WO2016131893A1 - Polypeptides de fusion de l'incrétine - Google Patents

Polypeptides de fusion de l'incrétine Download PDF

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Publication number
WO2016131893A1
WO2016131893A1 PCT/EP2016/053400 EP2016053400W WO2016131893A1 WO 2016131893 A1 WO2016131893 A1 WO 2016131893A1 EP 2016053400 W EP2016053400 W EP 2016053400W WO 2016131893 A1 WO2016131893 A1 WO 2016131893A1
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WIPO (PCT)
Prior art keywords
seq
polypeptide
modified
lipid
amino acid
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PCT/EP2016/053400
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English (en)
Inventor
Isabelle SERMADIRAS
Peter Ravn
Maria A BEDNAREK
Arthur Suckow
Monika Papworth
Elise Bernard
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Medimmune Limited
Medimmune, Llc
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Publication of WO2016131893A1 publication Critical patent/WO2016131893A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • 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.
  • 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.
  • GLP glucagon-like peptide
  • Glucagon-like peptide- 1 derives from pre-pro glucagon, 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 glucagon-like peptide-1
  • GLP-2 glucagon- like peptide-2
  • OXM oxyntomodulin
  • 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-1 (7-36) amide or GLP-1 (7-37) acid are biologically active forms of GLP- 1, that demonstrate essentially equivalent activity at the GLP-1 receptor.
  • 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.
  • GLP-1 and GLP-1 analogues acting as agonists at the GLP-1 receptor, have been shown to be effective hypoglycemic control, e.g., type-2 diabetes.
  • Certain GLP-1 analogues 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).
  • 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 ⁇ -cell mass, and a decrease in gastric acid secretion.
  • GIP glucose-dependent insulinotropic polypeptide
  • Xi X 2 E G X 5 X 6 X 7 S X9 Xio S I L X 14 X15 X 16 X 1 A I X 20 X 21 F X 23 X 24 X 25 L X 27 X 28 X 29 X 30 (SEQ ID NO: 1); wherein Xi is Y or H; X 2 is S or G; X5 is T or M; X 6 is F or H; X 7 is T or I; X9 is D or L; Xio is L or Y; X 14 is K, L, or R; X15 is E or D; X 16 is R, E, K, or I; X 17 is E or Q; X 20 is D or E; X 21 is E, L, or A; X 23 is I, V or A; X 24 is A, E, or N; X 25 is W, D, E, L, F, I, S, N, G, M, H, K, or R; X 27
  • Xi X 2 E G X5 X 6 X 7 S X 9 Xio Xn X 12 X 13 X 14 X 15 X1 ⁇ 2 X17 A I X20 E F V N X 25 L X 27 X 28 X 29 X 30 (SEQ ID NO: 2); wherein Xi is Y or H; X 2 is S or G; X5 is T or M; X 6 is F or H; X 7 is T or I; X 9 is D or L; Xio is L or Y; Xn is S, A, or R, X 12 is S, K, I, A, M, or Q; X 13 is Y, L, A, or H; X 14 is K, L, or R; X 15 is E or D; X 16 is R, E, K, or I; X 17 is E or Q; X 20 is D or E; X 25 is W, D, E, L, F, I, S, N, G,
  • X 12 is I or K. In some embodiments, X 12 is I. In some embodiments, X 13 is L. In certain embodiments of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2, X 10 is L. In some embodiments, X 14 is K. In some embodiments, X 20 is D.
  • YSEGTFTSDLSILKE X16 X17 AIDEFVNWLLKGG (SEQ ID NO: 461); wherein X16 is I, M, K, S, R, or T and X17 is Q, H, N, K, D or Y.
  • X16 is M and X17 is H, X16 is K and X17 is N, X16 is S and X17 is Q, or X16 is I and X 17 is K. In certain other embodiments of SEQ ID NO: 461, X16 is I and X17 is Q, X16 is R and X17 is D, or X16 is T and X17 is Y.
  • YSEGTFTSDLSIL X 14 ERQAIDEFVNWLLKGG (SEQ ID NO: 3); wherein X 14 is L, K, or R.
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 276 (combo 0062), SEQ ID NO: 285 (combo 0094), SEQ ID NO: 291 (combo 0095), SEQ ID NO: 295 (combo 0154), SEQ ID NO: 302 (combo 0099), SEQ ID NO: 303 (combo 0101), SEQ ID NO: 304 (combo 0104), SEQ ID NO: 9 (IP0574_pm), SEQ ID NO: 10 (combo0005_pm), and SEQ ID NO: 15 (combo0011_pm).
  • X10 is L.
  • X 14 is R.
  • X 17 is Q.
  • X 17 is E.
  • X 14 is R, and X 17 is Q or E.
  • X 20 is D.
  • X 10 is L.
  • X 12 is I or K.
  • X 13 is L.
  • X 14 is R.
  • X 17 is Q.
  • X 17 is E.
  • X 14 is R and X 17 is Q or E.
  • X 20 is D.
  • a polypeptide of SEQ ID NO: 459 or SEQ ID NO: 460 further comprises the amino acid sequence PSSGA PPPX (SEQ ID NO: 477) fused to the C Terminus of the polypeptide, wherein X is no amino acid, any natural amino acid, a lipid-modified K, or a maleimide-modified K.
  • X is S (SEQ ID NO: 146) or G (SEQ ID NO: 479).
  • the polypeptide is produced synthetically.
  • the unnatural amino acid of X 2 is Aib.
  • the polypeptide further comprises a lipid-modified K at the C terminus of the polypeptide.
  • one or two of X 10 , X 12 , X 13 , X 14 , X 17 , X 18 , X 2 o, X 2 i, X 2 4, X 2 5, or the C terminus is a lipid-modified K.
  • one or two of X 10 , Xi2, X21, or the C terminus is a lipid-modified K.
  • the lipid-modified K comprises a gamma glutamine ( ⁇ ) and/or a PEG 4 linker.
  • the lipid is selected from the group consisting of a nonanoic (C9), decanoic (CIO), undecanoic (Cl l), lauric (C12), tridecanoic (C13), myristic (C14), pentadecanoic (C15), palmitic (C16), heptadecanoic (C17), stearic (C18), nonadecanoic (C19), and any combination thereof.
  • the lipid is selected from the group consisting of a undecanoic (Cl l), lauric (C12), tridecanoic (C13), myristic (C14), pentadecanoic (C15), palmitic (C16) heptadecanoic (C17), stearic (C18), nonadecanoic (CI 9), and any combination thereof.
  • the lipid is palmitic (C16).
  • the lipid-modified K is K(yE-palm), K(PEG 4 -palm), or PEG 4 - K(yE-palm).
  • an unnatural amino acid is selected from the group consisting of Aib, aMeGlu, aMePhe, aMeLeu, Nle, and any combination thereof.
  • the polypeptide comprises at least 2, at least 3, or at least 4 unnatural amino acids.
  • X 13 is aMeLeu.
  • X 6 is Nle
  • X n is Aib
  • X 17 is Aib
  • X 18 is Aib
  • X 27 is Nle.
  • X 13 is a lipid-modified K
  • X 22 is aMePhe
  • X 2 5 is aMePhe.
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 309 (gl681), SEQ ID NO: 310 (gl711), SEQ ID NO: 311 (gl677), SEQ ID NO: 313 (gl685), SEQ ID NO: 315 (gl734), SEQ ID NO: 378 (gl931), SEQ ID NO: 320 (gl896), SEQ ID NO: 322 (g2017), SEQ ID NO: 323 (gl 897), SEQ ID NO: 324 (g2018), SEQ ID NO: 326 (g2020), SEQ ID NO: 327 (g2021), SEQ ID NO: 328 (g2022), SEQ ID NO: 330 (gl898), SEQ ID NO: 331 (g2024), SEQ ID NO: 334 (gl 899), SEQ ID NO: 335 (g2286), SEQ ID NO: 342 (gl577), SEQ ID NO: 345 (gl72)
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 311 (gl677), SEQ ID NO: 345 (gl721), SEQ ID NO: 315 (gl734), SEQ ID NO: 415 (gl897), SEQ ID NO: 331 (g2024), and SEQ ID NO: 350 (g2029).
  • the polypeptide is a synthetically produced polypeptide conjugated to a heterologous moiety selected from the group consisting of a lipid moiety, 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.
  • a heterologous moiety selected from the group consisting of a lipid moiety, an organic polymer, an inorganic polymer, a polyethylene glycol (PEG), biotin, an albumin, a human serum albumin (HSA),
  • the heterologous moiety is an Fc domain.
  • the synthetically produced polypeptide is conjugated to the Fc domain via a free cysteine.
  • the synthetically produced polypeptide is conjugated at position 239C, 268C, or 442C of the Fc domain.
  • the synthetically produced polypeptide is conjugated to the Fc domain via a maleimide group.
  • the polypeptide comprises a maleimide-modified K and in certain embodiments, one of X 24 , X 2 8, or the C terminus is a maleimide-modified K conjugated to the Fc domain.
  • the maleimide-modified K comprises a PEG 4 linker.
  • the polypeptide is lipidated and is conjugated to the Fc domain.
  • X 24 or the C terminus is a maleimide-modified K conjugated to an Fc domain and Xio is a palmitoylated K.
  • the C terminus is a maleimide-modified K conjugated to an Fc domain and X 12 is a palmitoylated K.
  • X 28 or the C terminus is a maleimide-modified K conjugated to an Fc domain and X 13 is a palmitoylated K.
  • the palmitoylated K is K(yE-palm).
  • X 24 or the C terminus is a maleimide- modified K conjugated to an Fc domain and one of X 10 , X 12 , X 21 , or X 24 is K(PEG 2 -PEG 2 -stear).
  • the C terminus is a maleimide-modified K conjugated to an Fc domain and one of X 10 , X 12 , or X 21 , is K(PEG 2 -PEG 2 -stear).
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 436 (combo0051), SEQ ID NO: 437 (combo0052), SEQ ID NO: 438 (combo077), SEQ ID NO: 439 (combo0112), SEQ ID NO: 443 (combo0211), SEQ ID NO: 444 (combo0212), SEQ ID NO: 445 (combo0213), SEQ ID NO: 446 (combo0214), SEQ ID NO: 447 (combo0215), and SEQ ID NO: 452 (combo0121).
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 437 (combo0052).
  • the polypeptide can further comprise a heterologous moiety attached thereto.
  • the heterologous moiety comprises a heterologous polypeptide that is fused thereto via a peptide bond.
  • the heterologous polypeptide comprises a linker, a hinge, an Fc domain, or a combination thereof.
  • the linker comprises (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the linker comprises the amino acid sequence: G GGGGS GGGGS GGGGS GGGGS A (SEQ ID NO: 111), A PPGGS GGGGS GGGGS A (SEQ ID NO: 112), GT GGGGS GGGGS GGGGS A (SEQ ID NO: 113), G GGGGS GGGGS GGGGS A (SEQ ID NO: 114), G GGGGS A (SEQ ID NO: 115), G GGGGS GGGGS A (SEQ ID NO: 116), G GGGGS GGGGS GGGGS A (SEQ ID NO: 117), G KGGGS GGGGS GGGGS GGGGS A (SEQ ID NO: 118), G GGGGS GGGGS GGGGS A (SEQ ID NO: 119), G GGGGGGGGGGGGGGGG A (SEQ ID NO: 120), GG SGSTA SSGSG SATGG GGAA (SEQ ID NO: 197), AAAGG SGSTA SSG
  • the linker comprises AAA GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 121), AAA PAPAP APAPA PAPAP APAPA G (SEQ ID NO: 122), AAA GGKGG GGKGG GGKGG GGKGG G (SEQ ID NO: 474), AAA GKGKGKGKGK GKGKGKGKGKGKGK G (SEQ ID NO: 123), any variant thereof, or any fragment thereof.
  • the linker comprises AAA GGGGS GGGGS GGGGS (SEQ ID NO: 121).
  • the linker comprises (PA) n and wherein n is between 1 and 20. In certain embodiments, n is between 5 and 15.
  • the linker comprises AAA PAPAP APAPA PAPAP APAPA G (SEQ ID NO: 122).
  • the hinge comprises an amino acid sequence of an IgGl hinge, an IgG4 hinge, a fragment thereof, a variant thereof, or any combination thereof.
  • the hinge comprises ESKYGPPCPPCPAPEAA (SEQ ID NO: 124), THTCPPCPAPEF (SEQ ID NO: 125), THTCPPC (SEQ ID NO: 126), CPPCPAPEF (SEQ ID NO: 127), TYTCPPCPAPEF (SEQ ID NO: 128), TSTCPPCPAPEF (SEQ ID NO: 129), PPCPPCPAPEF (SEQ ID NO: 130), ESKYGPPCPPCPAPEF (SEQ ID NO: 131), APEF (SEQ ID NO: 132), ESKYGPPCPPC (SEQ ID NO: 133), THTCPPCPAPELL (SEQ ID NO: 134), CPPC (SEQ ID NO: 135), any variant thereof, any fragment thereof, or any combination thereof.
  • the hinge comprises ESKYGPPCPPCPAPEAA (S
  • the Fc region comprises an IgGl Fc region, an IgGl-TM 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.
  • the polypeptide comprises a TrpCage sequence.
  • TrpCage sequence is PSSGA PPPS (SEQ ID NO: 146) or PSSGA PPPG (SEQ ID NO: 479).
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 302 (combo0099), SEQ ID NO: 303 (comboOlOl), SEQ ID NO: 304 (combo0104), SEQ ID NO: 295 (combo0154), SEQ ID NO: 299 (combo0102), SEQ ID NO: 300 (combo0105), SEQ ID NO: 291 (combo0095), SEQ ID NO: 285 (combo0094), SEQ ID NO: 61 (IP0574), SEQ ID NO: 62 (combo0005), SEQ ID NO: 67 (comboOOl l), SEQ ID NO: 108 (IP0644), SEQ ID NO: 109 (IP0646), and SEQ ID NO: 110 (combo0062).
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 302 (combo0099) and SEQ ID NO: 303 (comboOlOl).
  • the heterologous moiety is a lipid moiety, 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.
  • PEG polyethylene glycol
  • HSA human serum albumin
  • 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,
  • the heterologous moiety is polyethylene glycol (PEG). In certain embodiments, the heterologous moiety is a lipid moiety. In certain embodiments, the heterologous moiety comprises a polyethylene glycol (PEG) attached to a lipid moiety. In certain embodiments, the heterologous moiety is attached via a lysine or a cysteine residue.
  • a polypeptide as provided herein can bind to a GIP receptor, bind to a GLP-1 receptor, or bind to both a GIP receptor and a GLP-1 receptor.
  • the polypeptide binds to a GIP receptor.
  • the GIP receptor is a mouse GIP receptor, a rat GIP receptor, a non-human primate GIP receptor, or a human GIP receptor.
  • the polypeptide binds to a GLP-1 receptor.
  • the GLP-1 receptor is a mouse GLP-1 receptor, a rat GLP-1 receptor, a non-human primate GLP-1 receptor, or a human GLP-1 receptor.
  • the polypeptide is an agonist of a GLP-1 receptor, an agonist of a GIP receptor, or an agonist of both a GLP-1 and a GIP receptor.
  • the polypeptide is an agonist of a human GIP or 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, less than 2 pM, or less than 1 pM.
  • the polypeptide is an agonist
  • a polypeptide as provided herein can bind to a rat endogenous cell line INS-IE, with an EC50 in the cAMP assay 1 of less than 25,000 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, or less than 5 pM.
  • a polypeptide as provided herein is an agonist in both a knock out GLP-lr cell line and a knock out GIPr cell line, with an EC50 in the cAMP assay 1 of less than 350,000 pM, less than 100,000 pM, 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, or less than 5 pM.
  • Certain aspects provide for an isolated polynucleotide encoding any of the above polypeptides, a vector comprising the polynucleotide, and a host cell comprising the polynucleotide or the vector. Certain aspects provide for a method of making the polypeptide, comprising culturing the host cell under conditions allowing expression of the polypeptide, and recovering the polypeptide.
  • compositions comprising any of the above polypeptides, and a carrier. And, certain aspects provide for a kit comprising the composition.
  • Certain aspects provide for 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 a polypeptide or composition described herein.
  • the disease or condition is diabetes.
  • the disease or condition is type-2 diabetes.
  • the administration further improves glycemic control, provides body weight control, improves ⁇ -cell function and mass, reduces the rate of gastric acid secretion and gastric emptying, or any combination thereof.
  • the polypeptide or composition is administered orally or by injection. In certain embodiments, the injection is administered subcutaneously or intravenously.
  • the polypeptide or composition is administered once per week. In certain embodiments, the polypeptide or composition is administered once per day. In certain embodiments, the method of treating or preventing further comprises administering one or more additional therapies.
  • 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.
  • the subject is human.
  • FIGURE 1 shows the structure of an exemplary GIP/GLP-1 agonist polypeptide as provided herein.
  • FIGURE 2 shows an alignment of various incretin peptides.
  • FIGURE 3 shows the effect of the TrpCage on body weight reduction in lean rats.
  • FIGURE 4 is a representation of the results mini-library of X14 (based on IP0281).
  • FIGURE 5 shows the results of a lean mice PK study on the IP0574 (SEQ ID NO: 61) and
  • IP0640 (SEQ ID NO: 220).
  • FIGURE 6 is a representation of the results of mini-library X16X17 (based on IP0574).
  • FIGURE 7A shows the results of an ipGTT in lean mice with certain agonist polypeptides disclosed herein (blood glucose).
  • FIGURE 7B shows the results of an ipGTT in lean mice with certain agonist polypeptides disclosed herein (blood glucose AUC).
  • FIGURE 8A shows the results of an ipGTT in wild type mice with certain agonist polypeptides disclosed herein (blood glucose).
  • FIGURE 8B shows the results of an ipGTT in GLP-lr knock out mice with certain agonist polypeptides disclosed herein (blood glucose)
  • FIGURE 8C shows the results of an ipGTT in wild type mice with certain agonist polypeptides disclosed herein (blood glucose AUC).
  • FIGURE 8D shows the results of an ipGTT in GLP-lr knock out mice with certain agonist polypeptides disclosed herein (blood glucose AUC).
  • FIGURE 9 illustrates various representative examples of lipids, lipid moieties, and linkers.
  • a or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • 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 produced by any means.
  • 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.
  • amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • 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, and/or modification by unnatural amino acids.
  • polypeptide 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 recombinant fusion polypeptide or a synthetic conjugated 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. Chemical modifications intended to improve metabolic stability of peptides can involve additional chemical manipulation following synthesis of the main peptide chain. Examples of manipulation include lipidation and/or PEGylation.
  • a polypeptide as provided herein can be multimeric.
  • 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 disulfide bonds, hydrogen bonds, or other covalent or non-covalent linkages.
  • fragment 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 and deletion fragments that exhibit desirable properties during expression, purification, and or administration to a subject.
  • variants can refer to a polypeptide that differs from a reference 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 fused or conjugated to a heterologous amino acid sequence or other moiety, e.g., for increasing half-life, solubility, or stability.
  • moieties to be conjugated or fused to a polypeptide 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 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.
  • Synthetically produced peptides described herein can be of any length, e.g., any number of amino acids in length, e.g., about 5 amino acids to about 200 amino acids in length, about 10 amino acids to about 150 amino acids in length, about 20 amino acids to about 100 amino acids in length, about 30 amino acids to about 75 amino acids in length, or about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, or about 100 amino acids in length.
  • any number of amino acids in length e.g., about 5 amino acids to about 200 amino acids in length, about 10 amino acids to about 150 amino acids in length, about 20 amino acids to about 100 amino acids in length, about 30 amino acids to about 75 amino acids in length, or about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, or about 100 amino acids in length.
  • amino acid refers to naturally occurring and unnatural amino acids (also referred to herein as “non-naturally occurring amino acids”), e.g., amino acid analogues and amino acid mimetics that function similarly to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogues refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogues can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.
  • amino acid and “amino acid residue” are used interchangeably throughout. Substitution refers to the replacement of a naturally occurring amino acid either with another naturally occurring amino acid or with an unnatural amino acid. For example, during chemical synthesis of a synthetic peptide, the native amino acid can be readily replaced by an unnatural amino acid or another naturally occurring amino acid.
  • lipid-modified amino acid and “lipidated amino acid” are used interchangeably herein, and refer to an amino acid, typically a lysine (K) or cysteine (C), which has a lipid or lipid moiety attached.
  • lipidated polypeptide refers to a peptide or polypeptide that includes one or more lipid-modified amino acids.
  • Figure 9 illustrates various representative examples of lipids, lipid moieties, and linkers.
  • a lipidated polypeptide comprises one or more, e.g., one or two, attached lipids or lipid moieties.
  • maleimide-modified amino acid refers to an amino acid, typically a lysine (K) or cysteine (C), which has a maleimide group attached. The attachment may be direct or through a linker such as PEG 4 .
  • a "maleimide-modified amino acid” is also a "lipid-modified amino acid,” such as where a lipid or lipid moiety is attached to the amino acid or attached to the amino acid via a linker and the maleimide group is indirectly attached to the amino acid via the lipid.
  • the terms “maleimide-modified polypeptide,” “maleimide polypeptide,” and the like refer to a peptide or polypeptide that includes one or more maleimide- modified amino acids.
  • a maleimide-modified amino acid is used to conjugate an agonist polypeptide to another protein.
  • sequence identity 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.
  • 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.
  • 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).
  • BLASTN for nucleotide sequence comparison
  • BLASTP for polypeptide sequence comparison
  • polynucleotide and “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).
  • mRNA messenger RNA
  • pDNA plasmid DNA
  • 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)).
  • PNA peptide nucleic acids
  • nucleic acid refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
  • 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.
  • 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.
  • a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.
  • 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.
  • 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.
  • 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 cerevisiae, 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).
  • fungal cells e.g., yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomyces pombe
  • insect cells e.g., Sf-9
  • mammalian cells e.g., HEK293F,
  • compositions and “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.
  • 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.
  • 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 ⁇ -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 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.
  • 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.
  • the subject is a human subject.
  • a "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.
  • 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%, 99%, 100%, or more 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%, 99%, 100%, or more 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.
  • BSA-cAMP assay bovine serum albumin
  • the term “native GIP” refers to naturally-occurring GIP, e.g., human GIP (i.e., GIPi_4 2 ), comprising the sequence of SEQ ID NO: 6, 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: 4), or active fragments thereof, or a mixture of those two compounds.
  • GIP GLP-1
  • GLP-1 native human GLP-1
  • GIP human GIP
  • GLP-1 human GLP- 1.
  • TrpCage refers to the native extension of the peptide Exendin-4, which is 9 amino acids long and folds back on W25. Without being bound by theory, it is believed to protect the peptide from proteolysis.
  • TrpCage sequences and TrpCage related and/or derived sequences include, but are not limited to, PSSGA PPG (SEQ ID NO: 138), PSSGA PPPGE G (SEQ ID NO: 139), PSSGA PPEGG (SEQ ID NO: 140), PSSGA PPCS (SEQ ID NO: 141), PSSGA PPPSC (SEQ ID NO: 142), PGGGA PPPGC (SEQ ID NO: 143), PSSGR PPPS (SEQ ID NO: 145), PSSGA PPPX wherein X can be no amino acid or any natural amino acid (SEQ ID NO: 476) or PSSGA PPPX wherein X can also be a lipid- modified K or maleimide-modified K (SEQ ID NO: 477), PSSGA PPPS (SEQ ID NO: 146), and PSSGA PPPG (SEQ ID NO: 479).
  • PSSGA PPG SEQ ID NO: 138
  • PSSGA PPPGE G SEQ ID
  • polypeptides that bind both to a GIP receptor and to a GLP-1 receptor.
  • 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 ⁇ -cell mass, or decreased body fat.
  • GIP/GLP-1 agonist polypeptides as provided herein can further possess optimized solubility, formulatability, and stability.
  • GIP/GLP-1 agonist polypeptides as provided herein are active at the human GLP-1 and human GIP receptors.
  • 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.
  • 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.
  • GIP/GLP-1 agonist peptides provided herein are fusion proteins or conjugated 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.
  • GIP/GLP-1 agonist peptides represent an optimized family of dual agonists that exhibit improved potency in recombinant over-expressing human GIP receptor (hGIPr) and human GLP-1 receptor (hGLP-lr) cell lines, as well as in the rat endogenous ⁇ -cell line INS- IE, which expresses both GIPr and GLP-lr. Further, it has been discovered that for certain GIP/GLP-1 agonist peptides, their half-life can be extended by the addition of an Fc domain.
  • this disclosure provides a GIP/GLP-1 agonist polypeptide comprising a peptide sequence derived from the "AID" peptide.
  • 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).
  • GIP/GLP-1 agonist polypeptides are based on the peptide moiety of IP0306. Representative examples of agonist polypeptides based on the peptide moiety of IP0306 are shown in Table B.
  • GIP/GLP-1 agonist polypeptides and/or their Fc fusions are improved over, such as more potent than, IP0306_pm (SEQ ID NO: 7) and/or IP0306 (SEQ ID NO: 59).
  • Xi X 2 E G X 5 X 6 X 7 S X 9 Xio S I L X 14 X 15 X 16 X 1 A I X 20 X 21 F X 23 X 24 X 25 L X 27 X 28 X 29 X 30 (SEQ ID NO: 1); wherein Xi is Y or H; X 2 is S or G; 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 L or Y; X 14 is K, L, or R; X ⁇ is E or D; X 16 is R, E, K, or I; X 17 is E or Q; X 20 is D or E; X 21 is E, L, or A; X 23 is I, V or A; X 24 is A, E, or N; X 25 is W, D, E, L, F, I, S, N, G, M, H, K, or R; X 27 is
  • This disclosure further provides an isolated polypeptide comprising the amino acid sequence:
  • Xi X 2 E G X 5 X 6 X 7 S Xg Xio Xii X 12 X 13 X 14 X 15 Xi6 Xn A I X 2 o E F V N X 25 L X 27 X 28 X 29 X 30 (SEQ ID NO: 2); wherein Xi is Y or H; X 2 is S or G; 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 L or Y; Xn is S, A, or R, X 12 is S, K, I, A, M, or Q; X 13 is Y, L, A, or H; X 14 is K, L, or R; X 15 is E or D; X 16 is R, E, K, or I; X 17 is E or Q; X 20 is D or E; X 25 is W, D, E, L, F, I, S, N, G
  • X 12 is I or K. In certain embodiments, X 13 is L. In certain embodiments, X 14 is R. In certain embodiments, X 17 is Q. In certain embodiments, X 14 is R and X 17 is Q or X 17 is E.
  • X 10 is L.
  • X 14 is K.
  • X 20 is D.
  • X M can be Y, M, I, H, A, or V
  • X i6 can be Y
  • X 17 can be I or L
  • X 2 o can be O
  • X 24 can be S
  • X 27 can be M
  • X 2 8 can be R.
  • X 24 X 25 L X 27 X 28 X 29 X 30 (SEQ ID NO: 468); wherein Xi is Y or H; X 2 is S or G; 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 L or Y; X 14 is K, L, R, Y, M, I, H, A, or V; X 15 is E or D; X 16 is R, E, K, I, or Y; X 17 is I, E, Q, or L; X 20 is D, E, or O; X 21 is E or A; X 23 is I, V or A; X 24 is A, N or S; X 25 is W, D, E, L, F, I, S, N, G, M, H, K, or R; X 27 is L, A, or M; X 28 is A, K, R, or G; X 29 is G, Q, or A;
  • X M can be Y, M, I, H, A, or V
  • X 16 can be Y
  • X i7 can be I or L
  • X 20 can be Q
  • X 27 can be V
  • X 28 can be R.
  • Xi is Y or H
  • X 2 is S or G
  • X5 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 L or Y
  • Xn is S, A, or R
  • X 12 is S, K, I, A, M, or Q
  • X 13 is Y, L, A, or H
  • X 14 is K, L, R, Y, M, I, H, A, or V
  • X 15 is E or D
  • X 16 is R, E, K, I, or Y
  • X 17 is I, E, Q, or L
  • X 2 o is D, E, or Q
  • X 25 is W, D, E, L, F, I, S, N, G, M, H, K, or R
  • X 27 is L, A or V
  • X 28 is A, K, R, or
  • X 12 is I or K. In certain embodiments, X 13 is L. In certain embodiments, X 14 is R. In certain embodiments, X 17 is Q. In certain embodiments, X 14 is R and X 17 is Q or X 17 is E.
  • Xio is L.
  • X 14 is K.
  • X 20 is D.
  • X 14 is R
  • X 17 is E
  • X 24 is A
  • X 28 is R.
  • X 14 is R
  • X 17 is E
  • X 28 is R
  • X i7 is I.
  • This disclosure also provides for an isolated polypeptide comprising the amino acid sequence: YSEGTFTSDLSILKE X16 X17 AIDEFVNWLLKGG
  • X16 is M and X17 is H, X16 is K and X17 is N, X16 is S and X17 is Q, or X16 is I and X17 is K.
  • X16 is I and X17 is Q, X16 is R and X17 is D, or X16 is T and X17 is Y.
  • YSEGTFTSDLSIL X 14 ERQAIDEFVNWLLKGG (SEQ ID NO: 3); wherein X 14 is L, K, or R.
  • Certain embodiments are directed to an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 276 (combo 0062), SEQ ID NO: 285 (combo 0094), SEQ ID NO: 291 (combo 0095), SEQ ID NO: 295 (combo 0154), SEQ ID NO: 302 (combo 0099), SEQ ID NO: 303 (combo 0101), SEQ ID NO: 304 (combo 0104), SEQ ID NO: 9 (IP0574_pm), SEQ ID NO: 10 (combo0005_pm), SEQ ID NO: 15 (combo0011_pm), and any combination thereof.
  • lipidation of amino acid residues and/or substitution of unnatural amino acids for native amino acids can occur at native amino acid residues, e.g., at positions that are susceptible to proteolytic cleavage.
  • GIP/GLP-1 agonist polypeptides are provided with the selective and strategic positioning of the lipidation of one or more amino acid residues.
  • a GIP/GLP-1 agonist polypeptide corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 468, or SEQ ID NO: 469 is lipidated at any one of positions X 10 to X 30 .
  • a lipidated polypeptide comprises at least one lipidated amino acid residue.
  • the lipidated polypeptide comprises at least two lipidated amino acid residues.
  • the lipidated polypeptide contains only one lipidated amino acid residue.
  • the lipidated polypeptide is a synthetically produced polypeptide.
  • the lipidated synthetic polypeptide comprises at least one substitution of an unnatural amino acid for a native amino acid residue.
  • this disclosure provides an isolated lipidated polypeptide comprising the amino acid sequence:
  • Xi X 2 E G X 5 X 6 X 7 S Xg Xio Xii X 12 X 13 X 14 X 15 Xi6 Xn is X19 X20 X21 X22 X 23 X24 X25 X26 X27 X 2 8 X29 X30 (SEQ ID NO: 470); wherein Xi is Y or H; X 2 is S, G, or an unnatural amino acid; X5 is T or M; X 6 is F or H; X 7 is T or I; X 9 is D or L; Xi 0 is L, Y, or a lipid-modified K; Xn is S or a lipid-modified K; Xi 2 is I or a lipid-modified K; X13 is L or a lipid-modified K; X14 is K, L, R, Y, M, I, H, A, V, or a lipid- modified K; X
  • This disclosure further provides an isolated lipidated polypeptide comprising the amino acid sequence:
  • Xi 2 is I or K. In certain embodiments, Xi 3 is L. In certain embodiments, Xi 4 is R. In certain embodiments, Xi 7 is Q. In certain embodiments, Xi 4 is R and Xi 7 is Q or Xi 7 is E.
  • X 10 is L.
  • X 14 is K.
  • X 20 is D.
  • X 14 is R, X 17 is E, X 24 is A, and/or X 28 is R.
  • SEQ ID NO: 471 In certain embodiments of SEQ ID NO: 471, X 14 is R, X 17 is E, and/or X 28 is R. In certain embodiments of SEQ ID NO: 470 or SEQ ID NO: 471, X i7 is I.
  • the lipidated peptide comprises at least one substitution of an unnatural amino acid for a native amino acid residue. In other embodiments, a lipidated peptide comprises at least two, three, four, five or more substitutions of unnatural amino acids.
  • a GIP/GLP-1 agonist polypeptide comprises a maleimide-modified amino acid to which, for example, a heterologous moiety such as an Fc domain or region, as described elsewhere herein, is conjugated via the maleimide group.
  • the maleimide-modified amino acid for example a maleimide-modified K, comprises a linker.
  • the linker is PEG 4 .
  • This disclosure further provides an isolated polypeptide comprising the amino acid sequence: Xi X 2 E G X 5 X 6 X7 S X9 X10 X11 X 12 X 13 X 14 X 15 X1 ⁇ 2 X17 Xi8 X19 3 ⁇ 4o
  • X 12 is I or K. In certain embodiments, X 13 is L. In certain embodiments, X 14 is R. In certain embodiments, X 17 is Q. In certain embodiments, X 14 is R and X 17 is Q or X 17 is E.
  • X 10 is L.
  • X 14 is K.
  • X 20 is D.
  • X 14 is R
  • X 17 is E
  • X 24 is A
  • X 28 is R.
  • X 14 is R
  • X 17 is E
  • X 28 is R
  • X 17 is I.
  • a lipidated polypeptide comprises a lipid-modified K at the C terminus of the polypeptide.
  • the lipidated polypeptide comprises a TrpCage sequence, such as PSSGA PPPX (SEQ ID NO: 477) fused to the C terminus of the polypeptide wherein X is no amino acid, any natural amino acid, a lipid-modified K, or a maleimide-modified K.
  • the TrpCage sequence is PSSGA PPPS (SEQ ID NO: 146), or PSSGA PPPG (SEQ ID NO: 479).
  • the polypeptide comprising a TrpCage comprises a lipid-modified K at the C terminus of the polypeptide.
  • the polypeptide comprises a lipid- modified K at one or two of positions X 10 , Xi2, Xi3, Xi4, Xi7, Xi8, X20, X 2 i, X24, X 2 5, or the C terminus.
  • a polypeptide comprising the sequence of any one of SEQ ID NO: 459, SEQ ID NO: 460, SEQ ID NO: 470, or SEQ ID NO: 471, one or two of X 10 , X 12 , X 21 , or the C terminus is a lipid-modified K.
  • a lipid-modified K comprises a linker, for example, gamma glutamine ( ⁇ ) or PEG 4 .
  • the lipid or lipid moiety can range in length from C9 (nonanoic) to C19 (nonadecanoic).
  • the lipid is one or more of nonanoic (C9), decanoic (CIO), undecanoic (Cl l), lauric (C12), tridecanoic (C13), myristic (C14), pentadecanoic (C15), palmitic (C16), heptadecanoic (C17), stearic (C18), and nonadecanoic (C19).
  • lipidation with longer chains increases potency.
  • the lipid or lipid moiety ranges in length from Cl l to CI 9, i.e., undecanoic (Cl l), lauric (C12), tridecanoic (C13), myristic (C14), pentadecanoic (C15), palmitic (C16), heptadecanoic (C17), stearic (C18), and nonadecanoic (C19).
  • palmitoylation (CI 6) increases in vitro potency and thus the lipidated polypeptide is palmitoylated.
  • the lipid-modified K is K(yE-palm), K(PEG 4 -palm), or PEG 4 -K(yE-palm).
  • unnatural amino acids include a-aminoisobutyric acid (Aib), alpha-methyl glutamine (aMeGlu), alpha-methyl phenylalanine (aMePhe), alpha-methyl leucine (aMeLeu), and norleucine (Nle).
  • the unnatural amino acid can be Aib, aMeGlu, aMePhe, aMeLeu, Nle, or any combination thereof.
  • the polypeptide comprises at least 2, at least 3, at least 4, at least 5, or at least 6 unnatural amino acids.
  • X 13 is aMeLeu.
  • X 6 is Nle
  • X n is Aib
  • X n is Aib
  • Xi 8 is Aib
  • X 27 is Nle.
  • X 13 is a lipid- modified K
  • X 22 is aMePhe
  • X 2 5 is aMePhe.
  • a GIP/GLP-1 agonist polypeptide can be described by the substitution of unnatural amino acids, independent of lipidation although the polypeptide can be lipidated and/or maleimide-modified at positions and in the manner described elsewhere herein. [0326] Accordingly, this disclosure provides an isolated polypeptide comprising the amino acid sequence:
  • the polypeptide comprises 1, 2, 3, 4, 5, or more unnatural amino acids.
  • one or more of X 2 , X 6 , X11, Xi 3 , or X 22 is an unnatural amino acid.
  • X 12 is I or K. In certain embodiments, X 13 is L. In certain embodiments, X 14 is R. In certain embodiments, X 17 is Q. In certain embodiments, X 14 is R and X 17 is Q or X 17 is E. [0329] In certain embodiments of either SEQ ID NO: 472 or SEQ ID NO: 473, X i0 is L. In certain embodiments of either SEQ ID NO: 472 or SEQ ID NO: 473, X 14 is K. In certain embodiments of either SEQ ID NO: 472 or SEQ ID NO: 473, X 20 is D.
  • X 14 is R
  • X 17 is E
  • X 24 is A
  • X 28 is R.
  • X 14 is R
  • X 17 is E
  • X 28 is R
  • X i7 is I.
  • the polypeptide comprises 1, 2, 3, 4, 5, or more unnatural amino acids.
  • one or more of X 2 , X 6 , Xii, X13, or X 22 is an unnatural amino acid.
  • X i3 is aMeLeu.
  • X 6 is Nle
  • X n is Aib
  • X i7 is Aib
  • Xi8 is Aib
  • X 27 is Nle.
  • Certain embodiments are directed to an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 309 (gl681), SEQ ID NO: 310 (gl711), SEQ ID NO: 311 (gl677), SEQ ID NO: 313 (gl685), SEQ ID NO: 315 (gl734), SEQ ID NO: 378 (gl931), SEQ ID NO: 320 (gl896), SEQ ID NO: 322 (g2017), SEQ ID NO: 323 (gl897), SEQ ID NO: 324 (g2018), SEQ ID NO: 326 (g2020), SEQ ID NO: 327 (g2021), SEQ ID NO: 328 (g2022), SEQ ID NO: 330 (gl898), SEQ ID NO: 331 (g2024), SEQ ID NO: 334 (gl899), SEQ ID NO: 335 (g2286), SEQ ID NO: 342 (gl577), SEQ ID NO: 30
  • certain embodiments are directed to an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 311 (gl677), SEQ ID NO: 345 (gl721), SEQ ID NO: 315 (gl734), SEQ ID NO: 415 (gl897), SEQ ID NO: 331 (g2024), SEQ ID NO: 350 (g2029), and any combination thereof.
  • a GIP/GLP-1 agonist polypeptide disclosed herein is a synthetically produced polypeptide conjugated to a heterologous moiety.
  • the heterologous moiety is a lipid moiety, 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- Fnlll scaffold, an epitope tag, a recombinant polypeptide polymer, a cytokine, or a combination of two or more of the recited moieties.
  • the synthetic polypeptide is conjugated to an Fc domain which is described elsewhere in detail herein.
  • the agonist polypeptide is conjugated to the Fc via a free cysteine of the Fc domain.
  • the free cysteine is at position 239C, 268C, or 442C of the Fc domain.
  • the polypeptide is conjugated to the Fc domain via a maleimide group and a free cysteine of the Fc domain.
  • position X 24 , X 28 , and/or the C terminus is a maleimide- modified K conjugated to an Fc domain.
  • the polypeptide comprises a maleimide- modified K at the C terminus conjugated to an Fc domain.
  • the maleimide group is indirectly attached to the amino acid via a lipid.
  • the maleimide-modified amino acid comprises a linker such as PEG 4 .
  • the polypeptide is lipidated and is conjugated to an Fc domain.
  • the polypeptide comprises a maleimide-modified K at X 24 or at the C terminus conjugated to an Fc domain and Xio is a palmitoylated K.
  • the polypeptide comprises a maleimide-modified K at the C terminus conjugated to an Fc domain and X 10 is a palmitoylated K.
  • the polypeptide comprises a maleimide-modified K at the C terminus conjugated to an Fc domain and Xi2 is a palmitoylated K. In certain embodiments of SEQ ID NO: 459 or SEQ ID NO: 460, the polypeptide comprises a maleimide-modified K at X 28 or at the C terminus conjugated to an Fc domain and X 13 is a palmitoylated K. In certain embodiments of SEQ ID NO: 459 or SEQ ID NO: 460, the polypeptide comprises a maleimide-modified K at the C terminus conjugated to an Fc domain and X 13 is a palmitoylated K.
  • Xio is K(yE-palm).
  • position X 24 or the C terminus is a maleimide-modified K conjugated to an Fc domain and one of X 10 , X 12 , X 21 , or X 24 is K(PEG 2 -PEG 2 -stear).
  • the C terminus is a maleimide-modified K conjugated to an Fc domain and one of X 10 , X 12 , or X 21 , is K(PEG 2 -PEG 2 -stear).
  • Certain embodiments are directed to an isolated polypeptide comprising the amino acid sequence SEQ ID NO: 436 (combo0051), SEQ ID NO: 437 (combo0052), SEQ ID NO: 438 (combo077), SEQ ID NO: 439 (combo0112), SEQ ID NO: 443 (combo0211), SEQ ID NO: 444 (combo0212), SEQ ID NO: 445 (combo0213), SEQ ID NO: 446 (combo0214), SEQ ID NO: 447 (combo0215), SEQ ID NO: 452 (combo0121), or any combination thereof.
  • the isolated polypeptide comprises the amino acid sequence SEQ ID NO: 437 (combo0052).
  • a GIP/GLP-1 agonist polypeptide comprises a TrpCage sequence or a TrpCage-related and/or TrpCage-derived sequence.
  • the TrpCage is attached by a peptide bond directly to the C-terminal end of the agonist polypeptide.
  • the TrpCage is attached between an agonist polypeptide and a heterologous moiety such as a linker, hinge, and/or an Fc domain.
  • such a sequence comprises the amino acid sequence PSSGA PPG (SEQ ID NO: 138), PSSGA PPPGE G (SEQ ID NO: 139), PSSGA PPEGG (SEQ ID NO: 140), PSSGA PPCS (SEQ ID NO: 141), PSSGA PPPSC (SEQ ID NO: 142), PGGGA PPPGC (SEQ ID NO: 143), PSSGR PPPS (SEQ ID NO: 145), or PSSGA PPPS (SEQ ID NO: 146).
  • the sequence comprises PSSGA PPPX (SEQ ID NO: 476), wherein X is no amino acid or any natural amino acid.
  • the sequence comprises PSSGA PPPX (SEQ ID NO: 477), wherein X is no amino acid, any natural amino acid, a lipid- modified K, or a maleimide-modified K.
  • the sequence is PSSGA PPPS (SEQ ID NO: 146) or PSSGA PPPG (SEQ ID NO: 479).
  • any one or more GIP/GLP-1 agonist peptides as described above can be fused to one or more heterologous moieties.
  • any one or more GIP/GLP-1 agonist peptides as described above can be fused to one or more additional heterologous polypeptide domains.
  • additional polypeptide regions can facilitate, e.g., activity, efficacy, stability, or in vivo half-life.
  • a heterologous polypeptide domain can comprise a linker, a hinge, an Fc domain, or a combination thereof.
  • the agonist peptide is fused to the additional heterologous polypeptide via a peptide bond.
  • Linkers used in various GIP/GLP-1 agonist polypeptides provided herein can facilitate formation of a desired structure.
  • the linker is a polypeptide linker.
  • a polypeptide linker can comprise 1-50 amino acids, 1-25 amino acids, 25-50 amino acids, or 30-50 amino acids.
  • 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.
  • certain linkers do not comprise any Serine residues.
  • 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.
  • 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.
  • the linker comprises (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • linkers comprise the amino acid sequence: G GGGGS GGGGS GGGGS GGGGS A (SEQ ID NO: 111), A PPGGS GGGGS GGGGS GGGGS A (SEQ ID NO: 112), GT GGGGS GGGGS GGGGS A (SEQ ID NO: 113), G GGGGS GGGGS GGGGS A (SEQ ID NO: 114), G GGGGS A (SEQ ID NO: 115), G GGGGS GGGGS A (SEQ ID NO: 116), G GGGGS GGGGS GGGGS A (SEQ ID NO: 117), G KGGGS GGGGS GGGGS GGGGS A (SEQ ID NO: 118), G GGGGS GGGGS GGGGS GGGGSA (SEQ ID NO: 119), G GGGGGGGGGGGGGGGG A (SEQ ID NO: 120), any combination thereof, any fragment thereof, or any variant thereof.
  • the linker comprises the amino acid sequence GG SGSTA SSGSG SATGG GGAA (SEQ ID NO: 197), AAAGG SGSTA SSGSG SATGG GGAA (SEQ ID NO: 198), APPGG SGSTA SSGSG SATGG GGAA (SEQ ID NO: 199), AAA GGGGS GGGGS GGGGS (SEQ ID NO: 121), AAA PAPAP APAPA PAPAP APAPA G (SEQ ID NO: 122), AAA GKGKGKGKGKGK GKGKGKGKGKGKGKGKGKGK G (SEQ ID NO: 123), AAA GGKGG GGKGG GGKGG GGKGG G (SEQ ID NO: 474), AAA PKPAP KPAPK PAPKP APKPA G (SEQ ID NO: 475), any combination thereof, any fragment thereof, or any variant thereof.
  • the linker comprises the amino acid sequence AAA GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 121), AAA PAPAP APAPA PAPAP APAPA G (SEQ ID NO: 122), AAA GGKGG GGKGG GGKGG GGKGG G (SEQ ID NO: 474), any combination thereof, any fragment thereof, or any variant thereof.
  • the linker comprises the amino acid sequence AAA GGGGS GGGGS GGGGS (SEQ ID NO: 121), any fragment thereof, or any variant thereof.
  • the linker comprises (PA) n wherein n is between 1 and 20. In certain embodiments, n is between 5 and 15.
  • the linker comprises the amino acid sequence AAA PAPAP APAPA PAPAP APAPA G (SEQ ID NO: 122), any fragment thereof, or any variant thereof. [0344] In certain aspects, the linker can comprise a combination of any of the linkers disclosed herein.
  • Hinges used in various GIP/GLP-1 agonist polypeptides provided herein can facilitate formation of a desired structure.
  • the hinge comprises an amino acid sequence of an IgGl hinge, an IgG4 hinge, a fragment thereof, a variant thereof, or any combination thereof.
  • the hinge comprises the amino acid sequence ESKYGPPCPPCPAPEAA (SEQ ID NO: 124), THTCPPCPAPEF (SEQ ID NO: 125), THTCPPC (SEQ ID NO: 126), CPPCPAPEF (SEQ ID NO: 127), TYTCPPCPAPEF (SEQ ID NO: 128), TSTCPPCPAPEF (SEQ ID NO: 129), PPCPPCPAPEF (SEQ ID NO: 130), ESKYGPPCPPCPAPEF (SEQ ID NO: 131), APEF (SEQ ID NO: 132), ESKYGPPCPPC (SEQ ID NO: 133), THTCPPCPAPELL (SEQ ID NO: 134), CPPC (SEQ ID NO: 135), any variant thereof, any fragment thereof, or any combination thereof.
  • the hinge comprises the amino acid sequence CPPC (SEQ ID NO: 135), any fragment thereof, or any variant thereof.
  • 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.
  • the Fc region is a native immunoglobulin Fc region.
  • Fc domain and IgG Fc domain 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.
  • an Fc domain can comprise the entire second constant domain CH2 and the third constant domain CH3.
  • 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).
  • the IgG Fc domain is an IgGl domain (SEQ ID NO: 202), in some aspects the IgG Fc domain is an IgG4 Fc domain (SEQ ID NO: 203).
  • 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.
  • 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.
  • the Fc domain comprises a free cysteine that can be used for site specific conjugation to an agonist polypeptide disclosed herein.
  • the Fc domain can be conjugated to the polypeptide via the free cysteine and a maleimide group attached to the polypeptide.
  • the Fc domain comprises a free cysteine group at one or more of position 239C, 268C, or 442C, which can be used for conjugation.
  • GIP/GLP-1 agonist polypeptides provided herein can comprise a "TM" Fc domain.
  • 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.
  • 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.
  • the GIP/GLP-1 agonist polypeptides provided herein can comprise a "TM" ( ⁇ ) Fc domain.
  • This Fc domain comprises the set of mutations as described above for the "TM" Fc domain, but also includes deletion of the terminal Lysine residue.
  • a human IgGlTM ( ⁇ ) Fc domain is shown as SEQ ID NO: 483.
  • the GIP/GLP-1 agonist polypeptides provided herein can comprise a "YTE" Fc domain (SEQ ID NO: 205).
  • 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. 281:23514-24 (2006) and U.S. Patent No. 7,083,784, which are hereby incorporated by reference in their entireties.
  • GIP/GLP-1 agonist polypeptides provided herein can comprise a "TM-YTE IgG Fc domain.”
  • 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.
  • GIP/GLP-1 agonist polypeptides provided herein can comprise an Fc domain with additional mutations to provide additional stability.
  • the variant IgG Fc domain can comprise, alone or in addition to YTE and/or TM mutations one or more of the following mutations:
  • 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: 466.
  • 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, an IgTM ( ⁇ ) Fc domain, or any fragment thereof, or any variant thereof, or any combination thereof.
  • 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, an IgTM ( ⁇ ) Fc domain, or any fragment thereof, or any variant thereof, or any combination thereof.
  • 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 C.
  • a GIP/GLP-1 agonist polypeptide comprising a linker, a hinge, and an Fc domain comprises the amino acid sequence of SEQ ID NO: 302 (combo0099), SEQ ID NO: 303 (comboOlOl), SEQ ID NO: 304 (combo0104), SEQ ID NO: 295 (combo0154), SEQ ID NO: 299 (combo0102), SEQ ID NO: 300 (combo0105), SEQ ID NO: 291 (combo0095), SEQ ID NO: 285 (combo0094), SEQ ID NO: 61 (IP0574), SEQ ID NO: 62 (combo0005), SEQ ID NO: 67 (comboOOl l), SEQ ID NO: 108 (IP0644), SEQ
  • a GIP/GLP-1 agonist polypeptide as described above can form multimers.
  • 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.
  • two or more monomers can be identical, resulting in, e.g., a homodimer.
  • the two or more polypeptide monomers can be different, resulting in a, e.g., a heterodimer.
  • 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.
  • 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
  • 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.
  • GCGr glucagon receptor
  • 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.
  • 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.
  • 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.
  • GIP/GLP-1 agonist polypeptides as disclosed are agonists in a rat endogenous cell line INS- IE, with an EC50 in the cAMP assay 1 of less than 25,000 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, or less than 5 pM.
  • GIP/GLP-1 agonist polypeptides as disclosed are agonists in both a knock out GLP-lr cell line and a knock out GIPr cell line, with an EC50 in the cAMP assay 1 of less than 350,000 pM, less than 100,000 pM, 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, or less than 5 pM.
  • GIP/GLP-1 agonist polypeptides provided herein can be made by any suitable method.
  • 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.
  • 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.
  • the recombinant expression of a GIP/GLP-1 agonist polypeptide, derivative, analogue 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.
  • 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.
  • host cells are provided that contain a polynucleotide encoding a GIP/GLP-1 agonist polypeptide operably linked to a heterologous promoter.
  • 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
  • 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.
  • GIP/GLP-1 agonist polypeptide 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.
  • chromatography e.g. , ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • 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.
  • a GIP/GLP-1 agonist polypeptide can be characterized in a variety of ways.
  • a GIP/GLP-1 agonist polypeptide can be assayed for potency in a cAMP assay as described elsewhere herein.
  • GIP/GLP-1 agonist polypeptides provided herein can be stabilized via amino acid modifications.
  • the carboxyl group of the C-terminal amino acid is amidated.
  • the C-terminal amino acid is amidated glycine.
  • the C-terminal glycine is the unmodified acid.
  • GIP/GLP-1 agonist polypeptides are provided in which one or more amino acid residues are acylated.
  • GIP/GLP-1 agonist polypeptides provided herein contain one or more lysine residues, in which a lipid moiety is attached to the N(epsilon) group.
  • a linker is incorporated between lysine and the lipid 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 can be used such as addition of cholesterol or myristoyl groups.
  • 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 lipid, 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- Fnlll scaffold, an epitope tag, a recombinant polypeptide polymer, a cytokine, and a combination of two or more of such moieties.
  • a GIP/GLP-1 agonist polypeptide can be fused with or conjugated to 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 attached to proteins, either through recombinant gene fusion and expression or by chemical conjugation. Proteins that are suitable as partners for fusion or conjugation 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 al, Diabetes 53:2492-2500 (2004); P.
  • 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 al., 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.
  • heterologous moieties can be conjugated to GIP/GLP-1 agonist polypeptides to further stabilize or increase half-life.
  • 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).
  • PEG polyethylene glycol
  • a "pegylated GIP/GLP-1 agonist polypeptide" has a PEG chain covalently bound thereto.
  • GIP/GLP-1 agonist polypeptides e.g., pegylation
  • Derivatization of GIP/GLP-1 agonist polypeptides can be done at the lysine that is lipidated, 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.
  • polyethylene glycol 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.
  • 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 can be carried out using an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
  • 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).
  • polyethylene glycol such as a reactive ester or aldehyde derivative of PEG
  • GIP/GLP-1 agonist polypeptides provided herein possess one or more criteria of acceptable solubility, ease in formulatability, plasma stability, and improved pharmacokinetic properties.
  • GIP/GLP-1 agonist polypeptides as disclosed are soluble in standard buffers over a broad pH range.
  • 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 rV-cleavage products. Cleavage products arise from the action of proteases, e.g., DPP IV in plasma.
  • 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.
  • 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.
  • 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.
  • the composition can contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. Once formulated, compositions of the disclosure can be administered directly to the subject.
  • Carriers that can be used with compositions of the disclosure 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.
  • 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.
  • compositions 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.
  • 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.
  • 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 ⁇ -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 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, or a composition thereof, as disclosed herein. Further provided is a GIP/GLP-1 agonist polypeptide, or composition thereof, for treatment of a hypoglycemic condition, e.g., type-2 diabetes. Further provided is use of a GIP/GLP-1 agonist polypeptide, or composition thereof, as provided herein in the manufacture of a medicament for the treatment of a hypoglycemic condition, e.g., type-2 diabetes.
  • GIP/GLP-1 agonist polypeptides provided herein, or compositions thereof can be administered for glycemic control, promoting insulin production, promoting ⁇ -cell mass, promoting weight loss, or reducing excess body weight.
  • GIP/GLP-1 agonist polypeptides provided herein, or compositions thereof 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, atherosclerosis, arteriosclerosis, coronary heart disease, or peripheral artery disease, stroke, respiratory dysfunction, or renal disease.
  • Treatment is an approach for obtaining beneficial or desired clinical results.
  • beneficial or desired clinical results from the disclosed GIP/GLP-1 agonist polypeptides include, without limitation, stabilized serum glucose and serum insulin levels, increased ⁇ -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.
  • the route of administration of GIP/GLP-1 agonist polypeptides provided herein, or compositions thereof can be, for example, oral, parenteral, by inhalation or topical.
  • 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, or compositions thereof can be administered as a single dose or as multiple doses.
  • a GIP/GLP-1 agonist polypeptide, or a composition thereof is administered orally or by subcutaneous injection.
  • 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, once a week, 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).
  • the amount of a GIP/GLP-1 agonist polypeptide, or a composition thereof, 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, or a composition thereof 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.
  • GIP/GLP-1 agonist polypeptide or a composition thereof, 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.
  • GIP/GLP-1 agonist polypeptides provided herein, or compositions thereof can be administered once per day via injection.
  • GIP/GLP-1 agonist polypeptides provided herein, or compositions thereof can be administered once per week via injection.
  • a GIP/GLP1 agonist polypeptide as provided herein, or a composition thereof 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.
  • 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 comprising GIP/GLP-1 agonist polypeptides, which can be used to perform the methods described herein.
  • 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.
  • Dual-active GIP/GLP-1 agonist polypeptides were prepared according to the following methods.
  • GIP/GLP-1 dual agonist peptides were designed from the AID backbone peptides. A combination of rational substitutions and mini-library randomized mutations were made to allow specificity at GLP-lr and GIPr.
  • the backbone peptides can be subjected to random mutagenesis, expressed in phage, and selected based on binding to the various receptors.
  • peptide-linker-hinge-Fc CH2 and CH3 regions, from either IgGl or IgG4, with various mutations to improve stability and reduce effector function, such as IgGl-TM, or IgGl-FQQ, as described elsewhere herein.
  • peptide-linker-hinge-Fc CH2 and CH3 regions, from either IgGl or IgG4, with various mutations to improve stability and reduce effector function, 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 Table C.
  • An exemplary bivalent GIP/GLP-1 agonist polypeptide is shown in FIG. 1.
  • Proteins were purified using an automated system by affinity chromatography, where the Fc region of the protein binds to the column matrix. Columns were equilibrated in lx DPBS. Elution of the bound material was performedat low pH.
  • 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 HEK293 cells or CHO cells by standard methods.
  • GIPr and GLP-lr were studied in immortalized rat ⁇ -cell lines (INS- IE or INS-1 832/3). Peptide activation of these various receptors results in downstream production of cAMP second messenger that can be measured in a functional activity assay.
  • BSA-based assay medium 0.1% bovine serum albumin (BSA) in Hanks Balanced Salt Solution (GIBCO or Sigma), containing 0.5 mM IBMX (Sigma # 17018);
  • 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.
  • serum albumin that corresponds to the receptor being tested
  • serum albumin 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 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 e.g., human serum albumin for testing cAMP activity at hGIPr or hGLP-lr
  • BRAVO
  • test samples were made directly in 5 assay medium in black shallow- well u-bottom 384-well plate (Corning # 3676) using a non-contact liquid dispenser (ECHOTM, Labcyte). All sample dilutions were made in duplicate.
  • 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 2.0 x 10 5 cells /ml).
  • a 5 cell suspension was added to assay plates already containing 5 serially diluted test samples and the plates incubated at room temperature for 30 minutes.
  • 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.
  • anti-cAMP cryptate donor fluorophore
  • cAMP-d2 acceptor fluorophore
  • TrpCage is partly responsible for the extended half-life of Ex-4 compared to GLP-1.
  • the effect of TrpCage addition on several peptides was studied (see Table E).
  • CD rats were injected subcutaneously at day 0 with 1.5 mg/kg, 1.6 mg/kg, 1.2 mg/kg, or 1.5 mg/kg of IP0201, IP0295, IP0280, and IP0268, respectively.
  • the doses were normalized on the rat h-GLP-lr potency. Serum samples were taken over time (6 animals per time point). The serum samples were then measured for activity at the h-GLP-lr by the cAMP assay in HBSS/HEPES 0.1% BSA, as described above. The serum concentrations were calculated from the activity.
  • a TrpCage was added to IP0306.
  • the AAAGG SGSTA SSGSG SATGG GGAA (SEQ ID NO: 198) linker of IP0306 was also replaced by the AAA GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 121) linker and the THT motif of the hinge was also removed (see Table F). This generated the sequence IP0573 (SEQ ID NO: 60). These changes improved the in vitro potency of the molecule in the endogenous ⁇ -cell line as shown in Table 4. Table F.
  • Example 4 Recombinant GIP/GLP-1 dual agonist peptide Fc fusions analogues 13] Modifications were made to IP0306 to increase the potency in the low expressing cell GIPr cell line (C3), to achieve a more potent and balanced dual agonist. Using a rational design approach, several residues were mutated back to native GIP or Ex-4 sequence (see Table G). Point mutations often did not affect the potencies in the recombinant cell lines. However, when the point mutations are combined (comboOOl l; SEQ ID NO: 67), the potency was improved, especially on GLP-lr (see Table 5). Table G. Native GIP or Ex-4 -based mutations.
  • mini-libraries were used to identify novel single point mutations in search of peptides with improved potency. Mini-libraries were cloned, expressed and screened directly from the CHO cell supernatant for the dual agonist IP0281 to increase its cross-reactivity on the recombinant m-GIPr cell line.
  • Mini-library X14 was generated using site directed mutagenesis. Two complementary nucleotides were designed and synthesized:
  • IP0574 (SEQ ID NO: 61) was further characterized in the mouse and rat GLP-lr and GIPr overexpressing cell lines (see Table 8).
  • the peptide moiety portion of the dual-agonist Fc Fusion IP0574 (SEQ ID NO: 61) was subjected to further optimization using mini-libraries.
  • a mini-library was constructed of the two consecutive residues XI 6X17 on IP0574 (see Figure 6). The aim of the library was to find a balanced dual agonist (more potent on GIPr and less potent on GLP-lr compared to the parent).
  • I16Q17 mutant is less potent than IP0574 but more balanced (see Table 9). It is thus contemplated that in combination with other mutations, 116 might lead to a balanced and potent dual-agonist.
  • Example 5 Effect of recombinant fusion linker on potency. 22] It was discovered that the linker influences potency on the low expressing h-GIPr cell line and in the endogenous rat cell lines (see Table 11). Changing the linker can increase the potency 10-fold.
  • Example 6 in vivo stability of IP0574 and IP0640.
  • IP0574 stability was analyzed in a mouse PK study alongside IP0640 (GIP-TrpCage Fc) and appeared to be more stable.
  • Preliminary pharmacokinetics in mouse was examined as follows: C57 B1/6J mice were injected with 3 mg/kg (subcutaneously) with either IP0640 or IP0574, and serum samples were taken over time (3 animals per time point) (see Table 14). The serum samples were then measured for activity at the h-GIPr by the cAMP assay in HBSS/HEPES 0.1% BSA, as described above. The results are shown in Figure 5. The results show that the GIP/GLP-1 agonist polypeptide Fc fusion IP0574 is twice more stable than the GIP-TrpCage Fc fusion IP0640.
  • N-a-Fmoc-L-amino acids were purchased from Bachem AG, Switzerland, Iris Biotech AG, Germany or Pharmaron, China, and NovaSyn ® TGR (TentaGel Rink) and NovaSyn® TGA (TentaGel Wang) from Novabiochem, Merck Biosciences, Darmstadt, Germany.
  • the following amino acid derivatives were used: Fomc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)- OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH.
  • peptides were prepared as C-terminal carboxamides on NovaSyn ® TGR resin (0.24 mmole/g). Fmoc- amino acids were coupled to the resin with aid of HCTU/DIPEA in NMP at ambient temperature, and then the residual free a-amino groups capped with acetic anhydride/pyridine. Fmoc-deprotection was performed using piperidine in DMF (20% v/v) at ambient temperature.
  • Peptides were cleaved from the resin using a cocktail consisting of TFA (95% v/v), TIPS (2.5% v/v) and water (2.5% v/v). Subsequently, cleavage solutes were combined, concentrated by rotary evaporation and peptides precipitated with cold diethyl ether and filtered.
  • Conjugated molecules were made using site-specific conjugation technology where the synthetic peptide was conjugated to specific cysteines engineered into the Fc.
  • the Fc was first reduced (in solution in PBS at 5-10 mg/ml) using tris(2- carboxyethyl)phosphine (TCEP).
  • TCEP tris(2- carboxyethyl)phosphine
  • the peptide was then added to the de-capped antibody.
  • the peptide/Fc solution was incubated at 20 °C for 1 hour and the conjugation reaction was quenched by the addition of N- acetylcysteine (NAC) and subsequent incubation at 20 °C for 15 minutes.
  • NAC N- acetylcysteine
  • the peptide Fc conjugate was then affinity purified using protein A to separate the Fc from the unconjugated free peptide.
  • the positive fractions were combined and buffer exchanged against PBS using dialysis overnight.
  • the free synthetic peptides can be more potent than recombinant peptide Fc fusions (e.g., about 5 times more potent on GLP-lr and about 20/30 times more potent on GIPr in vitro; Table 15).
  • Palmitoylation at certain positions of the dual-agonist peptides was examined (Table N). Palmitoylation at certain positions, (e.g, 10, 12, 13, 17, 18, 21 and 40) was observed to increase the potency on both receptors (Table 16).
  • Analogues of g 1681 were synthesized and tested (Table O and Table 17). Notable analogues included: gl721 (SEQ ID NO: 345), gl722 (SEQ ID NO: 341) and g2029 (SEQ ID NO: 350).
  • Unnatural amino acids as described elsewhere herein can be incorporated into an agonist peptide to increase its stability in vivo (e.g., to protect against proteolytic cleavage).
  • Table P and Table 18 show representative sequences and functional testing data, respectively.
  • L13aMeLeu e.g., gl726; SEQ ID NO: 362.
  • F6Nle e.g., g2008; SEQ ID NO: 380
  • Sl lAib e.g., g2033; SEQ ID NO: 391. This property could be used to change the balance of GIPr/GLP-lr activation.
  • GIP >50700 27 35 22 691 16 gl681 3.0 2 8.0 2 33 2 gl718 25.5 2 19.7 2
  • SEQ ID NO: 400 Compound gl736 (lipidation at position 10 and W25aMePhe; SEQ ID NO:393) and compound gl682 (lipidation at C terminus and W25aMePhe; SEQ ID NO: 403) retain high potency and are expected to be more stable in vivo.
  • Lipids of different lengths can be attached to the peptides. Maximum activation of GLP- lr was observed with lipids between C16 and Cl l. For GIPr, higher activation was observed as lipid length increased. Table R and Table 20.
  • Table S and Table 21 show additional representative example peptide sequences and functional data, respectively, of synthetic peptides having lipidation or PEGylation.
  • Lipidated peptides can also be conjugated to a protein using maleimide.
  • the precursor peptides retain high potency in vitro as shown in Table U and Table 23.
  • Peptides can also be synthesised with lipid that contains a maleimide group. These constructs were observed to generally be less potent (Table W and Table 25)
  • Example 11 Endogenous cell lines screen
  • the synthetic dual GIP/GLP-1 agonist peptides were observed to be more potent than native GLP-1 in rat beta cells (INS- IE cell line) and have the characteristics of balanced dual agonists.
  • Recombinant dual GIP/GLP-1 agonist peptides are almost as potent as native GLP-1, but are not as balanced as the synthetic duals.
  • INS- IE rat immortalized pancreatic ⁇ cell line
  • INS-IE cells express both GLP-lr and GIPr endogenously.
  • the synthetic dual GIP/GLP- 1 peptides were observed to be more potent than GLP-1 native peptide in this cell line (Table Y and Table 27).
  • the recombinant peptides combo0099 (SEQ ID NO: 302) and comboOlOl (SEQ ID NO: 303) are only 2 fold less potent than native GLP-1 in INS- IE.
  • Knock out cell lines were generated to isolate the response of each receptor to various compounds disclosed herein and to confirm that the dual agonist molecules are balanced.
  • Knock out cell lines data shows that the synthetic duals are balanced dual (i.e., similar potency in WT and both KO cell lines), while the recombinant fusions and conjugates tested are more active through GLP-lr activation than GIPr activation (Table Z and Table 28).
  • Example 12 In vivo characterization
  • Lean C57B1/6J mice were purchased from Jackson Laboratories at 8 weeks of age, maintained on a standard chow diet (LabDiet; #5008) and allowed to acclimatize for at least 1 week prior to glucose tolerance testing and for 2 weeks prior to food intake studies. GLP-1 KO mice have previously been described (Scrocchi, Brown et al., Nature Medicine. 1996). They were also maintained on a standard chow diet (LabDiet; #5008). For DIO mouse studies, lean C57B1/6J mice were purchased from Jackson at 8 weeks of age and placed on a 60% high fat diet (Research Diets, RD12492) for 16-20 weeks prior to the initiation of dosing.
  • mice on high fat diet for 12-18 weeks were purchased directly from Jackson laboratories.
  • the db/db mice were purchased from Jackson laboratories at 6 weeks of age, maintained on standard chow diet and allowed to acclimatize for 2-3 weeks prior to the initiation of dosing. Prior to all single and repeated dosing studies, mice were sham dosed 3-5 times with saline. ipGTT
  • mice were grouped based on body weight and typically fasted for 6 hours prior to intraperitoneal injection of glucose at a dose of 2 g/kg and a dose volume of 5-10 mL/kg. Compounds were dosed subcutaneously at the indicated dose and a dose volume of 5-10 mL/kg either 4 or 24 hours prior to the administration of glucose. Glucose was measured from tail blood at the specified time points using the Bayer Ascensia Breeze 2 blood glucose monitoring system.
  • the glucose lowering effect could be caused by GLP-lr and/or GIPr activation.
  • GLP-lr knock out mice the glucose lowering effect is mediated through GIPr activation only.
  • the recombinant GLP-1 Fc fusion IP118; HGEGTFTSDVSSYLEEQAAKEFIAWLVKGG GGGGGSGGGGSGGGGS AESKYGPPCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
  • Example 13 Summary 1] Table AA, Table BB, and Table 29 show a summary of the sequences and results of GLP/GLP-1 agonist polypeptide fusions disclosed herein.
  • hGlucagonR hGLP-lR low expressing INS-IE INS1 832/3 hGJPR (Rat ⁇ -Cell) (Rat ⁇ -Cell)
  • Table CC and Table 30 show a summary of the sequences and results of GLP/GLP-1 agonist polypeptide conjugates disclosed herein. Table CC.

Abstract

La présente invention concerne des polypeptides agonistes doubles GIP/GLP-1, des protéines de fusion, et des protéines conjuguées et synthétiques pour le traitement d'états hypoglycémiques, par ex.,, le diabète de type 2.
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