSQGT FTSDY SKLLE EEAVR LFIEW LVKGP 16

YSEGT FTSDL SKLLE EEAVR LFIEW LVKGP 17

YSEGT FTSDY SSYME EEAVR LFIEW LVKGP 18

YSEGT FTSDY SI AMD EEAVR LFIEW LVKGP 19

YSEGT FTSDY SKLLE GQAAK LFIEW LVKGP 20

YSEGT FTSDY SKLLE KIHQQ LFIEW LVKGP 21

YSEGT FTSDY SKLLE EEAQR LFIEW LVKGP 22

YSEGT FTSDY SKLLE EEAVR EFIAW LVKGP 23

YSEGT FTSDY SKLLE EEAVR DFVNW LVKGP 24

_SEGT FTSDY SKLLE EEAVR LFIEW LVKGP 25

YSEGT FTSDY SKLLD EEAVR LFIEW LVKGP 26

YSEGT FTSDY SKLLE KEAVR LFIEW LVKGP 27

YSEGT FTSDY SKLLE EEAVQ LFIEW LVKGP 28

YSEGT FTSDY SKLLE EEAVR LFVEW LVKGP 29

YSEGT FTSDY SKLLE EEAVR LFIEW LLKGP 30

YSEGT FTSDY SKLLE EEAVR LFIEW LVKQP 31

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGK 32

YSEGT FTSDY SKLLE EEAVR LFIEW LLAG 191

YSEGT FTSDY SKLLE EEHVR LFIEW LVKGP 192

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 193

YSEGT FTSDY SKLLE EEAVR LFIEW LLAQK 194

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 195

YSEGT FTSDY SKLLE KEAVR LFIEW LLKGP 196

YSEGT FTSDY SKLLE KEAQR LFIEW LLKGP 197

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 198

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP PSSGA PPPS 199

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP SSGAP PPS 200

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGG PSSGA PPPS 201

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 202

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 203

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 204

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 205

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 206

YSEGT FTSDY SKLLE KEAER LFIEW LLKGP 207 YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 208

YSEGT FTSDY SKLLE EECVR LFIEW LVKGG PGGGA PPPGC 209

YSEGT FTSDY SKLLE EEAVR CFIEW LVKGG PGGGA PPCGG 210

YSEGT FTSDY SKLLE EEAVR CFIEC LVKGP 211

YSEGT FTSDY SKLLE EEAVR LFICW LVCGP 212

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGG PSSGA PPPS 213

YSEGT FTSDY SKLLE EEAVR CFIEW LVKGG PSSGA PPCS 214

YSEGT FTSDY SKLLE EEAVR LFIEW LLAG 215

YSEGT FISDY SKLLE EEAVR DFVEW LKAGG 216

YSEGT FTSDY SKLLE EEAVR LFIEW LLAG 217

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 218

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 219

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 220

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 221

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGG 222

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGG 223

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGG 224

YSEGT FTSDY SKLLE KEAQR LFIEW LLAGG 225

HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS 226

HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS 298

HGEGT FTSDL SKQME EECVR LFIEW LKNGG PSSGA PPPGC 299

YSEGM HTSDF SKLLE EEAVR LFIEW LLAGG 227

YSEGT HTSLF SKLLE EEAVR LFIEW LLAGG 228

YSEGT FTSDF SKLLE EEAVR LFIEF LLAGG 229

YSEGT FTSDF SKLLE EEAVR LFIEW LLAGG 230

YSEGT FTSDY SKLLE EEAVR LFIEH LLAGG 231

YSEGT FTSDY RILLE EEAVR LFIEW LLAGG 232

YSEGT FTSDY RILKE EEAVR LFIEW LLAGG 233

YSEGT FTSDY MMLKE EEAVR LFIEW LLAGG 234

YSEGT FTSDY RMLKE EEAVR LFIEW LLAGG 235

YSEGT FTSLY SKLKE EEAVR LFIEW LLAGG 236

YSEGM HTSDL SKLLE EEAVR LFIEW LLAGG 237

YSEGT FTSDY SIYLD EEAVR LFIEW LLAGG 238

YSEGT FTSLY SKLLE EEAVR LFIEW LLAGG 239

YSEGT HTSDF SKLLE EEAVR LFIEW LLAGG 240

YSEGT FTSDY SKLLE EEAVR LFIEF LLAGG 241 YSEGT FTSDY SKLLE EEAVR LFIEY LLAGG 242

YSEGT FTSLY SKLLE EEAVR LFIEY LLAGG 243

YSEGT FTSLY RILLE EEAVR LFIEW LLAGG 244

YSEGT FTSDY SKLLE EEAVR LFIEW LLKGG 245

YSEGT FTSDY SKLLE EEAVR LFIEW LVKGP 246

YSEGT HTSDL SKLLE EEAVR LFIEW LLAGG 247

YSEGT HTSDL RILLE EEAVR LFIEK LLAGG 248

YSEGT FTSDY SKLLE EEAIR LFIEW LLAGG 249

YSEGT FTSLY RIHKE EEAVR LFIEK LLAGG 250

YGEGT FTSDY SKLLE EEAVR LFIEW LVKGP 251

YSEGT FTSDY SKLLE EEANR LFIEW LLAGG 252

YSEGT FTSDY SKLLE EEASR LFIEW LLAGG 253

HSQGT FTSDY SKILE EEAVR LFIEW LLAGG 254

YSEGT FTSDY SKLLE EEAVR LFIEW LLAGG 255

YSEGT FTSDY SKLLE EEATR LFIEW LLAGG 256

[0083] In further aspects, this disclosure provides a GIP/GLP-1 agonist polypeptide comprising a peptide sequence derived from the "AID" peptide (SEQ ID NO:33). The AID peptide was selected from a phage display library of peptides based on GLP-1, by binding to GLP-lr and GIPr and confirmed in a cAMP selectivity assay. AID has 7 amino acid substitutions relative to human GLP-1 (1-29).

[0084] According to this aspect, this disclosure provides an isolated polypeptide comprising the amino acid sequence:

Xi X ₂ E G T F T S D X ₃ S X ₄ X ₅ X ₆ ? _{8 9} A I D E F Xio X _n Xi ₂ L L Xi ₃ Xi ₄ Xi ₅ (SEQ ID NO: 190);

where Xi is H or Y; X ₂ is G or S; X ₃ is V or Y; X ₄ is S, K, or I; X ₅ is Y, L, or A; X ₆ is M or L; X ₇ is E or D; Xg is R or E; X9 is Q or E; X ₁₀ is I or V; Xn is A or N; X ₁₂ is W or D; X ₁₃ is A, K, or G; X ₁₄ is G or Q; and X ₁₅ is no amino acid, G, or K. In certain aspects, Xi is Y and X ₂ is S. In certain aspects, X ₁₀ is I, Xn is A, and X ₁₂ is W; or Xi is Y, X ₂ is S X ₁₀ is I, Xn is A, and Xi ₂ is W. In certain further aspects related to any of the aspects above, X ₁₄ is G and X ₁₅ is no amino acid or G. In certain further aspects related to any of the aspects above, X ₇ is E. In certain further aspects related to any of the aspects above, X ₈ is R and X ₉ is Q. In certain further aspects related to any of the aspects above, X ₁ is K. In certain further aspects related to any of the aspects above, X ₃ is V. In certain further aspects related to any of the aspects above, X ₄ is S, X ₅ is Y, and X ₆ is M.

[0085] The disclosure also provides an isolated polypeptide comprising the amino acid sequence:

Xi X ₂ E G X ₅ X ₆ X ₇ S X9 X ₁₀ X11 X ₁₂ X ₁₃ Xi ₄ X15 i6 X17 I X ₂₀ X ₂₁ F X ₂₃ X ₂₄ X ₂₅ L X ₂₇ X ₂₈ X ₂₉ X ₃₀ (SEQ ID NO: 463); wherein Xi is H or Y; X ₂ is G or S; X5 is T or M; X ₆ is F or H; X ₇ is T or I; X9 is D or L; X ₁₀ is V, F, L, or Y; X _n is S, A, or R; X ₁₂ is S, K, A, M, Q or I; X ₁₃ is Y, L, H, or A; X ₁₄ is M, A, K, I, or L; X^ is E or D; X ₁₆ is R, K, or E; X ₁₇ is Q or E; X ₂₀ is D or E; X ₂₁ is E or A; X ₂₃ is I, A, or V; X ₂₄ is A or N; X ₂₅ is W, E, L, F, I , S, N, G, M, H, K or D; X ₂₇ is L or A; X ₂ 8 is A, K, or G; X29 is G, A, or Q; and X ₃ o is no amino acid, G, A, E, or K.

[0086] In certain specific aspects, this disclosure provides an AID-related GIP/GLP-1 agonist polypeptide comprising any one or more of the amino acid sequences listed in Table 2.

Table 2: GIP/GLP-1 Agonist Peptides based on "AID"

Peptide SEQ ID NO

HGEGT FTSDV SSYME RQAID EFIAW LLAG ("AID") 33

HGEGT FTSDV SSYME RQAID EFIAW LLKGG 34

YSEGT FTSDV SSYME RQAID EFIAW LLAG 35

YSEGT FTSDV SSYME RQAID EFIAW LLKGG 36

YSEGT FTSDY SSYME RQAID EFIAW LLKGG 37

YSEGT FTSDV SKLLE RQAID EFIAW LLKGG 38

YSEGT FTSDV SI AMD RQAID EFIAW LLKGG 39

YSEGT FTSDV SSYME EEAID EFIAW LLKGG 40

YSEGT FTSDV SSYME RQAID EFVND LLKGG 41

YSEGT FTSDV SSYME RQAID EFIAW LLGGG 42

YSEGT FTSDV SSYME RQAID EFIAW LLAQK 43

YSEGT FTSDV SSYLE RQAID EFIAW LLKGG 257

YSEGT FTSDV SSYLE REAID EFIAW LLKGG 258

YSEGT FTSDV SSYLE RQAIE EFIAW LLKGG 259

YSEGT FTSDY SKLLE RQAID EFIAW LLKGG 260 YSEGT FTSDV SSYME RQAID EFIAW LLKQG 261

YSEGT FISDV SSYLE RQAID EFIAW LLKGG 262

YSEGT FTSDV SSYME RQAID EFIAW LLKGK 263

YSEGT FISDV SSYME RQAID EFIAW LLKGG 264

YSEGT FISDY SSYLE RQAID EFIAW LLKGG 265

YSEGT FTSDV SSYLE KQAID EFIAW LLKGG 266

YSEGT FTSDY SKLLE RQAID EFIAE LLKGG 267

YSEGT FTSDY SKLLE RQAID EFIAL LLKGG 268

YSEGT FTSDY SKLLE RQAID EFIAD LLKGG 269

YSEGT FTSDY AAAAE RQAID AFAAW LAAAA 270

YSEGT FTSDY SKLLE RQAID EFIAF LLKGG 271

YSEGT FTSDY SKLLE RQAID EFIAI LLKGG 272

YSEGT FTSDY SKLLE RQAID EFIAS LLKGG 273

YSEGT FTSDY SKLLE RQAID EFIAN LLKGG 274

YSEGT FTSDY SKLLE RQAID EFIAG LLKGG 275

YSEGT FTSDY SKLLE RQAID EFIAM LLKGE 276

YSEGT FTSDF SKLLE RQAID EFIAW LLKGG 277

YSEGM HTSDF SKLLE RQAID EFIAW LLKGG 278

YSEGT FTSDY RMLKE RQAID EFIAW LLKGG 279

YSEGT FTSDY RILKE RQAID EFIAW LLKGG 280

YSEGT FTSDY RQAID RQAID EFIAW LLKGG 281

YSEGT FTSDY SKLLE RQAID EFIAH LLKGG 282

YSEGT HTSDF SKLLE RQAID EFIAW LLKGG 283

YSEGT HTSLF SKLLE RQAID EFIAW LLKGG 284

YSEGT FTSLF SKLLE RQAID EFIAW LLKGG 285

YSEGT FTSLY RILLE RQAID EFIAW LLKGG 286

YSEGT FTSDY SIYLD RQAID EFIAW LLKGG 287

YSEGM HTSDL SKLLE RQAID EFIAW LLKGG 288

YSEGT FTSDY SKLLE RQAID EFIAH LLKGG 289

YSEGT FTSDY SKLLE RQAID EFIAW LLKGG 290

YSEGT HTSDL SKLLE RQAID EFIAW LLKGG 291

YSEGT FTSLY RIHKE RQAID EFIAK LLKGG 292

YSEGT HTSDL RILLE RQAID EFIAK LLKGG 293

YSEGT HTSDL RILLE RQAID EFIAF LLKGG 294 [0087] In further aspects, this disclosure provides a GIP/GLP-1 agonist polypeptide comprising a peptide sequence derived from the "AIS" peptide (SEQ ID NO:44). The AIS peptide was selected from a phage display library of peptides based on GLP-1, by binding to GLP-lr and GIPr and confirmed by cAMP selectivity assay. AIS has 9 amino acid substitutions relative to human GLP-1 (1-29).

[0088] In certain specific aspects, this disclosure provides an AlS-related GIP/GLP-1 agonist polypeptide comprising any one or more of the amino acid sequences listed in

Table 3.

Table 3: GIP/GLP-1 Agonist Peptides based on "AIS"

[0089] In further aspects, this disclosure provides a GIP/GLP-1 agonist polypeptide comprising a peptide sequence derived from the "SIR" peptide (SEQ ID NO:46) or the "KIR" peptide (SEQ ID NO:47). The SIR and KIR peptides were selected from a phage display library of peptides based on GLP-1, by binding to GLP-lr and GIPr and confirmed by cAMP selectivity assay. SIR has 7 amino acid substitutions relative to human GLP-1 (1- 29). KIR has 6 amino acid substitutions relative to human GLP-1 (1-29).

[0090] In certain specific aspects, this disclosure provides a SIR or KIR-related

GIP/GLP- 1 agonist polypeptide comprising any one or more of the amino acid sequences listed in Table 4.

Table 4: GIP/GLP-1 Agonist Peptides based on "SIR" or "KIR"

[0091] In further aspects, this disclosure provides a GIP/GLP-1 agonist polypeptide comprising a peptide sequence derived from the "LVR" peptide (SEQ ID NO:48). The LVR peptide was selected from a phage display library of peptides based on GLP-1, by binding to GLP-lr and GIPr and confirmed by cAMP selectivity assay. LVR has 6 amino acid substitutions relative to human GLP-1 (1-29). [0092] In certain specific aspects, this disclosure provides an LVR-related GIP/GLP-1 agonist polypeptide comprising any one or more of the amino acid sequences listed in Table 5.

Table 5: GIP/GLP-1 Agonist Peptides based on "LVR"

[0093] In further aspects, this disclosure provides a GIP/GLP-1 agonist polypeptide comprising a peptide sequence derived from GIP or GLP-1 sequences but with amino acids substitutions derived from GIP, GLP-1, and/or exendin-4.

[0094] In certain specific aspects, this disclosure provides an GIP/GLP-1 agonist polypeptide comprising any one or more of the amino acid sequences listed in Table 6.

Table 6: GIP/GLP-1 Agonist Peptides

Peptide SEQ ID NO

YSEGT FTSDV SSYLE GQAAK EFIAW LLAG 50

HGEGT FISDY SIAMD KIHQQ DFVNW LLAQK 51

HGEGT FTSDV SSYLE GQAAK EFIAW LLAG 52

YSEGT FTSDV SSYLE EQAAK EFIAW LLAG 53

YSEGT FTSDV SSYLE GQAAK EFIAW LVAGG 54

HGEGT FTSDV SSYLE EEAAK EFIAW LVAGG 55

YSEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS 56

YSEGT FTSDV SSYLE EEAVR EFIEW LLAGG 57

YSEGT FISDY SIAMD EEAVR DFVEW LKAGG 297

YSQGT FTSDY SKILE EEAVR LFIEW LVKGP 58

HGEGT FTSDV SSYLE GQAAK EFIAW LVKG 59

HGEGT FTSDV SSYLE EQAAK EFIAW LVKG 60

HGEGT FTSDV SSYLE EQAAK EFIAW LVKGG 61

HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS 62

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 63

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 64

YAEGT FISDY SIAMD KIHQQ DFVNW LLAQK GKKND WKHNI TQ 65

YSEGT FISDY SI ALA AAAVR DFVNW LLAGG 295 YSEGT FISDY SKLLA AAAVR DFVNW LLAGG 296

YSEGT HTSDL RILLE EEAVR LFIEF LLAGG 300

YSEGT FISDY SI ALA AAAQQ DFVNW LLAGG 301

YSEGT FISDY SI ALA AAAAK DFVNW LLAGG 302

YSEGT FISDY SI ALA AAAID DFVNW LLAGG 303

YSEGT FTSDY SKLLE AAAAA AFAAW LAAAA 304

YSEGT FTSDY AAAAE AAAAA AFAAW LAAAA 305

YSEGT FTSDY SI ALA AAAQQ DFVNW LLAGG 306

YSEGT FISDY SI ALA AAAQQ DFVNW LLKGG 307

YSEGT FTSDY SI ALA AAAQQ DFVNW LLKGG 308

YSEGT FTSDY SAAAE AAAAA AFAAW LAAAA 309

YSEGT FTSDY AAAAE AAAAA AFAAW LAKAA 310

YSEGT FISDY SIAMD KIHQQ DFVNW LLAGG 311

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 312

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 313

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 314

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 315

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 316

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK PSSGA PPPS 317

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 318

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 319

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 320

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 321

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK GAAND WKHNI TQ 322

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK GKKND WKHNI TQ 323

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 324

YSEGT FISDY SIAMD KIHQQ DFVNW LLAQK 325 95] In certain aspects, any one or more GIP/GLP-1 agonist peptides as described above can be fused to one or more additional heterologous polypeptide domains. Such additional polypeptide regions can facilitate, e.g., activity, efficacy, stability, or in vivo half-life. For example, a heterologous polypeptide domain can comprise a linker, a hinge, an Fc domain, or a combination thereof. [0096] Linkers used in various GIP/GLP-1 agonist polypeptides provided herein can facilitate formation of a desired structure. In some aspects, a polypeptide linker can comprise 1-50 amino acids, 1-25 amino acids, 25-50 amino acids, or 30-50 amino acids. Generally longer linkers correlate with higher activity (more flexible), but also decreased stability as the peptide becomes more exposed. Linkers can comprise, e.g., (Gly-Ser) _n , residues, where n is an integer of at least one, and up to, e.g., 4, 5, 6, 10, 20, 50, 100, or more, optionally with some Glu or Lys residues dispersed throughout to increase solubility. Alternatively, certain linkers do not comprise any Serine residues, e.g., where the linker is subject to O-linked glycosylation. In some aspects, linkers can contain cysteine residues, for example, if dimerization of linkers is used to bring two or more GIP/GLP-1 agonist polypeptides into a dimeric configuration. In some aspects, a GIP/GLP-1 agonist polypeptide can comprise at least one, two, three, four, or more linkers. The length and amino acid sequence of a linker can be readily selected and optimized.

[0097] In certain aspects, the linker comprises (GGGGS)n, wherein n 1, 2, 3, or 4. For example, certain specific linkers the amino acid sequence: G GGGGS GGGGS GGGGS GGGGS A (SEQ ID NO:66), A PPGGS GGGGS GGGGS GGGGS A (SEQ ID NO:67), GT GGGGS GGGGS GGGGS GGGGS A (SEQ ID NO:68), G GGGGS GGGGS GGGGS GGGGS A (SEQ ID NO: 69), G GGGGS A (SEQ ID NO:70), G GGGGS GGGGS A (SEQ ID NO:71), G GGGGS GGGGS GGGGS A (SEQ ID NO:72), G KGGGS GGGGS GGGGS GGGGS A (SEQ ID NO:73), G GGGGS GGGGS GGGGS GGGGSA (SEQ ID NO: 179), G GGGG GGGG GGGG GGGG A (SEQ ID NO: 180) any combination thereof, any fragment thereof, or any variant thereof. The peptide linkers may optionally contain a potential glycosylation site, for example, but not by way of limitation, a Threonine in SEQ ID NO:68 and the Lysine in SEQ ID NO:73).

[0098] In certain aspects, the linker comprises GG SGSTA SSGSG SATGG GGAA

(SEQ ID NO:74), any variant thereof, or any fragment thereof. For example, certain specific linkers the amino acid sequence: AAAGG SGSTA SSGSG SATGG GGAA (SEQ ID NO:75), APPGG SGSTA SSGSG SATGG GGAA (SEQ ID NO:76), any combination thereof, any fragment thereof, or any variant thereof.

[0099] Hinges used in various GIP/GLP-1 agonist polypeptides provided herein can facilitate formation of a desired structure. In certain aspects, the hinge comprises an amino acid sequence of an IgGl hinge, an IgG4 hinge, a fragment thereof, a variant thereof, or any combination thereof. In certain, non-limiting aspects, the hinge comprises the amino acid sequence ESKYGPPCPPCPAPEAA (SEQ ID NO:77), THTCPPCPAPEF (SEQ ID NO:78), THTCPPC (SEQ ID NO:79), CPPCPAPEF (SEQ ID NO:80), TYTCPPCPAPEF (SEQ ID NO:81), TSTCPPCPAPEF (SEQ ID NO:82), PPCPPCPAPEF (SEQ ID NO:83), ESKYGPPCPPCPAPEF (SEQ ID NO:84), APEF (SEQ ID NO:85), ESKYGPPCPPC (SEQ ID NO:86), THTCPPCPAPELL (SEQ ID NO:87), any variant thereof, any fragment thereof, or any combination thereof.

0] Fc regions used in various GIP/GLP-1 agonist polypeptides provided herein can facilitate formation of a desired structure and to enhance or eliminate various desired or undesired effector functions. In certain aspects the Fc region is a native immunoglobulin Fc region.

[0101] The terms "Fc domain" and "IgG Fc domain" as used herein refer to the portion of an immunoglobulin, e.g., an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region comprises the C-terminal half of two heavy chains of an IgG molecule that are linked by disulfide bonds. For example, an Fc domain can comprise the entire second constant domain CH2 and the third constant domain CH3.

[0102] In certain aspects, GIP/GLP-1 agonist polypeptides provided herein can comprise a "wild type IgG Fc domain," e.g., any naturally occurring IgG Fc region (any allele). In certain aspects the IgG Fc domain is an IgGl domain (SEQ ID NO: 181), in some aspects the IgG Fc domain is an IgG4 Fc domain (SEQ ID NO: 182).

[0103] In certain aspects, GIP/GLP-1 agonist polypeptides provided herein can comprise a "variant IgG Fc domain," an IgG Fc domain comprising one or more amino acid substitutions, deletions, insertions or modifications introduced at any position within the Fc domain. In certain aspects a variant IgG Fc domain comprises one or more amino acid substitutions resulting in decreased or ablated binding affinity for an FcyR and/or Clq as compared to the wild type Fc domain not comprising the one or more amino acid substitutions.

[0104] In certain aspects, GIP/GLP-1 agonist polypeptides provided herein can comprise a "TM" Fc domain. The terms "TM" or "TM mutant" refer to a set of mutations in an IgG Fc domain that result in ablation of effector function, namely elimination of the Fc domain's ability to mediate antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In certain aspects, a TM mutant can comprise a combination of three "TM mutations": L234F, L235E, and P331S, where the numbering is according to the EU index as in Kabat. These mutations cause a profound decrease Fc domain binding to human FcyRI (CD64), FcyRIIA (CD32A), FcyRIII (CD16) and Clq. See, e.g., US 2011/0059078 and Oganesyan et al. Acta Crystallographica D (54:700-704 (2008), which are hereby incorporated by reference in their entireties. A human IgGl Fc domain comprising the TM mutations is shown as SEQ ID NO: 183.

[0105] In certain aspects, GIP/GLP-1 agonist polypeptides provided herein can comprise a "YTE" Fc domain. The terms "YTE" or "YTE mutant" refer to a set of mutations in an IgGl Fc domain that results in an increase in the binding to human FcRn and improves the serum half-life of the antibody having the mutation. A YTE mutant comprises a combination of three "YTE mutations": M252Y, S254T, and T256E, wherein the numbering is according to the EU index as in Kabat, introduced into the heavy chain of an IgG. See U.S. Patent No. 7,658,921, which is incorporated by reference herein. The YTE mutant has been shown to increase the serum half-life of antibodies compared to wild-type versions of the same antibody. See, e.g., Dall'Acqua et al., J. Biol. Chem. 287:23514-24 (2006) and U.S. Patent No. 7,083,784, which are hereby incorporated by reference in their entireties. A human IgGl Fc domain comprising the YTE mutations is shown as SEQ ID NO: 184.

[0106] In certain aspects, GIP/GLP-1 agonist polypeptides provided herein can comprise a "TM-YTE IgG Fc domain." The term "TM-YTE IgG Fc domain" refers to an IgG Fc domain comprising one or more of the three "TM" mutations (L234F/L235E/P331S) and one or more of the three "YTE" mutations (M252Y/S254T/T256E), where all the numbering is according to the EU index as in Kabat. A human IgGl Fc domain comprising the TM mutations and the YTE mutations is shown as SEQ ID NO: 186.

[0107] In certain aspects, GIP/GLP-1 agonist polypeptides provided herein can comprise an Fc domain with additional mutations to provide additional stability. In some aspects the variant IgG Fc domain can comprise, alone or in addition to YTE and/or TM mutations one or more of the following mutations:

(i) a phenylalanine (F) amino acid at EU position 234,

(ii) an alanine (A), asparagine (N), phenylalanine (F), a

glutamine (Q) or a valine (V) amino acid at EU position 235, and (iii) an alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N), or glutamine (Q) amino acid at EU position 322, or -in the alternative- an alanine (A) or glycine (G) amino acid at EU position 331.

[0108] In certain aspects, GIP/GLP-1 agonist polypeptides provided herein can comprise an "FQQ" mutation: an IgGl Fc domain with a phenylalanine (F) amino acid at EU position 234, a glutamine (Q) amino acid at EU position 235, and a glutamine (Q) amino acid at EU position 322. The FQQ mutation can be in combination with a YTE mutation, an TM mutation, or both a YTE mutation and a TM mutation. A human IgGl Fc domain comprising the FQQ mutations is shown as SEQ ID NO: 185.

[0109] In sum, a GIP/GLP-1 agonist polypeptide as provided herein can comprise, without limitation, an Fc domain, e.g., an IgGl Fc domain, an IgTM Fc domain, IgGl- FQQ Fc domain, an IgG4 Fc domain, a YTE Fc domain, any fragment thereof, any variant thereof, or any combination thereof.

[0110] In certain specific aspects, a GIP/GLP-1 agonist polypeptide comprising a linker, a hinge, and an Fc domain can comprise one or more of the G89 polypeptides provided in Table 7.

Table 7: G89-based GIP/GLP-1 Agonist Polypeptides

[0111] In certain specific aspects, a GIP/GLP-1 agonist polypeptide comprising a linker, a hinge, and an Fc domain can comprise one or more of the AID-based polypeptides provided in Table 8.

Table 8: AID-based GIP/GLP-1 Agonist Polypeptides

[0112] In certain specific aspects, a GIP/GLP-1 agonist polypeptide comprising a linker, a hinge, and an Fc domain can comprise one or more of the AIS-, SIR/KIR-, or LVR-based polypeptides provided in Table 9.

Table 9: AIS-, SIR/KIR-, or LVR-based GIP/GLP-1 Agonist Polypeptides

[0113] In certain specific aspects, a GIP/GLP-1 agonist polypeptide comprising a linker, a hinge, and an Fc domain can comprise one or more of the polypeptides provided in Table 10.

Table 10: GIP/GLP-1 Agonist Polypeptides

[0114] In certain aspects, a GIP/GLP-1 agonist polypeptide as described above can form multimers. For example, two or more GIP/GLP-1 monomer polypeptides can be joined through disulfide bonds, through cysteines contained, e.g., in the linker or hinge regions of two or more polypeptide monomers. In certain aspects two or more monomer can be identical, resulting in a homomonomer, e.g., a homodimer. Alternatively the two or more polypeptide monomers can be different, resulting in a heteromonomer, e.g., a heterodimer.

[0115] In certain aspects, GIP/GLP-1 agonist polypeptides as disclosed have desirable potencies at the GIP and GLP-1 receptors for controlling symptoms of a hypoglycemic condition, e.g., type-2 diabetes. In certain aspects, GIP/GLP-1 agonist polypeptides as disclosed exhibit in vitro potencies at the GLP-1 receptor as shown by an EC50 in the BSA-cAMP assay of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, less than 2 pM, or less than 1 pM.

[0116] In certain aspects, GIP/GLP-1 agonist polypeptides as disclosed exhibit in vitro potencies at the GIP receptor as shown by an EC50 in the BSA-cAMP assay of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, less than 2 pM, or less than 1 pM.

[0117] Activity at the glucagon receptor (GCGr) (which promotes glucose production) can be undesirable in the control of hypoglycemic conditions, e.g., type-2 diabetes. See, e.g, D'Alessio, D., Diabetes Obes. Metab. Suppl 7: 126-32 (2011). Accordingly, In certain aspects, a GIP/GLP-1 agonist polypeptide as disclosed herein exhibit reduced potency for GCGr relative to either GIPr or GLP-lr, or both GIPr and GLP-lr. For example, the polypeptide can exhibit an EC50 for GCGr, as measured by the BSA-cAMP assay, of at least 10-fold higher, at least 100-fold higher, at least 1000-fold higher, or at least 10,000 or more-fold higher than the polypeptide's EC50 for GIPr, GLP-lr, or both, as measured by the BSA-cAMP assay.

[0118] In certain aspects, GIP/GLP-1 agonist polypeptides as disclosed exhibit in vitro potencies at GCGr as shown by an EC50 in the BSA-cAMP assay of greater than 0.1 nM, greater than 1 nM, greater than 5 nM, greater than 10 nM, greater than 50 nM, greater than 100 nM, greater than 200 nM, greater than 300 nM, greater than 400 nM, greater than 500 nM, greater than 600 nM, greater than 700 nM, greater than 800 nM, greater than 900 nM, greater than 1000 nM, greater than 2000 nM, greater than 3000 nM, greater than 4000 nM, greater than 5000 nM, greater than 6000 nM, greater than 7000 nM, greater than 8000 nM, greater than 9000 nM, or greater than 10,000 nM.

[0119] Methods of making. This disclosure provides a method of making a GIP/GLP-1 agonist polypeptide. GIP/GLP-1 agonist polypeptides provided herein can be made by any suitable method. For example, GIP/GLP-1 agonist polypeptides provided herein can be produced recombinantly using a convenient vector/host cell combination as would be well known to the person of ordinary skill in the art. A variety of methods are available for recombinantly producing GIP/GLP-1 agonist polypeptides. Generally, a polynucleotide sequence encoding the GIP/GLP-1 agonist polypeptide is inserted into an appropriate expression vehicle, e.g., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. The nucleic acid encoding the GIP/GLP-1 agonist polypeptide is inserted into the vector in proper reading frame. The expression vector is then transfected into a suitable host cell that will express the GIP/GLP-1 agonist polypeptide. Suitable host cells include without limitation bacteria, yeast, or mammalian cells. A variety of commercially available host-expression vector systems can be utilized to express the GIP/GLP-1 agonist polypeptides described herein.

[0120] The recombinant expression of a GIP/GLP-1 agonist polypeptide, derivative, analog or fragment thereof as described herein can be accomplished through the construction of an expression vector containing a polynucleotide that encodes the polypeptide. Once a polynucleotide encoding the GIP/GLP-1 agonist polypeptide has been obtained, the vector for the production of the polypeptide can be produced by recombinant DNA technology using techniques well known in the art.

[0121] Thus, methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding a GIP/GLP-1 agonist polypeptide are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, replicable vectors are provided that comprise a nucleotide sequence encoding a GIP/GLP-1 agonist polypeptide, operably linked to a promoter.

[0122] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce a GIP/GLP- 1 agonist polypeptide. Thus, host cells are provided that contain a polynucleotide encoding a GIP/GLP-1 agonist polypeptide, operably linked to a heterologous promoter.

[0123] A variety of host-expression vector systems can be utilized to express a GIP/GLP-

1 agonist polypeptide. Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express a polypeptide comprising a GIP/GLP-1 agonist polypeptide in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a sequence or sequences encoding a GIP/GLP-1 agonist polypeptide; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing a sequence or sequences encoding a GIP/GLP-1 agonist polypeptide; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a sequence or sequences encoding a GIP/GLP-1 agonist polypeptide; plant cell systems infected with recombinant virus expression vectors (e.g. , cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. , Ti plasmid) containing a sequence or sequences encoding a GIP/GLP-1 agonist polypeptide; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells or from mammalian viruses.

[0124] A host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g. , cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO, CRL7030 and HsS78Bst cells.

[0125] For long-term, high-yield production of recombinant proteins, stable expression is often preferred. For example, cell lines that stably express a GIP/GLP-1 agonist polypeptide can be engineered using methods known in the art.

[0126] Once a GIP/GLP- 1 agonist polypeptide has been produced by recombinant expression, it can be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g. , ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0127] Alternatively, a GIP/GLP-1 agonist polypeptides provided herein can be chemically synthesized by methods well known to those of ordinary skill in the art, e.g., by solid phase synthesis as described by Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Solid phase peptide synthesis can be accomplished, e.g. , by using automated synthesizers, using standard reagents. [0128] A GIP/GLP-1 agonist polypeptide can be characterized in a variety of ways. In particular, a GIP/GLP-1 agonist polypeptide can be assayed for potency in a cAMP assay as described elsewhere herein.

[0129] Modifications, Conjugates, Fusions, and Derivations. In certain aspects,

GIP/GLP-1 agonist polypeptides provided herein are stabilized via amino acid modifications. In certain aspects, the carboxyl group of the C-terminal amino acid is amidated. In certain aspects, the C-terminal amino acid is amidated glycine. In certain aspects the C-terminal glycine is the unmodified acid. In certain aspects, GIP/GLP-1 agonist polypeptides are provided in which one or more amino acid residues are acylated. For example, in certain aspects GIP/GLP-1 agonist polypeptides provided herein contain one or more lysine residues, in which a palmitoyl moiety is attached to the N(epsilon) group. In certain aspects a linker is incorporated between lysine and the palmitoyl group. This linker can be a gamma glutamic acid group, or an alternative linker such as, but not limited to, beta alanine and aminohexanoic acid. Different acylation methods may be used such as addition of cholesterol or myristoyl groups.

[0130] A GIP/GLP-1 agonist polypeptide as disclosed herein can be associated with a heterologous moiety, e.g., to extend half-life. The heterologous moiety can be a protein, a peptide, a protein domain, a linker, an organic polymer, an inorganic polymer, a polyethylene glycol (PEG), biotin, an albumin, a human serum albumin (HSA), a HSA FcRn binding portion, an antibody, a domain of an antibody, an antibody fragment, a single chain antibody, a domain antibody, an albumin binding domain, an enzyme, a ligand, a receptor, a binding peptide, a non-FnIII scaffold, an epitope tag, a recombinant polypeptide polymer, a cytokine, and a combination of two or more of such moieties.

[0131] For example, a GIP/GLP-1 agonist polypeptide can be fused with a heterologous polypeptide, e.g., a linker, a hinge, an Fc, or a combination thereof, as described above or in additional ways. The peptides can be fused to proteins, either through recombinant gene fusion and expression or by chemical conjugation. Proteins that are suitable as partners for fusion include, without limitation, serum albumin, e.g., human serum albumin, antibodies and antibody fragments including fusion to the Fc portion of the antibodies (as described above). GLP-1 has been fused to these proteins with retention of potency (L. Baggio et a.l, Diabetes 53:2492-2500 (2004); P. Barrington et al.,. Diabetes, Obesity and Metabolism 73:426-433 (2011); P. Paulik et al, American Diabetes Association 2012, Poster 1946). Extended recombinant peptide sequences have also been described to give the peptide high molecular mass (V.Schellenberger et ah, Nature Biotechnol 27: 1186-1190 (2009). In certain aspects GIP/GLP- 1 agonist polypeptides are incorporated as the N-terminal part of a fusion protein, with the fusion partner, e.g., an Fc domain as described above, or an albumin domain, at the C-terminal end. GIP/GLP- 1 agonist polypeptides as described herein can also be fused to peptides or protein domains, such as 'Albudabs' that have affinity for human serum albumin (M.S. Dennis et ah, J Biol Chem 277:35035-35043 (2002); A. Walker et al, Protein Eng Design Selection 23:271-278 (2010)). Methods for fusing GIP/GLP- 1 agonist polypeptides as disclosed herein with a heterologous polypeptide, e.g., albumin or an Fc region, are well known to those of ordinary skill in the art.

[0132] Other heterologous moieties can be conjugated to GIP/GLP- 1 agonist polypeptides to further stabilize or increase half-life. For chemical fusion, certain aspects feature maintenance of a free N-terminus, but alternative points for derivatization can be made. A further alternative method is to derivatize the peptide with a large chemical moiety such as high molecular weight polyethylene glycol (PEG). A "pegylated GIP/GLP- 1 agonist polypeptide" has a PEG chain covalently bound thereto. Derivatization of GIP/GLP- 1 agonist polypeptides, e.g., pegylation, can be done at the lysine that is palmitoylated, or alternatively at a residue such as cysteine, that is substituted or incorporated by extension to allow derivatization. GIP/GLP- 1 agonist polypeptide formats above can be characterized in vitro and/or in vivo for relative potency and the balance between GLP-1 and GIP receptor activation.

[0133] The general term "polyethylene glycol chain" or "PEG chain", refers to mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH ₂ CH ₂ ) _n OH, where n is an integer of 3, 4, 5, 6, 7, 8, 9, or more. PEG chains include polymers of ethylene glycol with an average total molecular weight selected from the range of about 500 to about 40,000 Daltons. The average molecular weight of a PEG chain is indicated by a number. For example, PEG- 5,000 refers to polyethylene glycol chain having a total molecular weight average of about 5,000.

[0134] PEGylation can be carried out by any of the PEGylation reactions known in the art. See, e.g., Focus on Growth Factors, 3: 4-10, 1992 and European patent applications EP 0 154 316 and EP 0 401 384. PEGylation may be carried out using an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).

[0135] Methods for preparing a PEGylated GIP/GLP-1 agonist polypeptides generally include the steps of (a) reacting a GIP/GLP-1 agonist polypeptide or with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the molecule becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s).

Formulations and Pharmaceutical Compositions

[0136] In certain aspects, GIP/GLP-1 agonist polypeptides provided herein possess one or more criteria of acceptable solubility, ease in formulatability, plasma stability, and improved pharmacokinetic properties. In certain aspects, GIP/GLP-1 agonist polypeptides as disclosed are soluble in standard buffers over a broad pH range.

[0137] In certain aspects, GIP/GLP-1 agonist polypeptides as disclosed are acceptably stable against proteases in serum or plasma. Common degradation products of GIP or GLP-

1 include +1 products (acid) and the DPP IV-cleavage products. Cleavage products arise from the action of proteases, e.g., DPP IV in plasma. In certain aspects, GIP/GLP-1 agonist polypeptides as disclosed are remain stable in plasma at levels up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after 24, 36 hours, 48 hours, or more hours in plasma at 37°C.

[0138] Further provided are compositions, e.g., pharmaceutical compositions, that contain an effective amount of a GIP/GLP-1 agonist polypeptide as provided herein, formulated for the treatment of metabolic diseases, e.g., obesity.

[0139] Compositions of the disclosure can be formulated according to known methods.

Suitable preparation methods are described, for example, in Remington's Pharmaceutical Sciences, 19th Edition, A.R. Gennaro, ed., Mack Publishing Co., Easton, PA (1995), which is incorporated herein by reference in its entirety. Composition can be in a variety of forms, including, but not limited to an aqueous solution, an emulsion, a gel, a suspension, lyophilized form, or any other form known in the art. In addition, the composition can contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. Once formulated, compositions of the invention can be administered directly to the subject.

[0140] Carriers that can be used with compositions of the invention are well known in the art, and include, without limitation, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, and polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. Compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered. A resulting composition can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. Compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

Method of treating hypoglycemic conditions, e.g., type-2 diabetes, model systems.

[0141] GIP/GLP-1 agonist polypeptides can combine the effect of GIP and GLP-1 to provide one or more of prevention or modulation of hyperglycemia, promotion of insulin synthesis, inhibition of glucagon synthesis, an increase in β-cell mass, weight loss or weight maintenance (e.g., prevention of weight gain), reduction in food intake, modulation of gastric acid secretion, or modulation of gastric emptying.

[0142] This disclosure provides a method of treating a hypoglycemic condition, e.g., type-2 diabetes, comprising administering to a subject in need of treatment a GIP/GLP-1 agonist polypeptide as disclosed herein. Further provided is a GIP/GLP-1 agonist polypeptide for treatment of a hypoglycemic condition, e.g., type-2 diabetes. Further provided is use of a GIP/GLP-1 agonist polypeptide as provided herein in the manufacture of a medicament for the treatment of a hypoglycemic condition, e.g., type-2 diabetes.

[0143] GIP/GLP-1 agonist polypeptides provided herein can be administered for glycemic control, promoting insulin production, promoting β-cell mass, promoting weight loss, or reducing excess body weight. In addition, GIP/GLP-1 agonist polypeptides provided herein can be used for treatment of related disorders. Examples of related disorders include without limitation: insulin resistance, glucose intolerance, pre-diabetes, increased fasting glucose, hypertension, dyslipidemia (or a combination of these metabolic risk factors), glucagonomas, cardiovascular diseases such as congestive heart failure, atherosclerois, arteriosclerosis, coronary heart disease, or peripheral artery disease, stroke, respiratory dysfunction, or renal disease.

[0144] "Treatment" is an approach for obtaining beneficial or desired clinical results. As provided herein, beneficial or desired clinical results from the disclosed GIP/GLP- 1 agonist polypeptides include, without limitation, stabilized serum glucose and serum insulin levels, increased β-cell mass, or amelioration, palliation, stabilization, diminishment of weight gain. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures in certain aspects. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. By treatment is meant improved glycemic control in type-2 diabetes, and is not necessarily meant to imply complete cure of the relevant condition.

[0145] The route of administration of GIP/GLP-1 agonist polypeptides provided herein can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Another example of a form for administration is a solution for injection, in particular for intravenous or intraarterial injection or drip. GIP/GLP- 1 agonist polypeptides provided herein can be administered as a single dose or as multiple doses. In certain aspects, a GIP/GLP- 1 agonist polypeptide is administered orally or by subcutaneous injection.

[0146] Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed with a maintenance dose. These compositions can be administered at specific fixed or variable intervals, e.g., once a day, or on an "as needed" basis, e.g., based on patient- initiated blood glucose measurements. Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0147] The amount of a GIP/GLP-1 agonist polypeptide to be administered can be readily determined by one of ordinary skill in the art without undue experimentation given the disclosure herein. Factors influencing the mode of administration and the respective amount of a GIP/GLP-1 agonist polypeptide include, but are not limited to, the severity of the disease (e.g. , the extent of obesity), the subject's history, and the age, height, weight, health, and physical condition of the subject undergoing therapy. Similarly, the amount of a GIP/GLP-1 agonist polypeptide to be administered will be dependent upon the mode of administration and whether the subject will undergo a single dose or multiple doses of this agent. In certain aspects, GIP/GLP-1 agonist polypeptides provided herein can be administered once per day via injection.

[0148] In certain aspects a GIP/GLP1 agonist polypeptide as provided herein can be administered in combination with one or more additional therapies. The additional therapy can include one or more existing standard therapies type-2 diabetes or other hypoglycemic condition, or new therapies. In certain aspects, the one or more additional therapies can include, without limitation, blood sugar monitoring, diet modifications, exercise, insulin, a thiazolidinedione, a sulfonylurea, an incretin, metformin, a glyburide, a dipeptidyl peptidase 4 inhibitor, a bile acid sequestrant, or any combination thereof.

KITS

[0149] In yet other aspects, the present disclosure provides kits comprising GIP/GLP-1 agonist polypeptides, which can be used to perform the methods described herein. In certain aspects, a kit comprises a GIP/GLP-1 agonist polypeptide disclosed herein in one or more containers. A kit as provided herein can contain additional compositions for combination therapies. One skilled in the art will readily recognize that the disclosed GIP/GLP- 1 agonist polypeptides can be readily incorporated into one of the established kit formats that are well known in the art.

Examples

Example 1: Synthesis, modifications, and characterization of GIP/GLP-1 agonist

polypeptides

[0150] Dual-active GIP/GLP-1 agonist polypeptides were prepared according to the following methods. GIP/GLP dual agonist peptides were designed by starting with various different backbone peptides, including GLP-1, GIP, and Exendin-4. Rational substitutions were made to allow specificity at GLP-1 r and GIPr. Alternatively, the backbone peptides were subjected to random mutagenesis, expressed in phage, and selected based on binding to the various receptors.

[0151] A large number of peptides were produced, and were expressed as a fusion protein, extending from N-terminus to C-terminus: peptide-linker-hinge-Fc (CH2 and CH3 regions, from either IgGl or IgG4, with various mutations to improve stability and reduce effector functions, such as IgGl-TM, or IgGl-FQQ, as described elsewhere herein). Such a construct will form a dimer through disulfide linkages in the hinge region. Various peptide- linker-hinge-Fc combinations were constructed as shown in Tables 7-10. An exemplary bivalent GIP/GLP-1 agonist polypeptide composition is shown in FIG. 1.

[0152] The various GIP/GLP-1 agonist polypeptides were constructed and expressed in

CHO cells by standard methods using the following procedures. CEP6 cells (Medlmmune) were maintained in CD-CHO medium (Invitrogen) containing 100 mg/1 Hygromycin B and 25mg/l MSX. Cells were received one day prior to transfection at approximately 1.0 x 10 ⁶ viable cells/ml, having been passaged twice without Hygromycin B selection prior to delivery. The cells generally double in number after 24 hours reaching the required 2.0 x 10 ⁶ viable cells/ml for use on the day of transfection.

[0153] Aliquots of 30 ml of CEP6 cells at 2.0 x 10 ⁶ viable cells/ml were transferred into

125 ml flasks. Place The flasks were placed in a 37°C shaking incubator set to 140 rpm with 5% C0 ₂ and 80% humidity.

[0154] DNA was prepared by adding a total of 20 μg (total) of plasmid DNA to a 1.5 ml Eppendorf tube. The volume in each tube was made up to a total of 500 μΐ with 150 mM sodium chloride solution. The PEI was prepared by adding a 200 μΐ volume of PEI Max working solution (1 mg/ml) to a 1.5 ml Eppendorf tube. The volume in each tube was made up to a total of 500 μΐ with 150 mM sodium chloride solution. The PEI Max was added to the DNA and vortexed for 10 sec (DNA:PEI Max ratio 1: 10). The PEI Max/DNA complex was incubated at room temperature for 1 min, and then added to the cells.

[0155] Each flask was incubated in a 37°C shaking incubator set to 140 rpm with 5% C0 ₂ and 80% humidity. After a minimum of 4 hours, a 9 ml volume of feed (30% initial volume) was added.

[0156] The cultures were incubated for seven days in a 34°C shaking incubator set to 140 rpm with 5% C0 ₂ and 80% humidity. [0157] Following incubation, the CEP6 cells were pelleted by transferring the content of the flasks to 50 ml Falcon tubes and centrifuging at 1500 rpm for 20 min. The cells were harvested from the flasks by filtering the supernatant through a 0.22 μιη Steriflip filter.

[0158] Proteins were purified using the AKTAxpress by affinity chromatography with

MabSelectSuRe columns, where the Fc region of the protein binds to the column matrix. Columns were equilibrated in lx DPBS, (Gibco, Invitrogen. Cat No: 14190-094). During sample loading the protein binds to the column, with the unbound sample being washed. Elution of the bound material was done with 0.1M Sodium citrate, pH3 into collection blocks containing one fifth vol. 1M Tris-HCl, pH 9.0.

[0159] Finally, purified protein was buffer exchanged on PD10 columns equilibrated with

PBS.

Example 2: GIP and GLP-1 receptor mediated cAMP production

[0160] Biological activity of peptides in cell-based cAMP activity assay. The biological activity of GIP/GLP-1 agonist polypeptides produced by the method of Example 1 were tested for biological activity, e.g., stimulation of one or more cellular receptor responses, by the following methods. Stable cell lines expressing human, mouse, or rat GLP-1 receptor (GLP-lr), GIP receptor (GIPr) or glucagon receptor (GCGr) were generated in HEK293s or CHO cells by standard methods. For human GIPr, two different lines were used, shown as "hGIPr" and "hGIPr (C3)" in Table 11. Peptide activation of these various receptors results in downstream production of cAMP second messenger that can be measured in a functional activity assay.

[0161] One basic cAMP assays was performed, differing in the medium used for the assay, the "BSA-cAMP assay":

BSA-based assay medium: 0.1% bovine serum albumin (BSA) in

Hanks Balanced Salt Solution (GIBCO or Sigma), containing 0.5mM IBMX

(Sigma # 17018);

In certain aspects, the assay can also be carried out in medium supplemented with serum albumin that corresponds to the receptor being tested, e.g., human serum albumin for testing cAMP activity at hGIPr or hGLP-lr, rat serum albumin for testing cAMP activity at rGIPr or rGLP-lr, and so on. Low protein binding 384- well plates (Greiner # 781280) were used to perform eleven 1 in 5 serial dilutions of test samples that were made in assay medium. All sample dilutions were made in duplicate.

[0162] A frozen cryo-vial of cells expressing the receptor of interest was thawed rapidly in a water-bath, transferred to pre-warmed assay media and spun at 240xg for 5 minutes. Cells were re-suspended in assay buffer at an optimized concentration (e.g., hGLP-lr and hGIPr cells at 0.5xl0 ⁵ cells /ml).

[0163] From the dilution plate, a 5μί replica was stamped onto a black shallow- well u- bottom 384-well plate (Corning # 3676). To this, 5^L cell suspension was added and the plates incubated at room temperature for 30 minutes.

[0164] cAMP levels were measured using a commercially available cAMP dynamic 2

HTRF kit (Cisbio, Cat # 62AM4PEJ), following the two step protocol as per manufacturer's recommendations. In brief; anti-cAMP cryptate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were made up separately by diluting each 1/20 in conjugate & lysis buffer provided in the kit. 5μί anti-cAMP cryptate was added to all wells of the assay plate, and 5^L cAMP-d2 added to all wells except non-specific binding (NSB) wells, to which conjugate and lysis buffer was added. Plates were incubated at room temperature for one hour and then read on an Envision (Perkin Elmer) using excitation wavelength of 320nm and emission wavelengths of 620nm & 665nm. Data was transformed to % Delta F as described in manufacturer's guidelines and then transformed to percent activation of maximal native agonist response and analysed by 4-parameter logistic fit to determine EC50 values.

[0165] EC50 values for the GIP/GLP-1 agonist polypeptides disclosed herein (see Tables

7 to 10) were determined in cAMP assays are shown in Table 11. The results are compared to corresponding results for native GIP, GLP-1, and glucagon. We used unconstrained 4 parameter logistic fit of data, curve mid-point to determine EC50. This was tested on two occasions, not two batches. Table 11: cAMP activity of GIP/GLP-1 agonist polypeptides

GLPGP-lOOWOl

GLPGP-lOOWOl

GLPGP-lOOWOl

GLPGP-lOOWOl

GLPGP-lOOWOl

GLPGP-lOOWOl

GLPGP-lOOWOl

GLPGP-lOOWOl

GLPGP-lOOWOl

GLPGP-lOOWOl

GLPGP-lOOWOl

Ident. hGLPlr hGCGr hGIPr hGIPr (C3) mGLPlr mGIPr rGLPlr rGIPr

STDEV STDEV STDEV STDEV STDEV STDEV STDEV STDEV

8.4E-09 4.6E-09 54E-12 7.0E-08 5.1E-12 9.6E-09 2.8E-12

IP335 >0.00000548 2.8E-07 5.8E-12 >2.74E-06 2.0E-11 >2.74E-06 6.7E-12

N=2 2.4E-08 2. IE- 12 N=2 3.7E-12 N=2 4.7E-13

IP336 >0.00000451 2.0E-07 5.0E-12 >2.26E-06 1.2E-11 >2.26E-06 4.7E-12

N=2 2.0E-08 3.7E-13 N=2 1.6E-12 N=2 7.1E-15

IP337 >0.0000023 2.0E-07 6.2E-12 >1.15E-06 1.3E-11 >1.15E-06 5.2E-12

N=2 3.2E-08 4.5E-12 N=2 4.4E-12 N=2 4.3E-13

IP338 >0.00000189 1.9E-07 5.2E-12 >9.45E-07 1.2E-11 >9.45E-07 5.0E-12

N=2 2.2E-08 2.6E-12 N=2 4.3E-12 N=2 1.1E-12

IP344 >0.00000855 2.0E-07 5.3E-12 >8.55E-06 l . lE-11 >8.55E-06 6.2E-12

N=2 1.6E-08 34E-13 N=2 6.4E-13 N=2 2.5E-12

IP351 1.1E-07 1.3E-07 3.6E-12 8.2E-07 9.8E-12 2.1E-07 6.3E-12

4.0E-08 1.2E-08 1.7E-12 2.6E-07 4.0E-12 14E-07 4. IE- 13

IP386 >0.00000913 5.4E-07 3.4E-12 >1.66E-05 3.0E-11 >1.66E-05 8.5E-12

1.1E-05 2.5E-08 1.7E-12 N=l l. lE-11 N=2 4.8E-12

IP387 3.7E-07 2.2E-07 4.5E-12 >1.47E-06 1.9E-11 1.1E-07 7.5E-12

#DIV/0! #DIV/0! 2.7E-12 N=l 8.5E-12 2.0E-08 4.9E-12

IP388 >0.0000052745 2.7E-07 3.0E-12 >9.59E-06 1.9E-11 >9.59E-06 6.6E-12

6.1E-06 5.8E-08 5.7E-13 N=l 4.7E-12 N=2 3.4E-12

IP389 6.5E-08 >0.000000269 2.7E-12 1.3E-06 1.7E-11 9.9E-09 7.2E-12

3.4E-08 4.4E-08 1.3E-13 N=l 8.5E-13 8.3E-10 4. IE- 12

IP392 2.8E-06 5.3E-07 2.6E-12 >1.71E-05 3.1E-11 7.2E-07 7.6E-12

#DIV/0! 3.8E-08 2.3E-13 N=l 1.3E-11 4.9E-08 5.7E-12

IP398 9.5E-06 1.6E-06 3.7E-12 >1.72E-05 1.2E-11 1.4E-05 5.6E-12

1.1E-05 1.5E-07 1.7E-12 N=l 9.9E-13 2.8E-07 2.2E-13

IP402 3.1E-08 2.0E-07 3.8E-11 8.5E-07 3.3E-11 1.1E-08 6.8E-12

4.6E-09 1.1E-07 4.9E-11 3.5E-07 2.5E-11 3.8E-09 2.2E-12

IP419 >0.0000000259 >0.0000000259 1.2E-11

N=2 N=2 6. IE- 12

Blank fields in human assays means "not active," except column hGIPr (C3) wherein blank fields mean "not tested";

Blank fields in rodent assays means "not tested."

Where no "Batch" number is indicated, Batch "1" was tested.

Example 3: In vitro Characterization ofIP088

[0166] Certain of the GIP/GLP-1 agonist peptides tested were evaluated for further studies. Desirable properties included, without limitation, high potency, e.g., picomolar range EC50s for both human GIPr and human GLP-lr, EC50s for the human glucagon receptor (GCGr) of much lower potency, e.g., at least 100-fold, 1000-fold, or more lower potency for GCGr (e.g., in the micromolar range), and, to allow for in vivo studies in rodent models, high potency, e.g., picomolar-to-nanomolar range EC50s for either mouse or rat GIPr and GLP-lr. The GIP/GLP-1 dual agonist polypeptide IP088 was chosen for further analysis. FIG 2A shows an alignment if IP088 with Exendin-4, hGLP-1, and hGIP, as well as GLPla-Fc.

[0167] The potency of IP088 on hGLP-lr and hGIPr in comparison with hGLP-1 and hGIP is shown in FIGs 2B and 2C. Potency curves for the GLPla-Fc are also shown for comparison.

[0168] Preliminary pharmacokinetics in mouse was examined as follows: C57 B1/6J mice were injected with 1 mg/kg (subcutaneously) with either IP088 or GLPla-Fc, and serum samples were taken over time (3 animals per time point). The serum samples were then measured for activity at the hGLP-lr by the cAMP assay in HBSS/HEPES 0.1% BSA, as described above. The results are shown in FIG. 2D. The results show that the GIP/GLP-1 agonist polypeptide IP088 is stable, and maintains its activity in the nanomolar range for at least 48 hours in vivo.

[0169] More extended pharmacokinetic assays to determine protein stability and activity were performed as follows. CD male rats (n=3) were injected subcutaneously or intravenously with lmg/kg IP088 or GLPla-Fc. Periodic serum samples were obtained out to 168 hours. The serum samples were subjected to ELISA for protein (Fc) detection by the following method. ELISA plates (Costar 3690) were coated with Mouse anti-human IgG (JCD-10) 1/500 in PBS (final concentration 1 μ^πύ). 50 μΐ of diluted serum was added to each well and the plates were incubated overnight at 4°C. Following washing with PBS-Tween (PBS-T), the plates were blocked with 120 μΐ 1% BSA in PBS for 1 h at RT with gentle shaking. Following washing with PBS-T, standards, and dilutions of mouse serum samples were added (50 μΐ/well), and the plate was incubated for 2 h at RT with gentle shaking. Following washing 50 μΐ biotinylated mouse anti-human IgG (JCD-10) diluted 1/1000 in ELISA incubation buffer (final concentration 0.5 μ§/ηι1) was added to each well and the plates were incubated for 1 h at RT with gentle shaking. Following washing 50 μΐ of Streptavidin-HRP diluted 1/1000 in ELISA incubation buffer was added to each well, and the plates were incubated for 45 min at RT with gentle shaking. Following a final washing, 50 μΐ/well of TMB solution (Invitrogen #00-2023) was added to each well and the plates were incubate in the dark until desired blue color appeared (5-10 min). The reaction was stopped by addition of 50 μΐ of stop solution (1 M HC1). The results are plotted in FIG. 3A and FIG. 3B. IP088 and GLPla-Fc had equivalent PK profiles, as shown in FIG. 3C.

[0170] The serum samples obtained were also tested for potency against hGLP-lr by the cAMP assay as described above in HBSS/HEPES 0.1% BSA. The results are plotted in FIG. 3D and FIG. 3E. IP088 and GLPla-Fc had similar PK activity profiles, as shown in FIG. 3F.

[0171] In order to be able to carry out in vivo studies in rodents, IP088 was tested for potency against the mouse receptors mGIPr and mGLP-lr as well as the rat receptors rGIPr and rGLP-lr, using cells expressing the respective receptors in the cAMP assay described above. The relative potencies expressed as EC50s are shown in Table 12. IP088 showed high potency across species for GLP-lr and showed high potencies for human and rat GIPr, but showed relatively lower potency for mouse GIPr.

Table 12: Relative Potencies of IP088 against Human, Rat, and Mouse GLP-1 and

GIP Receptors

[0172] Further potency assays were carried out using the GLP-1 receptor from the cynomolgus monkey, using the cAMP assay described above, the assay was carried out with cells expressing cyno GLP-lr or cyno GCGr using culture medium containing 4.2% cyno serum albumin (Equitech Bio Ltd.). The results are shown in Table 13. Table 13: Potencies of IP088 against Cynomolgus Monkey GLP-1 and Glucagon

Receptors

Example 3: Oral Glucose Tolerance Test in Mouse Model

[0173] The effect of IP088 on glycemic control in a mouse model was carried out as follows. Twenty-one (21) male C57 male mice (11-12 weeks old) were purchased from Taconic, Denmark and acclimatized for 3 weeks before experimentation. Throughout the acclimatization period the animals are offered ad libitum chow and tap water. Mice were single housed one week prior to the study. On day 1 of the study animals were randomized into 4 groups based on body weight (n=5-6 per group), and were administered the following compounds subcutaneously four hours (IP088, IP040, and GLPla-Fc) or two hours (liraglutide) prior to each oral glucose tolerance test:

Group 5: IP040 (vehicle)

Group 6: Liraglutide (GLP-1 analog, Novo Nordisk) 0.2mg/kg S.C.

Group 7: GLPla-Fc (GLP-1 agonist) lmg/kg S.C.

Group 8: IP088 lmg/kg S.C.

[0174] A schematic summary of the study is shown in FIG. 4A. Oral glucose tolerance tests (OGTT) were performed on days 0 and 2, both after a 4-hour fast. At t = 0 the mice were administered an oral glucose load of 2g/kg glucose (Glucose 250m/ml, Fresenius Kabi, Sweden). Glucose was given as gavage via a gastrically placed tube connected to a syringe ensuring accurate dosing. Blood glucose was measured from tail vein blood before glucose (at t = -240, -120 and 0 min, baseline) and after glucose administration at t = 15, 30, 60 and 120 min. Blood samples (tail vein) were collected into 10 μΐ heparinized glass capillary tubes and immediately suspended in buffer (0.5ml of glucose/lactate system solution (EKF- diagnostics, Germany) and analyzed for glucose on the test day using a BIOSEN c-Line glucose meter (EKF-diagnostics, Germany) according to manufacturer's instructions.

[0175] The results are shown in FIG. 4B (day 0 OGTT) and 4C (day 2 OGTT). While this study could not distinguish the relative contribution of GLP-1 and GIP agonism, IP088 showed excellent glucose lowering effects, and was superior to GLPla-Fc at glycemic control.

[0176] The disclosure is not to be limited in scope by the specific aspects described, which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods that are functionally equivalent are within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0177] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.