WO2002046227A2 - Glp-1 fusion proteins - Google Patents

Glp-1 fusion proteins Download PDF

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WO2002046227A2
WO2002046227A2 PCT/US2001/043165 US0143165W WO0246227A2 WO 2002046227 A2 WO2002046227 A2 WO 2002046227A2 US 0143165 W US0143165 W US 0143165W WO 0246227 A2 WO0246227 A2 WO 0246227A2
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xaa
glu
glp
lys
asp
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PCT/US2001/043165
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French (fr)
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WO2002046227A3 (en
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Wolfgang Glaesner
Radmila Micanovic
Sheng-Hung Rainbow Tschang
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Eli Lilly And Company
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Priority to HU0302529A priority Critical patent/HU229218B1/en
Priority to AU2689702A priority patent/AU2689702A/en
Priority to SK50057-2011A priority patent/SK288088B6/en
Priority to IL15581201A priority patent/IL155812A0/en
Priority to DK01995845T priority patent/DK1355942T3/en
Priority to PL366208A priority patent/PL209550B1/en
Priority to EP01995845A priority patent/EP1355942B1/en
Priority to HU1300326A priority patent/HU230603B1/en
Priority to SK670-2003A priority patent/SK288342B6/en
Priority to KR1020037007541A priority patent/KR100942864B1/en
Priority to AU2002226897A priority patent/AU2002226897B2/en
Application filed by Eli Lilly And Company filed Critical Eli Lilly And Company
Priority to NZ525577A priority patent/NZ525577A/en
Priority to JP2002547963A priority patent/JP2004528014A/en
Priority to BR0116024-9A priority patent/BR0116024A/en
Priority to SI200130878T priority patent/SI1355942T1/en
Priority to EA200300644A priority patent/EA005584B1/en
Priority to UA2003065280A priority patent/UA81897C2/en
Priority to CA2434237A priority patent/CA2434237C/en
Priority to DK06118975T priority patent/DK1724284T3/en
Priority to US10/433,108 priority patent/US7271149B2/en
Priority to DE60135581T priority patent/DE60135581D1/en
Priority to MXPA03005036A priority patent/MXPA03005036A/en
Publication of WO2002046227A2 publication Critical patent/WO2002046227A2/en
Publication of WO2002046227A3 publication Critical patent/WO2002046227A3/en
Priority to IL155812A priority patent/IL155812A/en
Priority to NO20032565A priority patent/NO331273B1/en
Priority to HR20030455A priority patent/HRP20030455A2/en
Priority to HK04102243.7A priority patent/HK1061411A1/en
Priority to IL184429A priority patent/IL184429A/en
Priority to NO20101602A priority patent/NO20101602L/en
Priority to NO20111369A priority patent/NO332221B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • 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
    • C07K14/76—Albumins
    • 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
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)

Definitions

  • the present invention relates to glucagon-like peptides including analogs and derivatives thereof fused to proteins that have the effect of extending the in vivo half-life of the peptides. These fusion proteins can be used to treat non-insulin dependent diabetes mellitus as well as a variety of other conditions.
  • Glucagon- ike Peptide 1 (GLP-1) is a 37 amino acid peptide that is secreted by the L-cells of the intestine in response to food ingestion. It has been found to stimulate insulin secretion (insulinotropic action) , thereby causing glucose uptake by cells and decreased serum glucose levels [see, e . g. , Mo sov, S., (1992) Int . J. Peptide Protein
  • GLP-1 is poorly active. A subsequent endogenous cleavage between the 6 th and 7 th position produces a more potent biologically active GLP-1 (7- 37) OH peptide.
  • GLP-1 analogs and derivatives are known and are referred to herein as "GLP-1 compounds.”
  • GLP-1 analogs include the Exendins which are peptides found in the venom of the GILA-monster .
  • the Exendins have sequence homology to native GLP-1 and can bind the GLP-1 receptor and initiate the signal transduction cascade responsible for the numerous activities that have been attributed to GLP-1 (7-37) OH.
  • GLP-1 compounds have a variety of physiologically significant activities. For example, GLP-1 has been shown to stimulate insulin release, lower glucagon secretion, inhibit gastric emptying, and enhance glucose utilization. [Nauck, M.A., et al . (1993) Diabetologia 36:741-744; Gutniak, M. , et al . (1992) New England J. of Med. 326:1316- 1322; Nauck, M.A. , et al . , (1993) J. Clin . Invest . 91:301- 307] . GLP-1 shows the greatest promise as a treatment for non-insulin dependent diabetes mellitus (NIDDM) .
  • NIDDM non-insulin dependent diabetes mellitus
  • GLP-1 activity is controlled by blood glucose levels. When levels drop to a certain threshold level, GLP-1 is not active. Thus, there is no risk of hypoglycemia associated with treatment involving GLP-1.
  • the usefulness of therapy involving GLP-1 peptides has been limited by their fast clearance and short half-lives. For example, GLP-1 (7-37) has a serum half-life of only 3 to 5 minutes.
  • GLP-1 (7-36) amide has a time action of about 50 minutes when administered subcutaneously. Even analogs and derivatives that are resistant to endogenous protease cleavage, do not have half-lives long enough to avoid repeated administrations over a 24 hour period. Fast clearance of a therapeutic agent is inconvenient in cases where it is desired to maintain a high blood level of the agent over a prolonged period of time since repeated administrations will then be necessary. Furthermore, a long-acting compound is particularly important for diabetic patients whose past treatment regimen has involved taking only oral medication. These patients often have an extremely difficult time transitioning to a regimen that involves multiple injections of medication.
  • the present invention overcomes the problems associated with delivering a compound that has a short plasma half- life.
  • the compounds of the present invention encompass GLP- 1 compounds fused to another protein with a long circulating half-life such as the Fc portion of an immunoglobulin or albumin .
  • GLP-1 (7-37) OH has a tendency to undergo a conformational change from a primarily alpha helix structure to a primarily beta sheet structure. This beta sheet form results in aggregated material that is thought to be inactive. It was, therefore, surprising that biologically active GLP-1 fusion proteins with increased half-lives could be developed. This was especially unexpected given the difficulty of working with GLP-1 (7- 37) OH alone and the large size of the fusion partner relative to the small GLP-1 peptide attached.
  • Compounds of the present invention include heterologous fusion proteins comprising a first polypeptide with a N- terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) human albumin; b) human albumin analogs; and c) fragments of human albumin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide.
  • Compounds of the present invention also include a heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) human albumin; b) human albumin analogs; and c) fragments of human albumin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide via a peptide linker.
  • the peptide linker is selected from the group consisting of : a) a glycine rich peptide; b) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser] n where n is 1, 2, 3, 4, 5 or 6; and c) a peptide having the sequence [Gly-Gly-Gly-Gly- Ser] 3 .
  • Additional compounds of the present invention include a heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) the Fc portion of an immunoglobulin; b) an analog of the Fc portion of an immunoglobulin; and c) fragments of the Fc portion of an immunoglobulin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide.
  • the GLP-1 compound may be fused to the second polypeptide via a peptide linker.
  • the peptide linker is selected from the group consisting of: a) a glycine rich peptide; b) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser] n where n is i, 2, 3, 4, 5 or 6; and c) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser] 3 .
  • the GLP-1 compound that is part of the heterologous fusion protein have no more than 6 amino acids that are different from the corresponding amino acid in GLP-1 (7-37) OH, GLP-1 (7-36) OH, or Exendin-4.
  • the GLP-1 compound have no more than 5 amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH, GLP-1 (7-36) OH, or Exendin-4. It is most preferred that the GLP-1 compound have no more than 4, 3, or 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37) OH, GLP-1 (7-36) OH, or Exendin-4.
  • a GLP-1 compound that is part of the heterologous fusion protein has glycine or valine at position 8.
  • the present invention also includes polynucleotides encoding the heterologous fusion protein described herein, vectors comprising these polynucleotides and host cells transfected or transformed with the vectors described herein. Also included is a process for producing a heterologous fusion protein comprising the steps of transcribing and translating a polynucleotide described herein under conditions wherein the heterolgous fusion protein is expressed in detectable amounts.
  • the present invention also encompasses a method for normalizing blood glucose levels in a mammal in need thereof comprising the administration of a therapeutically effective amount of a heterologous fusion protein described herein.
  • Figure 1 IgGl Fc amino acid sequence encompassing the hinge region, CH2 and CH3 domains.
  • Figure 2 Human serum albumin amino acid sequence.
  • Figure 3 A.. SDS-PAGE gel and immunoblot of same gel illustrating the molecular weight of IgGl-Fc and GLP-l-Fc fusion proteins (Lane 1, MW standards; Lane 2, Purified Fc; lane 3, Mock transfected media; Lane 4, Val 8 -GLP-1-Fc; Lane 5, Exendin-4-Fc) B.
  • Figure 4 SDS-PAGE gel of purified Fc, albumin, and GLP-1 fusion proteins (Lane 1, MW standards; Lane 2, purified Fc; Lane 3, Val8-GLP-1-Fc; Lane 4, Exendin-4-Fc; Lane 5, MW standard; Lane 6, Val8-GLP-1-HSA; Lane 7, Exendin-4-HSA; Lane 8, Exendin-4- [Gly-Gly-Gly-Gly-Ser] 3 - HSA) .
  • Figure 5 Expression cloning vector containing the Fc regions illustrated in figure 1.
  • Figure 6 Expression cloning vector containing the albumin sequence illustrated in figure 2.
  • Figure 7 Expression cloning vector containing DNA encoding a 15 amino acid linker fused in frame and 5' of the albumin sequence illustrated in figure 2.
  • Figure 8 In vitro dose response activity of GLP-1 fusion proteins .
  • Figure 9 Pharmacokinetics of GLP-1 Fc and HSA fusion proteins .
  • Figure 10 Glucodynamic response to Exendin-Fc in two normal fasted dogs.
  • Figure 11 Insulinotropic response to Exendin-Fc in two normal fasted dogs .
  • Figure 12 DNA sequence encoding a human IgGl Fc region.
  • Figure 13 DNA sequence encoding a human albumin protein.
  • the heterologous fusion proteins of the present invention comprise a GLP-1 compound fused to human albumin, a human albumin analog, a human albumin fragment, the Fc portion of an immunoglobulin, an analog of the Fc portion of an immunoglobulin, or a fragment of the Fc portion of an immunoglobulin.
  • the C-terminus of the GLP-1 compound may be fused directly, or fused via a peptide linker, to the N- terminus of an albumin or Fc protein.
  • These heterologous fusion proteins are biologically active and have an increased half-life compared to native GLP-1.
  • GLP-1 compounds that make up part of the heterologous fusion protein encompass polypeptides having from about twenty-five to about thirty- nine naturally occurring or non-naturally occurring amino acids that have sufficient homology to native GLP-1 (7-37) OH such that they exhibit insulinotropic activity by binding to the GLP-1 receptor on ⁇ -cells in the pancreas.
  • a GLP-1 compound typically comprises a polypeptide having the amino acid sequence of GLP-1 (7-37) OH, an analog of GLP-1 (7-37)OH, a fragment of GLP-1 (7-37) OH or a fragment of a GLP-1 (7-37) OH analog.
  • GLP-1 (7-37) OH has the amino acid sequence of SEQ ID NO: 1:
  • GLP-1 (7- 37) OH has been assigned number residue 7 and the carboxy- terminus, number 37.
  • the other amino acids in the polypeptide are numbered consecutively, as shown in SEQ ID NO: 1.
  • position 12 is phenylalanine and position 22 is glycine.
  • GLP-1 compounds also encompass "GLP-1 fragments.”
  • GLP-1 fragment is a polypeptide obtained after truncation of one or more amino acids from the N-terminus and/or C- terminus of GLP-1 (7-37 ) OH or an analog or derivative thereof.
  • the nomenclature used to describe GLP-1 (7-37) OH is also applicable to GLP-1 fragments.
  • GLP-1 (9- 36) OH denotes a GLP-1 fragment obtained by truncating two amino acids from the N-terminus and one amino acid from the C-terminus. The amino acids in the fragment are denoted by the same number as the corresponding amino acid in GLP-1 (7- 37) OH.
  • the N-terminal glutamic acid in GLP- 1(9-36) OH is at position 9; position 12 is occupied by phenylalanine; and position 22 is occupied by glycine, as in GLP-1 (7-37) OH.
  • GLP-1 (7-36) OH the glycine at position 37 of GLP-1 (7-37) OH is deleted.
  • GLP-1 compounds also include polypeptides in which one or more amino acids have been added to the N-terminus and/or C-terminus of GLP-1 (7-37) OH, or fragments or analogs thereof. It is preferred that GLP-1 compounds of this type have up to about thirty-nine amino acids .
  • the amino acids in the "extended" GLP-1 compound are denoted by the same number as the corresponding amino acid in GLP-1 (7-37) OH.
  • the N-terminus amino acid of a GLP-1 compound obtained by adding two amino acids to the N-terminal of GLP- 1(7-37) OH is at position 5; and the C-terminus amino acid of a GLP-1 compound obtained by adding one amino acid to the C- terminus of GLP-1 (7-37) OH is at position 38.
  • position 12 is occupied by phenylalanine and position 22 is occupied by glycine in both of these "extended" GLP-1 compounds, as in GLP-1 (7-37) OH.
  • Amino acids 1-6 of an extended GLP-1 compound are preferably the same as or a conservative substitution of the amino acid at the corresponding position of GLP-1 (1-37) OH.
  • Amino acids 38-45 of an extended GLP-1 compound are preferably the same as or a conservative substitution of the amino acid at the corresponding position of glucagon or Exendin-4.
  • GLP-1 compounds of the present invention encompass "GLP-1 analogs.”
  • a GLP-1 analog has sufficient homology to GLP-1 (7-37) OH or a fragment of GLP-1 (7-37) OH such that the analog has insulinotropic activity.
  • a GLP-1 analog has the amino acid sequence of GLP-1 (7-37) OH or a fragment thereof, modified so that from one, two, three, four or five amino acids differ from the amino acid in the corresponding position of GLP-1 (7-37 ) OH or a fragment of GLP-1 (7-37) OH.
  • the substituting amino acid and its position is indicated prior to the parent structure.
  • Glu 22 -GLP-1 (7-37) OH designates a GLP-1 compound in which the glycine normally found at position 22 of GLP- 1(7-37) OH has been replaced with glutamic acid
  • Val 8 -Glu 22_ GLP-1 (7-37) OH designates a GLP-1 compound in which alanine normally found at position 8 and glycine normally found at position 22 of GLP-1 (7-37) OH have been replaced with valine and glutamic acid, respectively.
  • GLP-1 compounds of the present invention also include "GLP-1 derivatives.”
  • a GLP-1 derivative is defined as a molecule having the amino acid sequence of GLP-1 or of a GLP-1 analog, but additionally having chemical modification of one or more of its amino acid side groups, ⁇ -carbon atoms, terminal amino group, or terminal carboxylic acid group.
  • a chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties.
  • Modifications at amino acid side groups include, without limitation, acylation of lysine ⁇ -amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine.
  • Modifications of the terminal, amino group include, without limitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications.
  • Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C 1 -C 4 alkyl.
  • one or more side groups , or terminal groups may be protected by protective groups known to the ordinarily- skilled protein chemist.
  • the -carbon of an amino acid may be mono- or dimethylated.
  • Any GLP-1 compound can be part of the heterologous fusion proteins of the present invention as long as the GLP- 1 compound itself is able to bind and induce signaling through the GLP-1 receptor. GLP-1 receptor binding and signal transduction can be assessed using in vi tro assays such as those described in EP 619,322 and U.S. Patent No. 5,120,712, respectively. Numerous active GLP-1 fragments, analogs and derivatives are known in the art and any of these analogs and derivatives can also be part of the heterologous fusion proteins of the present invention. Some examples of novel GLP-1 analogs as well as GLP-1 analogs and derivatives known in the art are provided herein.
  • GLP-1 analogs and GLP-1 fragments known in the art include, for example, GLP-1 (7-34) and GLP-1 (7-35) , GLP-1 (7- 36), Gln 9 -GLP-l(7-37) , D-Gln 9 -GLP-1 (7-37) , Thr 16 -Lys 18 -GLP- 1(7-37), and Lys 18 -GLP-1 (7-37) .
  • GLP-1 analogs such as GLP- 1(7-34) and GLP-l(7-35) are disclosed in U.S. Patent No.
  • GLP-1 Biologically processed forms of GLP-1 which have insulinotropic properties, such as GLP-1 (7-36) are also known.
  • Other known biologically active GLP-1 compounds are disclosed in U.S. Patent No 5,977,071 to Hoffmann, et al . ' , U.S. Patent No. 5,545,618 to Buckley, et al . , and Adelhorst, et al . , J. Biol . Chem . 269 : 6275 (1994).
  • GLP-1 analogs are composed of GLP-1 analogs of formula I (SEQ ID NO: 2)
  • Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, lie, Val,
  • Xaa at position 9 is Glu, Asp, or Lys
  • Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, lie, Val, Glu, Asp, or Lys;
  • Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, lie, Val,
  • Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, lie, Tyr, Glu, Asp, Trp, or Lys
  • Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys
  • Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, lie, Val,
  • Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, or Lys
  • Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, lie, Val, Glu, Asp, Met, Trp, Tyr, or Lys
  • Xaa ' at position 21 is Glu, Asp, or Lys ;
  • Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys;
  • Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, or Lys;
  • Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, lie, Val,
  • Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, lie, Val,
  • Xaa at position 26 is Lys, Arg, Gin, Glu, Asp, or His
  • Xaa at position 27 is Leu, Glu, Asp, or Lys
  • Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, lie, Val,
  • Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys
  • Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, lie, Val
  • Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, lie,
  • Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His
  • Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, lie, Val,
  • Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His
  • Xaa at position 37 is Pro, Gly, Ala, Ser, Thr, Leu, lie,
  • Xaa at position 38 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted;
  • Xaa at position 39 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted;
  • Xaa at position 40 is Gly, Asp, Glu, or Lys, or is deleted;
  • Xaa at position 41 is Ala, Phe, Trp, Tyr, Glu, Asp, or Lys, or is deleted;
  • Xaa at position 42 is Ser, Pro, Lys, Glu, or Asp, or is deleted;
  • Xaa at position 43 is Ser, Pro, Glu, Asp, or Lys, or is deleted;
  • Xaa at position 44 is Gly, Pro, Glu, Asp, or Lys, or is deleted; and Xaa at position 45 is Ala, Ser, Val, Glu, Asp, or Lys, or is deleted; provided that when the amino acid at position 37, 38, 39, 40, 41, 42, 43, or 44 is deleted, then each amino acid downstream of that amino acid is also deleted.
  • the GLP-1 compound of formula I contain less than six amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH or Exendin-4. It is more preferred that less than five amino acids differ from the corresponding amino acid in GLP-1 (7-37) OH or Exendin-4. It is even more preferred that less than four amino acids differ from the corresponding amino acid in GLP- 1(7-37) OH or Exendin-4.
  • GLP-1 compounds of the present invention include derivatives of formula I such as a C-l-6-ester, or amide, or C-l-6-alkylamide, or C-l-6-dialkylamide thereof.
  • WO99/43706 describes derivatives of GLP-1 compounds of formula I and is incorporated by reference herein in its entirety.
  • the compounds of formula I derivatized as described in WO99/43706 and underivatized are encompassed by the present invention.
  • Another preferred group of GLP-1 compounds is composed of GLP-1 analogs of formula II (SEQ ID NO: 3):
  • Xaa at position J is : L-histidine , D-histidine, desamino- histidine , 2 -amino-histidine , ⁇ -hydroxy-histidine, ho ohistidine , -f luoromethyl-histidine or oc-methyl- histidine ;
  • Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr;
  • Xaa at position 9 is: Thr, Ser, Arg, Lys, Trp, Phe, Tyr,
  • Xaa at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His
  • Xaa at position 12 is: His, Trp, Phe, or Tyr;
  • Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
  • Xaa at position 18 is: His, Pro, Asp, Glu, Arg, Ser, Ala, or
  • Lys; Xaa at position 19 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or
  • Xaa at position 23 is: His, Asp, Lys, Glu, Gin, or Arg;
  • Xaa at position 24 is: Glu, Arg, Ala, or Lys;
  • Xaa at position 26 is: Trp, Tyr, Phe, Asp, Lys, Glu, or His;
  • Xaa at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or
  • Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
  • Xaa at position 31 is: Asp, Glu, Ser, Thr, Arg, Trp, or Lys;
  • Xaa at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu;
  • Xaa at position 34 is: Glu, Lys, or Asp;
  • Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
  • Xaa at position 36 is: Thr, Ser, Asp, Trp, Tyr, Phe, Arg,
  • R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr,
  • GLP-1 compounds are composed of GLP-1 analogs of formula III (SEQ ID NO: 4) :
  • Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine or -methyl- histidine;
  • Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr;
  • Xaa at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His;
  • Xaa at position 12 is: His, Trp, Phe, or Tyr
  • Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu, or Ala;
  • Xaa at position 22 Gly, Asp, Glu, Gin, Asn, Lys, Arg, or
  • Xaa at position 25 is: Asp, Lys, Glu, or His;
  • Xaa at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys;
  • Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
  • Xaa at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu;
  • Xaa at position 34 is: Glu, Lys, or Asp;
  • Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
  • Xaa at position 36 is: Arg, Glu, or His; R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
  • GLP-1 compounds are composed of GLP-1 analogs of formula IV (SEQ ID NO : 5):
  • Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine or ⁇ -methyl- histidine;
  • Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, Met, or Thr;
  • Xaa at position 12 is: His, Trp, Phe, or Tyr;
  • Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
  • Xaa at position 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or Cys;
  • Xaa at position 23 is: His, Asp, Lys, Glu, or Gin
  • Xaa at position 26 is: Asp, Lys, Glu, or His
  • Xaa at position 30 is: Ala, Glu, Asp, Ser, or His
  • Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His, or Glu;
  • R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
  • Another preferred group of GLP-1 compounds is composed of GLP-1 analogs of formula V (SEQ ID NO: 6) :
  • Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine or ⁇ -methyl- histidine;
  • Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr;
  • Xaa at position 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or
  • Preferred GLP-1 compounds of formula I, II, III, IV, and V comprise GLP-1 analogs or fragments of GLP-1 analogs wherein the analogs or fragments contain an amino acid other than alanine at position 8 (position 8 analogs) . It is preferable that these position 8 analogs contain one or more additional changes at positions 9, 11, 12, 16, 18, 22, 23, 24, 26, 27, 30, 31, 33, 34, 35, 36, and 37 compared to the corresponding amino acid of native GLP-1 (7-37) OH. It is also preferable that these analogs have 6 or fewer changes compared to the corresponding amino acids in native GLP-1 (7- 37) OH or GLP-1 (7-36) OH.
  • More preferred analogs have 5 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH or GLP-1 (7-36) OH or have 4 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH or GLP-1 (7-36) OH. It is even more preferable that these analogs have 3 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH or GLP- 1(7-36) OH. It is most preferable that these analogs have 2 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37 ) OH.
  • GLP-1 compounds of formula II, III, IV, and V have a reduced propensity to aggregate and generate insoluble forms. This is also important in the context of a fusion protein wherein the relatively small GLP-1 peptide must maintain an active conformation despite being fused to a much larger protein.
  • Preferred GLP-1 compounds of formula II, III, IV, and V encompassed by the fusion proteins of the present invention comprise GLP-1 analogs or fragments of GLP-1 analogs in which glycine at position 22 and preferably alanine at position 8 have been replaced with another amino acid.
  • position 8 is preferably glycine, valine, leucine, isolecine, serine, threonine or methionine and more preferably valine or glycine.
  • position 8 is preferably glycine, valine, leucine, isolecine, serine, threonine or methionine and more preferably valine or glycine.
  • GLP-1 compounds include GLP-1 analogs of formula IV (SEQ ID NO: 5) wherein the analogs have the sequence of GLP-1 (7-37) OH except that the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 30 is glutamic acid, aspartic acid, serine, or histidine and more preferably glutamatic acid.
  • GLP-1 compounds include GLP-1 analogs of formula IV (SEQ ID NO: 5) wherein the analogs have the sequence of GLP-1 (7-37) OH except that the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 37 is histidine, lysine, arginine, threonine, serine, glutamic acid, aspartic acid, tryptophan, tyrosine, phenylalanine and more preferably histidine.
  • GLP-1 compounds include GLP-1 analogs of formula IV (SEQ ID NO: 5) wherein the analogs have the sequence of GLP-1 (7-37) OH except that the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 22 is glutamic acid, lysine, aspartic acid, or arginine and more preferably glutamic acid or lysine and position 23 is lysine, arginine, glutamic acid, aspartic acid, and histidine and more preferably lysine or glutamic acid.
  • GLP-1 compounds include GLP-1 analogs of formula V (SEQ ID NO: 6) wherein the analogs have the sequence of GLP-1 (7-37) OH except that the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 22 is glutamic acid, lysine, aspartic acid, or arginine and more preferably glutamine acid or lysine and position 27 is alanine, lysine, arginine, tryptophan, tyrosine, phenylalanine, or histidine and more preferably alanine .
  • GLP-1 compounds include GLP-1 analogs of formula II wherein the analogs have the sequence of GLP- 1(7-37) OH except that the amino acid at position 8 and one, two, or three amino acids selected from the group consisting of position 9, position 11, position 12, position 16, position 18, position 22, position 23, position 24, position 26, position 27, position 30, position 31, position 33, position 34, position 35, position 36, and position 37, differ from the amino acid at the corresponding position of native GLP-1 (7-37 ) OH.
  • GLP-1 compounds of formula II include: Val 8 -GLP-l(7-37)OH, Gly 8 -GLP-1 (7-37 ) OH, Glu 22 -GLP-1 (7-37 ) OH, Asp22-GLP-l(7-37)OH, Arg 2 2_GLP-l (7-37 ) OH, Lys 2 2_GLP-l (7- 37)OH, Cys22_GLP-l(7-37)OH, Val 8 -Glu 22 -GLP-1 (7-37 ) OH, Val 8 - Asp 2 -GLP-l(7-37)OH, Val 8 -Arg22_GLP-l (7-37 ) OH, Val 8 -Lys 22 - GLP-l(7-37)OH, Val 8 -Cys 2 2-GLP-1 (7-37 ) OH, Gly 8 -Glu 2 2-GLP-1 (7- 37) OH, Gly 8 -Asp22-GLP-l(7-37)
  • GLP-1 analogs and derivatives for use in the present invention is composed of molecules of formula VI (SEQ ID NO : 7)
  • Ri is selected from the group consisting of L- histidine, D-histidine, desa ino-histidine, 2-amino- histidine, ⁇ -hydroxy-histidine, homohistidine, alpha- fluoromethyl-histidine, and alpha-methyl-histidine;
  • X is selected from the group consisting of Ala, Gly, Val, Thr, lie, and alpha-methyl-Ala;
  • Y is selected from the group consisting of Glu, Gin, Ala, Thr, Ser, and Gly;
  • Z is selected from the group consisting of Glu, Gin, Ala, Thr, Ser, and Gly; and R2 is Gly-OH.
  • GLP-1 compounds for use in the present invention is disclosed in WO 91/11457, and consists essentially of GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7- 36), or GLP-l(7-37), or the amide form thereof, and pharmaceutically-acceptable salts thereof, having at least one modification selected from the group consisting of:
  • the substituted amino acids can optionally be in the D-form and the amino acids substituted at position 7 can optionally be in the N-acylated or N-alkylated form.
  • DPP IV dipeptidyl-peptidase IV
  • GLP-1 analogs and derivatives that are protected from the activity of DPP IV in the context of a fusion protein are preferred, and fusion proteins wherein the GLP-1 compound is Gly 8 -GLP- l(7-37)OH, Val 8 -GLP-l(7-37)OH, ⁇ -methyl-Ala 8 -GLP-l (7-37 ) OH, or Gly 8 -Gln l-GLP-1 (7-37) OH are more preferred.
  • GLP-1 compounds for use in the present invention consists of the compounds of formula VII (SEQ ID NO: 8) claimed in U.S. Patent No. 5,512,549, which is expressly incorporated herein by reference.
  • R2 formula VII (SEQ ID NO: 8) wherein R! is selected from the group consisting of 4- imidazopropionyl, 4-imidazoacetyl, or 4-imidazo- ⁇ , ⁇ dimethyl-acetyl ; R2 is selected from the group consisting of C 6 -C1 0 unbranched acyl, or is absent; R 3 is selected from the group consisting of Gly-OH or NH2 ; and,
  • Xaa is Lys or Arg.
  • More preferred compounds of formula IV for use in the present invention are those in which Xaa is Arg and R2 is 6 ⁇ ⁇ o unbranched acyl. Even more preferred compounds of formula IV for use in the present invention are those in which Xaa is Arg, R is Cg-Cio unbranched acyl, and R 3 is Gly-OH. Other highly-preferred compounds of formula IV for use in the present invention are those in which Xaa is Arg,
  • R2 is Cg-Cio unbranched acyl
  • R 3 is Gly-OH
  • R ⁇ is 4- imidazopropionyl .
  • An especially preferred compound of formula IV for use in the present invention is that in which
  • Xaa is Arg
  • R 2 is Cs unbranched acyl
  • R 3 is Gly-OH
  • R! is 4-imidazopropionyl .
  • the GLP-1 compounds comprise GLP-1 analogs wherein the backbone for such analogs or fragments contains an amino acid other than alanine at position 8 (position 8 analogs) .
  • the backbone may also include L-histidine, D- histidine, or modified forms of histidine such as desamino- histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine, or ⁇ -methyl- histidine at position 7. It is preferable that these position 8 analogs contain one or more additional changes at positions 12, 16, 18, 19, 20, 22, 25, 27, 30, 33, and 37 compared to the corresponding amino acid of native GLP-1 (7- 37) OH. It is more preferable that these position 8 analogs contain one or more additional changes at positions 16, 18, 22, 25 and 33 compared to the corresponding amino acid of native GLP-1 (7-37) OH.
  • the GLP-1 analog is GLP-1 (7- 37) OH wherein the amino acid at position 12 is selected from the group consisting of tryptophan or tyrosine. It is more preferred that in addition to the substitution at position 12, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 12 and 8, the amino acid at position 22 is substituted with glutamic acid.
  • the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 16 is selected from the group consisting of tryptophan, isoleucine, leucine, phenylalanine, or tyrosine. It is more preferred that in addition to the substitution at position 16, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 16 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 16 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 16 and 8, the amino acid at position 37 is substituted with histidine.
  • the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 18 is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, leucine, or isoleucine, preferably tryptophan, tyrosine, and isoleucine. It is more preferred that in addition to the substitution at position 18, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 18 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 18 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 18 and 8, the amino acid at position 37 is substituted with histidine
  • the GLP-1 analog is GLP-1 (7-37 ) OH wherein the amino acid at position 19 is selected from the group consisting of tryptophan or phenylalanine, preferably tryptophan. It is more preferred that in addition to the substitution at position 19, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 19 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 19 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 19 and 8, the amino acid at position 37 is substituted with histidine
  • the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 20 is phenylalanine, tyrosine, or tryptophan. It is more preferred that in addition to the substitution at position 20, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 20 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 20 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 20 and 8, the amino acid at position 37 is substituted with histidine
  • the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 25 is selected from the group consisting of valine, isoleucine, and leucine, preferably valine. It is more preferred that in addition to the substitution at position 25, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 25 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 25 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 25 and 8, the amino acid at position 37 is substituted with histidine.
  • the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 27 is selected from the group consisting of isoleucine or alanine. It is more preferred that in addition to the substitution at position 27, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 27 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 27 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 27 and 8, the amino acid at position 37 is substituted with histidine
  • the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 33 is isoleucine. It is more preferred that in addition to the substitution at position 33, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 33 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 33 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 33 and 8, the amino acid at position 37 is substituted with histidine
  • the GLP-1 compounds have modifications at one or more of the following positions: 8, 12, 16, 18, 19, 20, 22, 25, 27, 30, 33, and 37. These GLP-1 compounds show increased potency compared with GLP-1 (7-37 ) OH and comprise the amino acid sequence of formula IX (SEQ ID NO: 12)
  • Xaa 7 is: L-histidine, D-histidine, desamino-histidine, 2- amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ -f luoromethyl-histidine, or ⁇ - methyl-histidine;
  • Xaa 8 is: Ala, Gly, Val, Leu, lie, Ser, or Thr;
  • Xaai 2 is: Phe, Trp, or Tyr;
  • Xaa i6 is: Val, Trp, lie, Leu, Phe, or Tyr;
  • Xaais is: Ser, Trp, Tyr, Phe, Lys, lie, Leu, Val;
  • Xaai 9 is: Tyr, Trp, or Phe;
  • Xaa 2 o is: Leu, Phe, Tyr, or Trp;
  • Xaa 2 is: Gly, Glu, Asp, or Lys;
  • Xaa 25 is: Ala,
  • Xaa 3 o is : Ala or Glu Xaa 33 is: Val, or lie; and Xaa 37 is: Gly, His, NH 2 , or is absent.
  • Some preferred GLP-1 compounds of formula IX include
  • GLP-1 (7-37) OH wherein position 8 is valine or glycine, position 22 is glutamic acid, position 16 is tyrosine, leucine or tryptophan, position 18 is tyrosine, tryptophan, or isoleucine, position 25 is valine and position 33 is isoleucine.
  • Other preferred GLP-1 compounds include the following: Val 8 -Tyr 16 -GLP-1 ( (7- 37) OH, Val 8 -Tyr 12 -Glu 22 -GLP-l(7-37)OH, Val 8 -Tyr 16 -Phe 19 -GLP- l(7-37)OH.
  • the GLP-1 compounds of the present invention also encompass Exendin compounds .
  • Exendin-3 and Exendin-4 are biologically active peptides first isolated from Helodermatidae lizard venoms and have been shown to bind the GLP-1 receptor and stimulate cAMP-dependent H + production in mammalian parietal cells.
  • Exendin-3 and Exendin-4 are both 39 amino acid peptides which are approximately 53% homologous to GLP-1. They act as potent agonists of GLP-1 activity.
  • an N-terminally truncated derivative of Exendin known as Exendin (9-39 amino acids) , is an inhibitor of Exendin-3, Exendin-4 and GLP-1.
  • Exendin compound typically comprises a polypeptide having the amino acid sequence of Exendin-3, Exendin-4, or an analog or fragment thereof.
  • Exendin-3 and Exendin-4 are disclosed in U.S. Patent No. 5,424,286.
  • Exendin-3 has the amino acid sequence of SEQ ID NO : 9:
  • Exendin-4 has the amino acid sequence of SEQ ID NO: 10: 7 8 9 10 11 12 13 14 15 16 17 His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- 18 19 20 21 22 23 24 25 26 27 28
  • GLP-1 compounds also include Exendin fragments which are polypeptides obtained after truncation of one or more amino acids from the N-terminus and/or C-terminus of Exendin or an Exendin analog. Furthermore, GLP-1 compounds include Exendin polypeptides in which one or more amino acids have been added to the N-terminus and/or C-terminus of Exendin or fragments thereof. Exendin compounds of this type have up to about forty-five amino acids.
  • GLP-1 compounds also include "Exendin analogs.”
  • An Exendin analog has sufficient homology to Exendin-4, Exendin-3, or a fragment thereof such that the analog has insulinotropic activity.
  • the activity of Exendin fragments and/or analogs can be assessed using in vi tro assays such as those described ' in EP 619,322 and U.S. Patent No. 5,120,712.
  • an Exendin analog has the amino acid sequence of Exendin-4 or a fragment thereof, modified so that from one, two, three, four or five amino acids differ from the amino acid in corresponding position of Exendin-4 or the fragment of Exendin-4.
  • the substituting amino acid and its position is indicated prior to the parent o structure.
  • Val -Exendin-4 designates an
  • Exendin compound in which the glycine normally found at position 8 of Exendin-4 has been replaced with valine has been replaced with valine.
  • GLP-1 compounds is composed of GLP-1/Exendin-4 analogs of formula VIII (SEQ ID NO: 11).
  • Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine or ⁇ -methyl- histidine;
  • Xaa at position 8 is: Gly, Ala, or Val;
  • Xaa at position 16 is: Leu or Val
  • Xaa at position 18 is Lys or Ser
  • Xaa at position 19 is: Gin or Tyr;
  • Xaa at position 20 is: Met or Leu; Xaa at position 22 is: Glu or Gin;
  • Xaa at position 23 is: Glu, or Gin;
  • Xaa at position 25 is: Val or Ala
  • Xaa at position 26 is: Arg or Lys
  • Xaa at position 27 is Leu or Glu
  • Xaa at position 30 is: Glu or Ala
  • Xaa at position 33 is: Val or Lys
  • Xaa at position 34 is: Asn or Lys
  • Xaa at position 36 is: Gly or Arg; and R at position 37 is: Gly, Pro, Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser, or is absent.
  • the activity of 18 different species that fall within this genus is provided in Table 6.
  • the GLP-1 fusion proteins of the present invention can comprise glycosylation sites.
  • Glycosylation is a chemical modification wherein sugar moieties are added to the protein at specific sites. Glycosylation of proteins play a role in ensuring the correct charge, confirmation, and stability of maturing protein and can target the protein to the cell surface and eventual secretion of the protein. Most importantly, glycosylation effects the in vivo clearance rate for many proteins.
  • Sugars can be O-linked or N-linked. Generally, O-linked sugars are added to the hydroxyl-group oxygen of serine and threonine, while N-linked sugars are added to the amide nitrogen of asparagine.
  • the consensus site for N-glycosylation is Asn XI X2 wherein XI is any amino acid except Pro and X2 is Ser or Thr.
  • GLP-1 compounds are generally not glycosylated in vivo; however, interestingly the GLP-1 fusion proteins of the present invention that comprise a GLP-1 compound with a C terminal extension fused to an Fc sequence is glycosylated at the last serine in the C terminal extension (SSGAPPPS*) and at threonine at position 11 in the N terminal region of Fc (AEPKSCDKTHT*CPPC . . . ) .
  • SSGAPPPS* serine in the C terminal extension
  • Heterologous Fc fusion proteins are generally not glycosylated in vivo; however, interestingly the GLP-1 fusion proteins of the present invention that comprise a GLP-1 compound with a C terminal extension fused to an Fc sequence is glycosylated at the last serine in the C terminal extension (SSGAPPPS*) and at threonine at position 11 in the N terminal region of Fc (AEPKSCDKTHT*
  • the GLP-1 compounds described above can be fused directly or via a peptide linker to the Fc portion of an immunoglobulin.
  • Immunoglobulins are molecules containing polypeptide chains held together by disulfide bonds, typically having two light chains and two heavy chains. In each chain, one domain (V) has a variable amino acid sequence depending on the antibody specificity of the molecule. The other domains (C) have a rather constant sequence common to molecules of the same class.
  • the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, this term refers to an antibody fragment which is obtained by removing the two antigen • binding regions (the Fab fragments) from the antibody. One way to remove the Fab fragments is to digest the immunoglobulin with papain protease.
  • the Fc portion is formed from approximately equal sized fragments of the constant region from both heavy chains, which associate through non-covalent interactions and disulfide bonds.
  • the Fc portion can include the hinge regions and extend through the CH2 and CH3 domains to the C-terminus of the antibody.
  • the Fc portion can further include one or more glycosylation sites.
  • the amino acid sequence of a representative Fc protein containing a hinge region, CH2 and CH3 domains, and one N-glycosylation site at position 82 is shown in Figure 1.
  • IgG is the most abundant immunoglobulin in serum.
  • IgG also has the longest half-life in serum of any immunoglobulin (23 days) . Unlike other immunoglobulins, IgG is efficiently recirculated following binding to an Fc receptor.
  • IgG subclasses GI, G2 , G3 , and G4 each of which have different effect or functions. Gl, G2 , and G3 can bind Clq and fix complement while G4 cannot. Even though G3 is able to bind Clq more efficiently than Gl, Gl is more effective at mediating complement-directed cell lysis. G2 fixes complement very inefficiently.
  • the Clq binding site in IgG is located at the carboxy terminal region of the CH2 domain.
  • IgG subclasses are capable of binding to Fc receptors (CD16, CD32, CD64) with Gl and G3 being more effective than G2 and G4.
  • the Fc receptor binding region of IgG is formed by residues located in both the hinge and the carboxy terminal regions of the CH2 domain.
  • IgA can exist both in a monomeric and dimeric form held together by a J-chain. IgA is the second most abundant Ig in serum, but it has a half-life of only 6 days. IgA has three effect or functions. It binds to an IgA specific receptor on macrophages and eosinophils, which drives phagocytosis and degranulation, respectively. It can also fix complement via an unknown alternative pathway.
  • IgM is expressed as either a pentamer or a hexamer, both of which are held together by a J-chain.
  • IgM has a serum half-life of 5 days. It binds weakly to Clq via a binding site located in its CH3 domain.
  • IgD has a half-life of 3 days in serum. It is unclear what effect or functions are attributable to this Ig.
  • IgE is a monomeric Ig and has a serum half-life of 2.5 days. IgE binds to two Fc receptors which drives degranulation and results in the release of proinflammatory agents.
  • the heterologous fusion proteins of the present invention may contain any of the isotypes described above or may contain mutated Fc regions wherein the complement and/or Fc receptor binding functions have been altered.
  • the heterologous fusion proteins of the present invention may contain the entire Fc portion of an immunoglobulin, fragments of the Fc portion of an immunoglobulin, or analogs thereof fused to a GLP-1 compound.
  • the fusion proteins of the present invention can consist of single chain proteins or as multi-chain polypeptides . Two or more Fc fusion proteins can be produced such that they interact through disulfide bonds that naturally form between Fc regions. These multimers can be homogeneous with respect to the GLP-1 compound or they may contain different GLP-1 compounds fused at the N- terminus of the Fc portion of the fusion protein.
  • the Fc or Fc-like region must serve to prolong the in vivo plasma half-life of the GLP-1 compound fused at the N-terminus. Furthermore, the fused, GLP-1 compound must retain some biological activity. An increase in half-life can be demonstrated using the method described in Example 7 wherein the half-life of the fusion protein is compared to the half-life of the GLP-1 compound alone. Biological activity can be determined by in vitro and in vivo methods known in the art . Representative biological assays are described in Examples 6, 8, and 9.
  • Fab fragments are rapidly degraded, it is believed that the relevant sequence for prolonging half-life reside in the CH2 and/or CH3 domains. Further, it has been shown in the literature that the catabolic rates of IgG variants that do not bind the high-affinity Fc receptor or Clq are indistinguishable from the rate of clearance of the parent wild-type antibody, indicating that the catabolic site is distinct from the sites involved in Fc receptor or Clq binding. [Wawrzynczak et al . , (1992) Molecular Immunology 29:221] . Site-directed mutagenesis studies using a murine IgGl Fc region suggested that the site of the IgGl Fc region that controls the catabolic rate is located at the CH2-CH3 domain interface.
  • Fc regions can be modified at the catabolic site to optimize the half-life of the fusion proteins. It is preferable that the Fc region used for the heterologous fusion proteins of the present invention be derived from an IgGl or an IgG4 Fc region. It is even more preferable that the Fc region be IgG4 or derived from IgG4. Preferably the IgG Fc region contains both the CH2 and CH3 regions including the hinge region.
  • Heterologous albumin fusion proteins
  • GLP-1 compounds described above can be fused directly or via a peptide linker to albumin or an analog, fragment, or derivative thereof.
  • albumin proteins making up part of the fusion proteins of the present invention can be derived from albumin cloned from any species.
  • human albumin and fragments and analogs thereof are preferred to reduce the risk of the fusion protein being immunogenic in humans.
  • Human serum albumin (HSA) consists of a single non- glycosylated polypeptide chain of 585 amino acids with a formula molecular weight of 66,500. The amino acid sequence of human HSA is shown in figure 2. [See Meloun, et al . (1975) FEBS Letters 58:136; Behrens, et al . (1975) Fed. Proc. 34:591; Lawn, et al .
  • EP 399,666 discloses albumin fragments that include HS (1-177) and HSA(l-200) and fragments between HSA(1-177) and HSA(l-200) .
  • the heterologous fusion proteins of the present invention include GLP-1 compounds that are coupled to any albumin protein including fragments, analogs, and derivatives wherein such fusion protein is biologically active and has a longer plasma half-life than the GLP-1 compound alone.
  • the albumin portion of the fusion protein need not necessarily have a plasma half-life equal to that of native human albumin. Fragments, analogs, and derivatives are known or can be generated that have longer half-lives or have half-lives intermediate to that of native human albumin and the GLP-1 compound of interest.
  • the heterologous fusion proteins of the present invention encompass proteins having conservative amino acid substitutions in the GLP-1 compound and/or the Fc or albumin portion of the fusion protein.
  • a "conservative substitution” is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape.
  • Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number carbon and heteroatoms in their side chains differs by no more than about four . They have approximately the same shape when the number of branches in their side chains differs by no more than one.
  • Amino acids with phenyl or substituted phenyl groups in their side chains are considered to have about the same size and shape. Except as otherwise specifically provided herein, conservative substitutions are preferably made with naturally occurring amino acids .
  • amino acid is used herein in its broadest sense, and includes naturally occurring amino acids as well as non-naturally occurring amino acids, including amino acid analogs and derivatives .
  • the latter includes molecules containing an amino acid moiety.
  • reference herein to an amino acid includes, for example, naturally occurring proteogenic L-amino acids; D- amino acids; chemically modified amino acids such as amino acid analogs and derivatives; naturally occurring non- proteogenic amino acids such as norleucine, ⁇ -alanine, ornithine, GABA, etc.; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids.
  • the term "proteogenic” indicates that the amino acid can be incorporated into a peptide, polypeptide, or protein in a cell through a metabolic pathway.
  • the incorporation of non-natural amino acids, including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the heterologous fusion proteins of the present invention can be advantageous in a number of different ways.
  • D-amino acid-containing peptides, etc. exhibit increased stability in vi tro or in vivo compared to L-amino acid-containing counterparts.
  • the construction of peptides, etc., incorporating D-amino acids can be particularly useful when greater intracellular stability is desired or required.
  • D-peptides, etc. are resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of the molecule, and prolonged lifetimes in vivo when such properties are desirable. Additionally, D-peptides, etc., cannot be processed efficiently for major histocompatibility complex class Il-restricted presentation to T helper cells, and are therefore, less likely to induce humoral immune responses in the whole organism.
  • hydropathic index of amino acids In addition to structure/function analyses of the various polypeptides encompassed by the present invention, there are numerous factors that can be considered when selecting amino acids for substitution.
  • One factor that can be considered in making such changes is the hydropathic index of amino acids.
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a protein has been discussed by Kyte and Doolittle (1982, J. Mol . Biol . , 157: 105-132). It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resultant protein. This, in turn, affects the interaction of the protein with molecules such as enzymes, substrates, receptors, ligands, DNA, antibodies, antigens, etc.
  • each amino acid has been assigned a hydropathic index as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate/glutamine/aspartate/asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • amino acids in a peptide, polypeptide, or protein can be substituted for other amino acids having a similar hydropathic index or score and produce a resultant peptide, etc., having similar or even improved biological activity.
  • amino acids having hydropathic indices within ⁇ 2 are substituted for one another. More preferred substitutions are those wherein the amino acids have hydropathic indices within ⁇ 1. Most preferred substitutions are those wherein the amino acids have hydropathic indices within ⁇ 0.5.
  • hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4) .
  • amino acids having hydropathic indices within ⁇ 2 are preferably substituted for one another, those within +1 are more preferred, and those within ⁇ 0.5 are most preferred.
  • amino acid substitutions in the fusion proteins of the present invention can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Furthermore, substitutions can be made based on secondary structure propensity. For example, a helical amino acid can be replaced with an amino acid that would preserve the helical structure. Exemplary substitutions that take various of the foregoing characteristics into consideration in order to produce conservative amino acid changes resulting in silent changes within the present peptides, etc., can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids .
  • heterologous fusion proteins of the present invention can be made by a variety of different methods, recombinant methods are preferred.
  • the following general molecular biology terms and abbreviations are defined below.
  • the terms and abbreviations used in this document have their normal meanings unless otherwise designated. For example, “°c” refers to degrees Celsius; “mmol” refers to millimole or millimoles; “mg” refers to milligrams; “ ⁇ g” refers to micrograms; “ml or mL” refers to milliliters; and “ ⁇ l or ⁇ L” refers to microliters.
  • Amino acids abbreviations are as. set forth in 37 C.F.R. ⁇ 1.822 (b) (2) (1994) .
  • Base pair refers to DNA or RNA.
  • the abbreviations A,C,G, and T correspond to the 5'- monophosphate forms of the deoxyribonucleosides (deoxy) adenosine, (deoxy) cytidine, (deoxy) guanosine, and thymidine, respectively, when they occur in DNA molecules.
  • the abbreviations U,C,G, and A correspond to the 5'- monophosphate forms of the ribonucleosides uridine, cytidine, guanosine, and adenosine, respectively when they occur in RNA molecules.
  • base pair may refer to a partnership of A with T or C with G.
  • heteroduplex base pair may refer to a partnership of A with U or C with G.
  • “Digestion” or “Restriction” of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (“sequence- specific endonucleases” ) .
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements were used as would be known to one of ordinary skill in the art.
  • Ligation refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments. Unless otherwise provided, ligation may be accomplished using known buffers and conditions with a DNA ligase, such as T4 DNA ligase.
  • Plasmid refers to an extrachromosomal (usually) self- replicating genetic element. Plasmids are generally designated by a lower case “p” followed by letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the -art and will be apparent to the ordinarily skilled artisan.
  • Recombinant DNA cloning vector refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
  • Recombinant DNA expression vector refers to any recombinant DNA cloning vector in which a promoter to control transcription of the inserted DNA has been incorporated.
  • Transcription refers to the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence. ⁇ F- ⁇ ⁇ > h 0 JD rt 3 rt ⁇ ⁇ £ rt Q JD fi P $ g ⁇ JD ⁇ hi JD ⁇ JD JD ⁇
  • TJ 0 N > rt JU TJ P JD JD ⁇ ⁇ JD P . ⁇ - ⁇ h- 1 rt rt ⁇ ⁇ Ch ⁇ ⁇ ⁇
  • “Complementary” or “Complementarity”, as used herein, refers to pairs of bases (purines and pyrimidines) that associate through hydrogen bonding in a double stranded nucleic acid.
  • the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil .
  • Hybridization refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing.
  • the conditions employed in the hybridization of two non-identical, but very similar, complementary nucleic acids varies with the degree -of complementarity of the two strands and the length of the strands. Such techniques and conditions are well known to practitioners in this field.
  • isolated amino acid sequence refers to any amino acid sequence, however, constructed or synthesized, which is locationally distinct from the naturally occurring sequence.
  • isolated DNA compound refers to any DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location in genomic DNA.
  • isolated nucleic acid compound refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.
  • Primer refers to a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation.
  • Promoter refers to a DNA sequence which directs transcription of DNA to RNA.
  • Probe refers to a nucleic acid compound or a fragment, thereof, which hybridizes with another nucleic acid compound.
  • An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5X SSC (750 mM sodium chloride, 75 mM sodium citrate) , 50 mM sodium phosphate at pH 7.6, 5X Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in IX SSC at about 37-50°C.
  • 5X SSC 750 mM sodium chloride, 75 mM sodium citrate
  • 50 mM sodium phosphate at pH 7.6
  • 5X Denhardt's solution 10% dextran sulfate
  • 20 mg/mL denatured sheared salmon sperm DNA followed by washing the filters in IX SSC at about 37-50°C.
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc., as necessary to accommodate factors such as probe length and the like.
  • PCR refers to the widely-known polymerase chain reaction employing a thermally-stable DNA polymerase.
  • Leader sequence refers to a sequence ' of amino acids which can be enzymatically or chemically removed to produce the desired polypeptide of interest.
  • “Secretion signal sequence” refers to a sequence of amino acids generally present at the N-terminal region of a larger polypeptide functioning to initiate association of that polypeptide with the cell membrane compartments like endoplasmic reticulum and secretion of that polypeptide through the plasma membrane .
  • Wild-type albumin and Immunoglobulin proteins can be obtained from a variety of sources. For example, these proteins can be obtained from a cDNA library prepared from tissue or cells which express the mRNA of interest at a detectable level . Libraries can be screened with probes designed using the published DNA or protein sequence for the particular protein of interest. For example, immunoglobulin light or heavy chain constant regions are described in Adams, et al . (1980) Biochemistry 19:2711-2719; Goughet, et al . (1980) Biochemistry 19:2702-2710; Dolby, et al . (1980) Proc. Natl. Acad. Sci.
  • Screening a cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al . , Molecular Cloning: A
  • PCR primers can be designed based on published sequences .
  • the full-length wild-type sequences cloned from a particular species can serve as a template to create analogs, fragments, and derivatives that retain the ability to confer a longer plasma half-life on the GLP-1 compound that is part of the fusion protein. It is preferred that the Fc and albumin portions of the heterologous fusion proteins of the present invention be derived from the native human sequence in order to reduce the risk of potential immunogenicity of the fusion protein in humans .
  • the immunoglobulin portion of a fusion protein encompassed by the present invention contain only an Fc fragment of the immunoglobulin.
  • an Fc fragment may contain the hinge region along with the CH2 and CH3 domains or some other combination thereof .
  • These Fc fragments can be generated using PCR techniques with primers designed to hybridize to sequences corresponding to the desired ends of the fragment.
  • PCR primers can be designed which are complementary to internal albumin sequences.
  • PCR primers can also be designed to create 5 restriction enzyme sites to facilitate cloning into expression vectors .
  • DNA encoding the GLP-1 compounds of the present invention can be made by a variety of different methods including cloning methods like those described above as well
  • primers can be designed to PCR native GLP-1 compounds and fragments thereof.
  • the gene encoding a fusion protein can then be
  • the in vivo function and stability of the heterologous fusion proteins of the present invention can be optimized by adding small peptide linkers to prevent potentially unwanted domain interactions . Although these linkers can potentially be any length and consist of any combination of amino acids,
  • the linkers should not contain amino acids with extremely bulky side chains or amino acids likely to introduce significant 35 secondary structure. It is preferred that the linker be serine-glycine rich and be less than 30 amino acids in length. It is more preferred that the linker be no more than 20 amino acids in length. It is even more preferred that the linker be no more than 15 amino acids in length.
  • a preferred linker contains repeats of the sequence Gly-Gly- Gly-Gly-Ser. It is preferred that there be between 2 and 6 repeats of this sequence. It is even more preferred that there be between 3 and 4 repeats of this sequence.
  • the DNA encoding wild-type GLP-1, albumin, and Fc polypeptides and fragments thereof can be mutated either before ligation or in the context of a cDNA encoding an entire fusion protein.
  • a variety of mutagenesis techniques are well known in the art.
  • a mutagenic PCR method utilizes strand overlap extension to create specific base mutations for the purposes of changing a specific amino acid sequence in the corresponding protein.
  • This PCR mutagenesis requires the use of four primers, two in the forward orientation (primers A and C) and two in the reverse orientation (primers B and D) .
  • a mutated gene is amplified from the wild-type template in two different stages.
  • the first reaction amplifies the gene in halves by performing an A to B reaction and a separate C to D reaction wherein the B and C primers target the area of the gene to be mutated. When aligning these primers with the target area, they contain mismatches for the bases that are targeted to be changed.
  • the reaction products are isolated and mixed for use as the template for the A to D reaction. This reaction then yields the full, mutated product.
  • a gene encoding an entire fusion protein is produced it can be cloned into an appropriate expression vector. Specific strategies that can be employed to make the GLP-1 fusion proteins of the present invention are described in example 1. General methods to recombinantly express the heterologous fusion proteins of the present invention :
  • Host cells are transfected or transformed with expression or cloning vectors described herein for heterologous fusion protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the culture conditions such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook, et al . , supra. Methods of transfection are known to the ordinarily skilled artisan, for example, CaP0 and electroporation.
  • Transformations into yeast are typically carried out according to the method of van Solingen et al . , J " Bact . 130(2): 946-7 (1977) and Hsiao et al . , Proc . Natl . Acad. Sci . USA 76(8): 3829-33 (1979).
  • other methods for introducing DNA into cells such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene or polyomithine, may also be used.
  • Suitable host cells for cloning or expressing the nucleic acid (e.g., DNA) in the vectors herein include prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes include but are not limited to eubacteria, such as Gram- negative or Gram-positive organisms, for example, Enterobacteriacea such as E. coli.
  • Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 3 1.446); E. coli XI 776 (ATCC 3 1.537); E. coli strain W3 110 (ATCC 27.325) and K5 772 (ATCC 53.635).
  • prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g.. E. coli, Enterobacter , Erwinia, Klebisella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigeila, as well as Bacilli such as B. subtilis and B. lichentforrais (e.g., B. licheniformis 4 1 P disclosed in DD266,7 10, published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces . These examples are illustrative rather than limiting.
  • Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes.
  • strain W3 110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1A2 , which has the complete genotype ronA; E. coli W3 110 strain 9E4, which has the complete genotype ton4 ptr3 ; E.
  • coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA, ptr3 phoA E15 (argF-lac) 169 degP ompT /can'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990.
  • in vivo methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for fusion protein vectors.
  • Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.
  • Others include Schizosaccharomyces pombe [Beach and Nurse, Nature 290: 140-3 (1981); EP 139,383 published 2 May 1995]; Muyveromyces hosts [U.S. Patent No. 4,943,529; Fleer, et al .
  • K lactis (MW98-8C, CBS683, CBS4574) [de Louvencourt et al . , J " . Bacteriol . 154(2): 737-42 (1983)]; K. fiagilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K wickeramii (ATCC 24,178), K waltii (ATCC 56,500), K. drosophilarum (ATCC 36.906) [Van den Berg et al . , Bio / 'Technology 8 (2) : 135-9 (1990)]; K. thermotoierans, and K.
  • Schwanniomyces such as Schwanniomyces occidentulis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991) , and Aspergillus hosts such as A. nidulans [Ballance et al., Biochem . Biophys . Res . Comm. 112(1): 284-9 (1983)]; . Tilburn, et al . , Gene 26(2-3): 205-21 (1983); Yelton, et al . , Proc . Natl . Acad.
  • Methylotropic yeasts are selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotoruia. A list of specific species that are exemplary of this class of yeast may be found in C. Antony, The Biochemistry of Methylotrophs 269 (1982) .
  • Suitable host cells for the expression of the fusion proteins of the present invention are derived from multicellular organisms.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sp, Spodoptera high5 as well as plant cells .
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line [293 or 293 cells subcloned for growth in suspension culture, Graham, et al . , J. Gen Virol .
  • the fusion proteins of the present invention may be recombinantly produced directly, or as a protein having a signal sequence or other additional sequences which create a specific cleavage site at the N-terminus of the mature fusion protein.
  • the signal sequence may be a component of the vector, or it may be a part of the fusion protein- • encoding DNA that is inserted into the vector.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lp ⁇ , or heat-stable enterotoxin II leaders.
  • the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyvero yces cc-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179), or the signal described in WO 90/13646.
  • yeast invertase leader alpha factor leader (including Saccharomyces and Kluyvero yces cc-factor leaders, the latter described in U.S. Patent No. 5,010,182)
  • acid phosphatase leader the C. albicans glucoamylase leader
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species as well as viral secretory leaders .
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells .
  • Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria, the 2u plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells .
  • Selection genes will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement autotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the fusion protein-encoding nucleic acid, such as DHFR or thymidine kinase.
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line ,deficient in DHFR activity, prepared and propagated as described [Urlaub and Chasin, Proc . Natl . Acad . Sci . USA, 77(7): 4216-20 (1980) ] .
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 [Stinchcomb, et al .
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEPC1 [Jones, Genetics 85: 23-33 (1977)].
  • Expression and cloning vectors usually contain a promoter operably linked to the fusion protein-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ - lactamase and lactose promoter systems [Chang, et al . , Nature 275(5681): 617-24 (1978); Goeddel, et al . , Nature 281(5732) : 544-8 (1979)], alkaline phosphatase, a tryptophan (up) promoter system [Goeddel, Nucleic Acids Res .
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the fusion protein.
  • suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman, et al . , J. Biol . Chem .
  • glycolytic enzymes such as enolase, glycer ldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase .
  • enolase such as enolase, glycer ldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase .
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • Transcription of fusion protein-encoding mRNA from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis- B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems .
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis- B virus and Simi
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription.
  • Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-ketoprotein, and insulin) . Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the fusion protein coding sequence but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms
  • Such sequences are commonly available from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNAs or cDNAs . These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the fusion protein.
  • Various forms of a fusion protein may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage.
  • Cells employed in expression of a fusion protein can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • the analogs can be isolated and purified.
  • the following procedures are exemplary of suitable purification procedures: fractionation on carboxymethyl cellulose; gel filtration such as Sephadex G-75; anion exchange resin such as DEAE or Mono-Q; cation exchange such as CM or Mono-S; protein A sepharose to remove contaminants such as IgG; metal chelating columns to bind epitope-tagged forms of the polypeptide; reversed-phase HPLC; chromatofocusing; silica gel; ethanol precipitation; and ammonium sulfate precipitation.
  • the purification step(s) selected will depend on the nature of the production process used and the particular fusion protein produced.
  • fusion proteins comprising an Fc fragment can be effectively purified using a Protein A or Protein G affinity matix.
  • Low or high pH buffers can be used to elute the fusion protein from the affinity matrix. Mild elution conditions will aid in preventing irreversible denaturation of the fusion protein.
  • Imidazole-containing buffers can also be used.
  • Example 3 describes some successful purification protocols for the fusion proteins of the present invention.
  • Characterization of the heterologous fusion proteins of the presen t inven ti on Numerous methods exist to characterize the fusion proteins of the present invention. Some of these methods include: SDS-PAGE coupled with protein staining methods or immunoblotting using anti-IgG or anti-HSA antibodies. Other methods include matrix assisted laser desporption/ionization- mass spectrometry (MALDI-MS) , liquid chromatography/mass spectrometry, isoelectric focusing, analytical anion exchange, chromatofocussing, and circular dichroism to name a few. A representative number of heterologous fusion proteins were characterized using SDS-PAGE coupled with immunoblotting as well as mass spectrometry (See examples 4 and 5 and figures 3 and 4) .
  • MALDI-MS matrix assisted laser desporption/ionization- mass spectrometry
  • table 3 illustrates the calculated molecular mass for a representative number of fusion proteins as well as the mass as determined by mass spectrometry.
  • Figures 3 and 4 illustrate molecular weights of a representative number of fusion proteins as determined by SDS PAGE. All heterologous fusion proteins tested were expressed and secreted transiently. In addition, the Ig ⁇ signal sequence was cleaved to yield proteins with the correct N-terminus.
  • table 3 illustrates that in some instances the mass determined by mass spectrometry is greater than expected. This is the result of glycosylation of the Fc portion and the C terminal extension. Enzymatic digestion of the fusion proteins followed by reversed-phase HPLC and mass spectrometry can identify peptide fractions that contain sugar moieties. These fractions can then be N-terminal amino acid sequenced to identify the potential glycosylation site. For example, characterization of Exendin-4-Fc (SEQ ID NO: 29) shows that the serine at position 39 and threonine at position 50 are 0- linked glycosylated and the asaparagine at position 122 is N- linked glycosylated.
  • GLP-1 fusion proteins were also tested for activity. Numerous methods exist to detect
  • GLP-1 activity in vi tro and in vivo see examples 6, 7, 8 , and
  • Table 4 illustrates GLP-1 receptor activity associated with several GLP-1 fusions. The numbers are
  • Glycerin is an example of an isotonicity- adjusting excipient.
  • Pharmaceutically acceptable means suitable for administration to a human or other animal and thus, does not contain toxic elements or undesirable contaminants and does not interfere with the activity of the active compounds therein.
  • a pharmaceutically-acceptable salt form of the heterologous fusion proteins of the present invention may be used in the present invention.
  • Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p- bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzole acid, acetic acid, and the like.
  • Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid.
  • Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like.
  • bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.
  • Administration may be via any route known to be effective by the physician of ordinary skill.
  • Peripheral, parenteral is one such method.
  • Parenteral administration is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump.
  • Peripheral parenteral routes can include intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration.
  • heterologous fusion proteins of the present invention may also be amenable to administration by oral, rectal, nasal, or lower respiratory routes, which are non-parenteral routes. Of these non-parenteral routes, the lower respiratory route and the oral route are preferred.
  • the fusion proteins of the present invention can be used to treat a wide variety of diseases and conditions.
  • the fusion proteins of the present invention primarily exert their biological effects by acting at a receptor, referred to as the "GLP-1 receptor.”
  • Subjects with diseases and/or conditions that respond favorably to GLP-1 receptor stimulation or to the administration of GLP-1 compounds can therefore be treated with the GLP-1 fusion proteins of the present invention. These subjects are said to "be in need of treatment with GLP-1 compounds" or "in need of GLP-1 receptor stimulation” .
  • non-insulin dependent diabetes insulin dependent diabetes
  • stroke see WO 00/16797
  • myocardial infarction see WO 98/08531
  • obesity see WO 98/19698
  • catabolic changes after surgery see U.S. Patent No. 6,006,753
  • functional dyspepsia see irritable bowel syndrome
  • WO 99/64060 subjects requiring prophylactic treatment with a GLP-1 compound, e.g., subjects at risk for developing non-insulin dependent diabetes (see WO 00/07617).
  • Subjects with impaired glucose tolerance or impaired fasting glucose subjects whose body weight is about 25% above normal body weight for the subject's height and body build, subjects with a partial pancreatectomy, subjects having one or more parents with non-insulin dependent diabetes, subjects who have had gestational diabetes and subjects who have, had acute or chronic pancreatitis are at risk for developing non-insulin dependent diabetes .
  • An "effective amount" of a GLP-1 compound is the quantity which results in a desired therapeutic and/or prophylactic effect without causing unacceptable side- effects when administered to a subject in need of GLP-1 receptor stimulation.
  • a “desired therapeutic effect” includes one or more of the following: 1) an amelioration of the symptom(s) associated with the disease or condition; 2) a delay in the onset of symptoms associated with the disease or condition; 3) increased longevity compared with the absence of the treatment; and 4) greater quality of life compared with the absence of the treatment.
  • an "effective amount" of a GLP-1 compound for the treatment of diabetes is the quantity that would result in greater control of blood glucose concentration than in the absence of treatment, thereby resulting in a delay in the onset of diabetic complications such as retinopathy, neuropathy or kidney disease.
  • an "effective amount" of a GLP-1 compound for the prevention of diabetes is the quantity that would delay, compared with the absence of treatment, the onset of elevated blood glucose levels that require treatment with anti-hypoglycaemic drugs such as sulfonyl ureas, thiazolidinediones, insulin and/or bisguanidines .
  • the dose of fusion protein effective to normalize a patient's blood glucose will depend on a number of factors, among which are included, without limitation, the subject's sex, weight and age, the severity of inability to regulate blood glucose, the route of administration and bioavailability, the pharmacokinetic profile of the fusion protein, the potency, and the formulation.
  • the present invention comprises GLP-1 compounds that have improved biochemical and biophysical properties by virtue of being fused to an albumin protein, an albumin fragment, an albumin analog, a Fc protein, a Fc fragment, or a Fc analog.
  • GLP-1 compounds that have improved biochemical and biophysical properties by virtue of being fused to an albumin protein, an albumin fragment, an albumin analog, a Fc protein, a Fc fragment, or a Fc analog.
  • These heterologous proteins can be successfully expressed in host cells, retain signaling activities associated with activation of the GLP-1 receptor, and have prolonged half-lives.
  • Example 1 Construction of DNA encoding .heterologous fus.ion pro g teins
  • Fc A Fc portion of human IgGl was isolated from a cDNA library and contains the full hinge region and the CH2 and CH3 domains. A fragment containing 696 base pairs of this Fc portion of human IgGl was subcloned into the Nhel and Eco47III sites of mammalian expression vector pJB02 to create pJB02/Fc (see Figure 5) . DNA encoding the Ig ⁇
  • the hybridization reaction was carried out using equivalent amounts of each oligonucleotide (1 pm/ ⁇ l final concentration for each oligo) .
  • the mixture of oligonucleotides was heated for 5 min at 100°C in ligation buffer (50 mM Tris-HCl, pH 7.5 , 10 mM MgCl 2 , 10 mM DTT, ImM ATP, 25 ⁇ g/i ⁇ il bovine serum albumin) and then cooled over at least 2 hours to 30°C.
  • ligation buffer 50 mM Tris-HCl, pH 7.5 , 10 mM MgCl 2 , 10 mM DTT, ImM ATP, 25 ⁇ g/i ⁇ il bovine serum albumin
  • the resulting hybridization product was ligated for 2 hours at room temperature or overnight at 16°C to the pJB02/Fc vector backbone which had been digested with Nhel and Eco47III .
  • the ligation products were used to transform competent XL-1 Blue cells (Stratagene) .
  • Recombinant plasmids were screened for the presence of peptide coding inserts by digesting clones with Ncol (encoding the Kozak sequence and first Met of the signal peptide) and sequenced.
  • the resulting expression plasmid used for transfection assays was denoted pJB02-V8-GLP-l-Fc ( Figure 5) .
  • Invitrogen (Catalog # H-M12523M-pcDNA3.1/GS) and used as a template to isolate the cDNA encoding human serum albumin (HSA) .
  • HSA cDNA was prepared using PCR wherein the DNA encoding the leader sequence as well as the six amino acid pro-peptide was removed from the 5' end.
  • stop codons were added directly at the 3 ' end of the HSA coding sequence.
  • restriction enzyme sites were engineered at the 5' and 3' end to facilitate cloning.
  • the HSA DNA sequence present in the original vector purchased from Invitrogen contained a single base change in the 3' region of the gene (position 667) compared to the native human sequence.
  • the Ig ⁇ leader sequence fused to the Val -GLP-1 (7-37) sequence was generated as discussed in Example la. This DNA was ligated into the Nhel and Fspl sites of pJB02-HSA to
  • the vector pJB02-HSA was prepared as discussed in
  • Example lb DNA encoding the linker sequence [GGGGS] 3 was ligated in frame to the 5 ' end of the HSA encoding DNA to create pJB02-linker-HSA ( Figure 7) .
  • Example la DNA encoding the IgK signal sequence fused to
  • Exending-4 was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides:
  • the hybridization reaction was carried out as described in Example la.
  • the hybridized product was ligated to the pJB02 vector which had been digested with Nhel and Eco47III as described in Example la to create pJB02-Exendin-4-Fc .
  • Example lb DNA encoding the lg ⁇ signal sequence fused to Exending-4 was generated by in vi tro hybridization of the same overlapping and complementary oligonucleotides described in Example Id. Hybridization reactions were also carried out as described above. DNA was cloned into unique Nhel and Fspl sites in pJB02-HSA to create pJB02-Exendin-4- HSA.
  • the plasmid pJB02-linker-HSA was constructed as described in Example lc .
  • DNA encoding the Ig ⁇ signal sequence fused to Exendin-4 and the 5 ' part of the linker sequence was generated as in Example Id. This DNA was cloned into unique Nhel and BspEI sites in pJB02-linker-HSA to create pJB02-Exendin-4-linker-HSA.
  • Example Ig Construction of DNA encoding Val -GLP-1/C-Ex-Fc The plasmid pJB02-Exendin-4-Fc was prepared as described in Example Id. The Exendin-4 encoding DNA was o excised from the vector with Agel and Eco47III. The Val - GLP-1/C-Ex encoding DNA was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides : 5 ' -CCGGTCACGTGGAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTGGA GGGCCAGGCCGCCA - 3' [SEQ ID NO: 22]
  • the hybridization reaction was carried out as described in Example la.
  • the hybridized product was ligated in place of Exendin-4 in the pJB02-Exendin-4-Fc expression vector to
  • the plasmid pJB02-Exendin-4-Fc was prepared as described in Example Id.
  • the Exendin-4 encoding DNA was o excised from the vector with Agel and Eco47III .
  • Glu -GLP-1 encoding DNA was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides :
  • the plasmid pJB02-Exendin-4-Fc was prepared as described in Example Id.
  • the Exendin-4 encoding DNA was
  • Glu GLP-1/C-Ex encoding DNA was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides :
  • the hybridization reaction was carried out as described in Example la.
  • the hybridized product was ligated in place of Exendin-4 in the pJB02-Exendin-4-Fc expression vector to o create pJB02-Val -Glu -GLP-1/C-Ex-Fc .
  • Example lj Construction of DNA encoding Gly -GLP-l-Fc The plasmid pJB02-Exendin-4-Fc was prepared as described in Example Id. The Exendin-4 encoding DNA was g excised from the vector with ⁇ grel and Eco47III . The Gly - GLP-1 encoding DNA was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides :
  • the hybridization reaction was carried out as described in Example la.
  • the hybridized product was ligated in place of Exendin-4 in the pJB02-Exendin-4-Fc expression vector to
  • Example 2 Expression of heterologous fusion proteins Expression of the fusion proteins encoded by the DNA constructs of Example 1 was carried out by transiently transfecting HEK 293EBNA cells (both adherent and suspension) . Cells were counted and seeded 24 hours prior to transfection. The transfection cocktail was prepared by mixing FuGeneTM6 transfection reagent (Roche Molecular Biochemicals, catalog # 1814443) with OptiMEM (Gibco/BRL) and incubating at room temperature for 5 min at which point
  • Tables 1 and 2 provide further transfection details. Table 1: Reagents used in transient transfections of 293EBNA cells .
  • conditioned medium fetal bovine serum
  • a CUNO filter system Approximately 4.5 liters of conditioned medium (fusion protein expression level approximately 20 ⁇ g/ml) from large- scale transfections was filtered using a CUNO filter system and concentrated to 250 ml using a ProFlux tangenti Qal flow filtration system with a 10 K filter membrane.
  • Val -GLP-l-Fc was captured with a 5 ml HiTrap protein A column in lx PBS, pH 7.4 at a flow rate of 2 ml/min and eluted with 50 mM citric acid pH 3.3. Fractions (1 ml) were collected in tubes containing 4 ml lx PBS and lOO ⁇ l 1 M Tris pH 8.
  • Val -GLP-l-Fc was eluted using a gradient from 5% B (0.1% TFA in acetonitrile) to 100% B in 70 min. Eluant fractions (3 mis/tube) were collected. Acetonitrile was removed by vacuum drying and 1 ml of H 2 0 was added. The purified sample (approximately 32 mis) was dialyzed twice against 4 liters of lx PBS pH7.4.
  • the dialyzed sample was then filtered using a MILLEX-GV 0.22 um Filter Unit and concentration was determined using absorption at 280 nm.
  • Linker-HSA Approximately 6.5 liters of conditioned medium (fusion protein expression level approximately lO ⁇ g/ml) was filtered using a CUNO filter system and concentrated to 380 is using a ProFlux tangential flow filtration system with a 10 K filter membrane. The fusion protein was captured using a 50 ml Fast Flow Q column (Pharmacia) in 20 mM Tris pH 7.4 at a flow rate of 5ml/min. Protein was eluted using a gradient: from 0% to 50% 20mM Tris pH 7.4, 1M NaCl in 10 CV, then to 100%B in 2 CV.
  • Fractions containing the fusion protein were pooled and subjected to C4 Reverse Phase Chromatography in 0.1% TFA water at a flow rate of 5 ml/min.
  • the fusion protein was eluted using a gradient from 20% B (0.1% TFA in acetonitrile) to 90% B in 120min. Fractions (3.5 ml/tube) were collected. Acetonitrile was removed by vacuum drying.
  • conditioned medium fetal bovine serum
  • fusion protein expression level approximately 8 ⁇ g/ml
  • Exendin-4-Fc was captured with a 5 ml HiTrap protein A column in lx PBS, pH 7.4 at a flow rate of 2 ml/min and eluted with 50 mM citric acid pH 3.3. Fractions containing the fusion protein were pooled, filtered, and dialyzed against 4 liters of 1 x PBS over night. The dialyzed sample was then applied to a Superdex 75 60/60 column in lx PBS pH7.4, 0.5M NaCI at a flow rate of 10 ml/min. Fractions (20 ml/tube) containing the fusion protein were collected, pooled, and concentrated to about 1 mg/ml . Concentrated samples were then filtered using a MILLEX-GV 0.22 um Filter Unit.
  • Example 3d purification of Exendin-4-HSA and Exendin-4- linker-HSA was captured with a 5 ml HiTrap protein A column in lx PBS, pH 7.4 at
  • conditioned medium fetal bovine serum
  • CUNO filter system Approximately 1.1 liters of conditioned medium (fusion protein expression level approximately 6 ⁇ g/ml) was filtered using a CUNO filter system and concentrated to 175 is using a ProFlux tangential flow filtration system with a 3 OK filter membrane .
  • the fusion protein was captured using a 5 ml HiTrap Q- sepharose column (Pharmacia) in 20 mM Tris pH 7.4 at a flow rate of 2 ml/min. Protein was eluted using a gradient from 0% to 50% 20mM Tris pH 7.4 , 1M NaCI in 12 CV and then to 100%B in 4 CV.
  • Fractions containing the fusion protein were pooled and subjected to C4 Reverse Phase Chromatography in 0.1% TFA water at a flow rate of 5 ml/min.
  • the fusion protein was eluted using a gradient from 10% B (0.1% TFA in acetonitrile) to 100% B in 70 min.
  • Fractions (10 ml/tube) containing the fusion protein were collected. Acetonitrile was removed using a vacuum dryer.
  • Example 4 Characterization of fusion proteins by SDS PAGE: SDS-PAGE followed by immunoblotting was used to analyze both purified fusion protein as well as conditioned medium from cells transfected with various fusion protein expression vectors. SDS-PAGE was performed on a Novex Powerease 500 system using Novex 16% Tris-Glycine Precast gels (EC6498) , running buffer (lOx, LC2675) and sample buffer (L2676) . Samples were reduced with 50 mM DTT and heated 3-5 min at 95°C prior to loading.
  • the primary antibodies were either a polycolonal goat anti-IgGl or rabbit anti-HSA.
  • the secondary antibody was either an anti-goat IgG HRP or an anti-rabbit IgG HRP .
  • the secondary antibody was diluted 1:5000.
  • Figure 3A compares purified Fc protein to conditioned
  • FIG. 8 media from pJB02-Val -GLP-l-Fc and pJB02-Exendin-4-Fc transfected cells.
  • the decrease in mobility is consistent with the increased size due to the GLP-1 portion of the fusion protein.
  • Figure 3B similarly compares purified HSA with conditioned media from cells transfected with pJB02-
  • the instrument was operated in linear mode for the analysis of the GLP-1 fusion proteins in question.
  • the linear detector is a device that detects ions that travel down the flight tube of the MALDI-ToF-MS instrument. It measures the ion abundance over time and sends a signal to the digitizer for conversion.
  • the digitizer is an analog- to-digital converter that allows the signal from the mass spectrometer to be transferred to the computer, where it is reconstructed into a usable m/z spectrum.
  • a recrystallized saturated sinapinic acid solution (diluted in 50/50 Acn / H 2 0 and 0.1% TFA) was utilized as the ionization matrix.
  • Sinapinic acid is a proper matrix ' for proteins above 10 kDa.
  • Mass appropriate reference proteins were used for internal and external calibration files in order to obtain accurate mass determinations for the samples analyzed. Samples were all analyzed using a 1:2 sample to matrix dilution. The instrument was initially set up under the following linear detector conditions:
  • CEx refers to a C-terminal extension and comprises the sequence of Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser .
  • Linker is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly- Gly- Gly-
  • Figure 8 represents in vi tro dose response curves for Val -
  • Table 5a and 5b provide the in vi tro activity of a large group of
  • GLP-1 analogs that can be fused to an Fc or an albumin protein to make biologically active fusion proteins . These activities are compared to GLP-1 (7-37) OH.
  • CEx refers to a C-terminal extension and comprises the sequence of Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser .
  • Linker is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly- Gly- Gly-
  • Gly-Gly-Ser C2 is Ser-Ser-Gly-Ala-Ser-Ser-Gly-Ala.
  • amino acid sequences of the fusion proteins described in Tables 3 and 4 are represented in SED ID NO: 13 to SEQ ID NO: 31.
  • Val 8 -GLP-1-Human serum albumin amino acid sequence is represented by SEQ ID NO: 13.
  • Val -GLP-1-Linker-Human serum albumin ammo acid sequence is represented by SEQ ID NO: 14. 1 HVEGTFTSDV SSYLEGQAAK EFIAWLVKGR GGGGGSGGGG SGGGGSDAHK SEVAHRFKDL
  • Exendin-4-Human serum albumin amino acid sequence is represented by SEQ ID NO: 16.
  • Exendin-4-Linker-Human serum albumin amino acid sequence is represented by SEQ ID NO: 17.
  • Val -GLP-1-Cex-IgGl ammo acid sequence is represented by
  • Val -Glu GLP-1-CEx-IgGl ammo acid sequence is represented by SEQ ID NO : 21 .
  • Gly -Glu GLP- 1-CEx-Linker- IgGl amino acid sequence is represented by SEQ ID NO : 23 .
  • Gly -Glu GLP-l-CEx-Linker-IgG4 amino acid sequence is represented by SEQ ID NO : 24 .
  • Gly -Glu GLP-l-2Linker-IgGl amino acid sequence is represented by SEQ ID NO: 26. 1 HGEGTFTSDV SSYLEEQAAK EFIAWLVKGR GGGGGSGGGG SGGGGSGGGG SGGGGSGGGGGG
  • Gly -Glu GLP-l-2CEx-IgGl ammo acid sequence is represented by SEQ ID NO : 27 . 1 HGEGTFTSDV SSYLEEQAAK EFIAWLVKGR GSSGAPPPSS SGAPPPSAEP KSCDKTHTCP
  • Gly -Glu -Val -lie GLP-1-CEx-Linker-IgGl amino acid sequence is represented by SEQ ID NO : 28 .
  • Exendin-4-IgGl amino acid sequence is represented by SEQ ID NO : 29 .
  • Exendin-4-C2-IgGl amino acid sequence is represented by SEQ ID NO : 30 .
  • Exendin-4-Linker-IgGl amino acid sequence is represent by SEQ ID NO : 31 .
  • GLP-1-HSA was performed in cynomologus monkeys. Monkeys
  • Val -GLP-1 were determined using a radioimmunoassay that utilizes a polyclonal antiserum whose primary specificity is
  • FIG. 8 Figure 9 depicts the plasma concentration of Val -GLP-l-Fc o and Val -GLP-1-Linker-HSA following a single intravenous dose to two cynomologus monkeys.
  • the Fc fusion protein had a half-life of approximately 45 hours and the albumin fusion had a half-life of approximately 87 hours.
  • Example 8: In vivo pharmacodynamics of Exendin-4-IgGl Two chronically cannulated normal male beagle dogs were studied after an overnight fast. Arterial and venous vascular access ports were accessed, and a catheter was inserted percutaneously into a cephalic vein and secured.
  • Gly 8 -Glu 22 -GLP-l-CEx-Linker-IgGl by subcutaneous (SC) or intravenous (IV) administration.
  • Plasma concentrations of Gly 8 -Glu 2 -GLP-l-CEx-Linker-IgGl immunoreactivity were determined by radioimmunoassay in samples collected from 30 minutes predose to 216 hours postdose for both the IV and SC groups . These concentrations were subsequently used to determine the reported pharmacokinetic parameters .
  • the mean elimination half-life of IV administered Gly 8 -Glu 22 -GLP-l-CEx-Linker-IgGl was approximately 55 hours and the total body clearance was 1.5 mL/h/kg.
  • the mean elimination half-life of SC administered Gly 8 -Glu 22 -GLP-l-CEx-Linker-IgGl was approximately 38 hours.

Abstract

The present invention relates to glucagon-like-1 compounds fused to proteins that have the effect of extending the in vivo half-life of the peptides. These fusion proteins can be used to treat non-insulin dependent diabetes mellitus as well as a variety of other conditions.

Description

GLP-1 FUSION PROTEINS
The present invention relates to glucagon-like peptides including analogs and derivatives thereof fused to proteins that have the effect of extending the in vivo half-life of the peptides. These fusion proteins can be used to treat non-insulin dependent diabetes mellitus as well as a variety of other conditions.
Glucagon- ike Peptide 1 (GLP-1) is a 37 amino acid peptide that is secreted by the L-cells of the intestine in response to food ingestion. It has been found to stimulate insulin secretion (insulinotropic action) , thereby causing glucose uptake by cells and decreased serum glucose levels [see, e . g. , Mo sov, S., (1992) Int . J. Peptide Protein
Research, 40:333-343]. -However, GLP-1 is poorly active. A subsequent endogenous cleavage between the 6th and 7th position produces a more potent biologically active GLP-1 (7- 37) OH peptide. Numerous GLP-1 analogs and derivatives are known and are referred to herein as "GLP-1 compounds."
These GLP-1 analogs include the Exendins which are peptides found in the venom of the GILA-monster . The Exendins have sequence homology to native GLP-1 and can bind the GLP-1 receptor and initiate the signal transduction cascade responsible for the numerous activities that have been attributed to GLP-1 (7-37) OH.
GLP-1 compounds have a variety of physiologically significant activities. For example, GLP-1 has been shown to stimulate insulin release, lower glucagon secretion, inhibit gastric emptying, and enhance glucose utilization. [Nauck, M.A., et al . (1993) Diabetologia 36:741-744; Gutniak, M. , et al . (1992) New England J. of Med. 326:1316- 1322; Nauck, M.A. , et al . , (1993) J. Clin . Invest . 91:301- 307] . GLP-1 shows the greatest promise as a treatment for non-insulin dependent diabetes mellitus (NIDDM) . There are numerous oral drugs on the market to treat the insulin resistance associated with NIDDM. As the disease progresses, however, patients must move to treatments that stimulate the release of insulin and eventually to treatments that involve injections of insulin. Current drugs which stimulate the release of insulin, however, can also cause hypoglycemia as can the actual administration of insulin. GLP-1 activity, however, is controlled by blood glucose levels. When levels drop to a certain threshold level, GLP-1 is not active. Thus, there is no risk of hypoglycemia associated with treatment involving GLP-1. However, the usefulness of therapy involving GLP-1 peptides has been limited by their fast clearance and short half-lives. For example, GLP-1 (7-37) has a serum half-life of only 3 to 5 minutes. GLP-1 (7-36) amide has a time action of about 50 minutes when administered subcutaneously. Even analogs and derivatives that are resistant to endogenous protease cleavage, do not have half-lives long enough to avoid repeated administrations over a 24 hour period. Fast clearance of a therapeutic agent is inconvenient in cases where it is desired to maintain a high blood level of the agent over a prolonged period of time since repeated administrations will then be necessary. Furthermore, a long-acting compound is particularly important for diabetic patients whose past treatment regimen has involved taking only oral medication. These patients often have an extremely difficult time transitioning to a regimen that involves multiple injections of medication.
The present invention overcomes the problems associated with delivering a compound that has a short plasma half- life. The compounds of the present invention encompass GLP- 1 compounds fused to another protein with a long circulating half-life such as the Fc portion of an immunoglobulin or albumin .
Generally, small therapeutic peptides are difficult to manipulate because even slight changes in their structure can affect stability and/or biological activity. This has been especially true for GLP-1 compounds currently in development. For example, GLP-1 (7-37) OH has a tendency to undergo a conformational change from a primarily alpha helix structure to a primarily beta sheet structure. This beta sheet form results in aggregated material that is thought to be inactive. It was, therefore, surprising that biologically active GLP-1 fusion proteins with increased half-lives could be developed. This was especially unexpected given the difficulty of working with GLP-1 (7- 37) OH alone and the large size of the fusion partner relative to the small GLP-1 peptide attached.
Compounds of the present invention include heterologous fusion proteins comprising a first polypeptide with a N- terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) human albumin; b) human albumin analogs; and c) fragments of human albumin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide.
Compounds of the present invention also include a heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) human albumin; b) human albumin analogs; and c) fragments of human albumin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide via a peptide linker. It is preferred that the peptide linker is selected from the group consisting of : a) a glycine rich peptide; b) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]n where n is 1, 2, 3, 4, 5 or 6; and c) a peptide having the sequence [Gly-Gly-Gly-Gly- Ser]3. Additional compounds of the present invention include a heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) the Fc portion of an immunoglobulin; b) an analog of the Fc portion of an immunoglobulin; and c) fragments of the Fc portion of an immunoglobulin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide. The GLP-1 compound may be fused to the second polypeptide via a peptide linker. It is preferable that the peptide linker is selected from the group consisting of: a) a glycine rich peptide; b) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]n where n is i, 2, 3, 4, 5 or 6; and c) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser] 3. It is generally preferred that the GLP-1 compound that is part of the heterologous fusion protein have no more than 6 amino acids that are different from the corresponding amino acid in GLP-1 (7-37) OH, GLP-1 (7-36) OH, or Exendin-4. It is even more preferred that the GLP-1 compound have no more than 5 amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH, GLP-1 (7-36) OH, or Exendin-4. It is most preferred that the GLP-1 compound have no more than 4, 3, or 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37) OH, GLP-1 (7-36) OH, or Exendin-4. Preferably, a GLP-1 compound that is part of the heterologous fusion protein has glycine or valine at position 8.
The present invention also includes polynucleotides encoding the heterologous fusion protein described herein, vectors comprising these polynucleotides and host cells transfected or transformed with the vectors described herein. Also included is a process for producing a heterologous fusion protein comprising the steps of transcribing and translating a polynucleotide described herein under conditions wherein the heterolgous fusion protein is expressed in detectable amounts.
The present invention also encompasses a method for normalizing blood glucose levels in a mammal in need thereof comprising the administration of a therapeutically effective amount of a heterologous fusion protein described herein.
The invention is further illustrated with reference to the following drawings: Figure 1: IgGl Fc amino acid sequence encompassing the hinge region, CH2 and CH3 domains.
Figure 2: Human serum albumin amino acid sequence.
Figure 3: A.. SDS-PAGE gel and immunoblot of same gel illustrating the molecular weight of IgGl-Fc and GLP-l-Fc fusion proteins (Lane 1, MW standards; Lane 2, Purified Fc; lane 3, Mock transfected media; Lane 4, Val8-GLP-1-Fc; Lane 5, Exendin-4-Fc) B. SDS-PAGE gel and immunoblot of same gel illustrating the molecular weight of human HSA and GLP-1-HSA fusion proteins (Lane 1, MW standards; Lane 2, Purified HSA; lane 3, Mock transfected media; Lane 4, Val8-GLP-1-HSA; Lane 5, Val8-GLP-l-[Gly-Gly-Gly-Gly-Ser]3-HSA; Lane 6, Exendin-4- HSA; Lane 7, Exendin-4- [Gly-Gly-Gly-Gly-Ser] 3-HSA) .
Figure 4: SDS-PAGE gel of purified Fc, albumin, and GLP-1 fusion proteins (Lane 1, MW standards; Lane 2, purified Fc; Lane 3, Val8-GLP-1-Fc; Lane 4, Exendin-4-Fc; Lane 5, MW standard; Lane 6, Val8-GLP-1-HSA; Lane 7, Exendin-4-HSA; Lane 8, Exendin-4- [Gly-Gly-Gly-Gly-Ser] 3- HSA) .
Figure 5 : Expression cloning vector containing the Fc regions illustrated in figure 1.
Figure 6: Expression cloning vector containing the albumin sequence illustrated in figure 2.
Figure 7 : Expression cloning vector containing DNA encoding a 15 amino acid linker fused in frame and 5' of the albumin sequence illustrated in figure 2.
Figure 8: In vitro dose response activity of GLP-1 fusion proteins .
Figure 9: Pharmacokinetics of GLP-1 Fc and HSA fusion proteins . Figure 10: Glucodynamic response to Exendin-Fc in two normal fasted dogs.
Figure 11: Insulinotropic response to Exendin-Fc in two normal fasted dogs .
Figure 12 : DNA sequence encoding a human IgGl Fc region.
Figure 13 : DNA sequence encoding a human albumin protein.
The heterologous fusion proteins of the present invention comprise a GLP-1 compound fused to human albumin, a human albumin analog, a human albumin fragment, the Fc portion of an immunoglobulin, an analog of the Fc portion of an immunoglobulin, or a fragment of the Fc portion of an immunoglobulin. The C-terminus of the GLP-1 compound may be fused directly, or fused via a peptide linker, to the N- terminus of an albumin or Fc protein. These heterologous fusion proteins are biologically active and have an increased half-life compared to native GLP-1.
It is preferred that the GLP-1 compounds that make up part of the heterologous fusion protein encompass polypeptides having from about twenty-five to about thirty- nine naturally occurring or non-naturally occurring amino acids that have sufficient homology to native GLP-1 (7-37) OH such that they exhibit insulinotropic activity by binding to the GLP-1 receptor on β-cells in the pancreas. A GLP-1 compound typically comprises a polypeptide having the amino acid sequence of GLP-1 (7-37) OH, an analog of GLP-1 (7-37)OH, a fragment of GLP-1 (7-37) OH or a fragment of a GLP-1 (7-37) OH analog. GLP-1 (7-37) OH has the amino acid sequence of SEQ ID NO: 1:
7 8 9 10 11 12 13 14 15 16 17 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser- 18 19 20 21 22 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- 29 30 31 32 33 34 35 36 37
Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1)
By custom in the art, the amino terminus of GLP-1 (7- 37) OH has been assigned number residue 7 and the carboxy- terminus, number 37. The other amino acids in the polypeptide are numbered consecutively, as shown in SEQ ID NO: 1. For example, position 12 is phenylalanine and position 22 is glycine. GLP-1 compounds also encompass "GLP-1 fragments." A
GLP-1 fragment is a polypeptide obtained after truncation of one or more amino acids from the N-terminus and/or C- terminus of GLP-1 (7-37 ) OH or an analog or derivative thereof. The nomenclature used to describe GLP-1 (7-37) OH is also applicable to GLP-1 fragments. For example, GLP-1 (9- 36) OH denotes a GLP-1 fragment obtained by truncating two amino acids from the N-terminus and one amino acid from the C-terminus. The amino acids in the fragment are denoted by the same number as the corresponding amino acid in GLP-1 (7- 37) OH. For example, the N-terminal glutamic acid in GLP- 1(9-36) OH is at position 9; position 12 is occupied by phenylalanine; and position 22 is occupied by glycine, as in GLP-1 (7-37) OH. For GLP-1 (7-36) OH, the glycine at position 37 of GLP-1 (7-37) OH is deleted. GLP-1 compounds also include polypeptides in which one or more amino acids have been added to the N-terminus and/or C-terminus of GLP-1 (7-37) OH, or fragments or analogs thereof. It is preferred that GLP-1 compounds of this type have up to about thirty-nine amino acids . The amino acids in the "extended" GLP-1 compound are denoted by the same number as the corresponding amino acid in GLP-1 (7-37) OH. For example, the N-terminus amino acid of a GLP-1 compound obtained by adding two amino acids to the N-terminal of GLP- 1(7-37) OH is at position 5; and the C-terminus amino acid of a GLP-1 compound obtained by adding one amino acid to the C- terminus of GLP-1 (7-37) OH is at position 38. Thus, position 12 is occupied by phenylalanine and position 22 is occupied by glycine in both of these "extended" GLP-1 compounds, as in GLP-1 (7-37) OH. Amino acids 1-6 of an extended GLP-1 compound are preferably the same as or a conservative substitution of the amino acid at the corresponding position of GLP-1 (1-37) OH. Amino acids 38-45 of an extended GLP-1 compound are preferably the same as or a conservative substitution of the amino acid at the corresponding position of glucagon or Exendin-4.
GLP-1 compounds of the present invention encompass "GLP-1 analogs." A GLP-1 analog has sufficient homology to GLP-1 (7-37) OH or a fragment of GLP-1 (7-37) OH such that the analog has insulinotropic activity. Preferably, a GLP-1 analog has the amino acid sequence of GLP-1 (7-37) OH or a fragment thereof, modified so that from one, two, three, four or five amino acids differ from the amino acid in the corresponding position of GLP-1 (7-37 ) OH or a fragment of GLP-1 (7-37) OH. In the nonmenclature used herein to designate GLP-1 compounds, the substituting amino acid and its position is indicated prior to the parent structure. For example, Glu22-GLP-1 (7-37) OH designates a GLP-1 compound in which the glycine normally found at position 22 of GLP- 1(7-37) OH has been replaced with glutamic acid; Val8-Glu22_ GLP-1 (7-37) OH designates a GLP-1 compound in which alanine normally found at position 8 and glycine normally found at position 22 of GLP-1 (7-37) OH have been replaced with valine and glutamic acid, respectively.
GLP-1 compounds of the present invention also include "GLP-1 derivatives." A GLP-1 derivative is defined as a molecule having the amino acid sequence of GLP-1 or of a GLP-1 analog, but additionally having chemical modification of one or more of its amino acid side groups, α-carbon atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine ε-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal, amino group include, without limitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Furthermore, one or more side groups , or terminal groups , may be protected by protective groups known to the ordinarily- skilled protein chemist. The -carbon of an amino acid may be mono- or dimethylated.
Any GLP-1 compound can be part of the heterologous fusion proteins of the present invention as long as the GLP- 1 compound itself is able to bind and induce signaling through the GLP-1 receptor. GLP-1 receptor binding and signal transduction can be assessed using in vi tro assays such as those described in EP 619,322 and U.S. Patent No. 5,120,712, respectively. Numerous active GLP-1 fragments, analogs and derivatives are known in the art and any of these analogs and derivatives can also be part of the heterologous fusion proteins of the present invention. Some examples of novel GLP-1 analogs as well as GLP-1 analogs and derivatives known in the art are provided herein.
Some GLP-1 analogs and GLP-1 fragments known in the art include, for example, GLP-1 (7-34) and GLP-1 (7-35) , GLP-1 (7- 36), Gln9-GLP-l(7-37) , D-Gln9-GLP-1 (7-37) , Thr16-Lys18-GLP- 1(7-37), and Lys18-GLP-1 (7-37) . GLP-1 analogs such as GLP- 1(7-34) and GLP-l(7-35) are disclosed in U.S. Patent No.
5,118,666. Biologically processed forms of GLP-1 which have insulinotropic properties, such as GLP-1 (7-36) are also known. Other known biologically active GLP-1 compounds are disclosed in U.S. Patent No 5,977,071 to Hoffmann, et al .', U.S. Patent No. 5,545,618 to Buckley, et al . , and Adelhorst, et al . , J. Biol . Chem . 269 : 6275 (1994).
A preferred group of GLP-1 analogs is composed of GLP-1 analogs of formula I (SEQ ID NO: 2)
7 8 9 10 11 12 13 14 15 16 17
His-Xaa-Xaa-Gly-Xaa-Phe-Thr-Xaa-Asp-Xaa-Xaa- 18 19 20 21 22 23 24 25 26 27 28 Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe- 29 30 31 32 33 34 35 36 37 38 39 Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-
40 41 42 43 44 45 Xaa—Xaa—Xaa—Xaa—Xaa—Xaa
Formula I (SEQ ID NO: 2)
wherein:
Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, lie, Val,
Glu, Asp, or Lys; Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, lie, Val, Glu, Asp, or Lys;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, lie, Val,
Glu, Asp, or Lys; Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, lie, Tyr, Glu, Asp, Trp, or Lys; Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys; Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, lie, Val,
Glu, Asp, Trp, Tyr, or Lys; Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, or Lys; Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, lie, Val, Glu, Asp, Met, Trp, Tyr, or Lys; Xaa 'at position 21 is Glu, Asp, or Lys ;
Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys; Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, or Lys; Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, lie, Val,
Arg, Glu, Asp, or Lys; Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, lie, Val,
Glu, Asp, or Lys; Xaa at position 26 is Lys, Arg, Gin, Glu, Asp, or His; Xaa at position 27 is Leu, Glu, Asp, or Lys; Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, lie, Val,
Glu, Asp, or Lys; Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys; Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, lie, Val,
Glu, Asp, or Lys; Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, lie,
Glu, Asp, or Lys; Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His; Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, lie, Val,
Glu, Asp, or Lys; Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His; Xaa at position 37 is Pro, Gly, Ala, Ser, Thr, Leu, lie,
Val, Glu, Asp, or Lys, or is deleted; Xaa at position 38 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted; Xaa at position 39 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted; Xaa at position 40 is Gly, Asp, Glu, or Lys, or is deleted; Xaa at position 41 is Ala, Phe, Trp, Tyr, Glu, Asp, or Lys, or is deleted; Xaa at position 42 is Ser, Pro, Lys, Glu, or Asp, or is deleted;
Xaa at position 43 is Ser, Pro, Glu, Asp, or Lys, or is deleted;
Xaa at position 44 is Gly, Pro, Glu, Asp, or Lys, or is deleted; and Xaa at position 45 is Ala, Ser, Val, Glu, Asp, or Lys, or is deleted; provided that when the amino acid at position 37, 38, 39, 40, 41, 42, 43, or 44 is deleted, then each amino acid downstream of that amino acid is also deleted.
It is preferred that the GLP-1 compound of formula I contain less than six amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH or Exendin-4. It is more preferred that less than five amino acids differ from the corresponding amino acid in GLP-1 (7-37) OH or Exendin-4. It is even more preferred that less than four amino acids differ from the corresponding amino acid in GLP- 1(7-37) OH or Exendin-4.
GLP-1 compounds of the present invention include derivatives of formula I such as a C-l-6-ester, or amide, or C-l-6-alkylamide, or C-l-6-dialkylamide thereof. WO99/43706 describes derivatives of GLP-1 compounds of formula I and is incorporated by reference herein in its entirety. The compounds of formula I derivatized as described in WO99/43706 and underivatized are encompassed by the present invention. Another preferred group of GLP-1 compounds is composed of GLP-1 analogs of formula II (SEQ ID NO: 3):
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Thr-Ser-Asp-Xaa-Ser- 18 19 20 21 22 23 24 25 26 27 28
Xaa-Xaa-Leu-Glu-Gly-Xaa-Xaa-Ala-Xaa-Xaa-Phe-
29 30 31 32 33 34 35 36 37 Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Xaa-R Formula II (SEQ ID NO: 3)
wherein :
Xaa at position J is : L-histidine , D-histidine, desamino- histidine , 2 -amino-histidine , β-hydroxy-histidine, ho ohistidine , -f luoromethyl-histidine or oc-methyl- histidine ; Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr;
Xaa at position 9 is: Thr, Ser, Arg, Lys, Trp, Phe, Tyr,
Glu, or His;
Xaa at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His; Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
Val, Tyr, Glu, or Ala;
Xaa at position 18 is: His, Pro, Asp, Glu, Arg, Ser, Ala, or
Lys; Xaa at position 19 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or
Cys ;
Xaa at position 23 is: His, Asp, Lys, Glu, Gin, or Arg;
Xaa at position 24 is: Glu, Arg, Ala, or Lys;
Xaa at position 26 is: Trp, Tyr, Phe, Asp, Lys, Glu, or His; Xaa at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or
Lys;
Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
Xaa at position 31 is: Asp, Glu, Ser, Thr, Arg, Trp, or Lys;
Xaa at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu; Xaa at position 34 is: Glu, Lys, or Asp;
Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
Asp, Gly, Pro, His, or Glu;
Xaa at position 36 is: Thr, Ser, Asp, Trp, Tyr, Phe, Arg,
Glu, or His; R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr,
Phe, His, Gly, Gly-Pro, or is deleted.
Another preferred group of GLP-1 compounds is composed of GLP-1 analogs of formula III (SEQ ID NO: 4) :
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Xaa-Xaa-Thr-Ser-Asp-Xaa-Ser-
18 19 20 21 22 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Xaa-Xaa-Xaa-Xaa-Lys-Xaa-Phe- 29 30 31 32 33 34 35 36 37 Ile-Xaa-Trp-Leu-Xaa-Xaa-Xaa-Xaa-R formula III (SEQ ID NO : 4)
wherein :
Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or -methyl- histidine;
Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr;
Xaa at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His;
Xaa at position 12 is: His, Trp, Phe, or Tyr; Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu, or Ala;
Xaa at position 22: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or
Cys; Xaa at position 23 is: His, Asp, Lys, Glu, or Gin; Xaa at position 24 is: Glu, His, Ala, or Lys;
Xaa at position 25 is: Asp, Lys, Glu, or His;
Xaa at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys;
Xaa at position 30 is: Ala, Glu, Asp, Ser, or His; Xaa at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu;
Xaa at position 34 is: Glu, Lys, or Asp;
Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
Asp, Gly, Pro, His, or Glu;
Xaa at position 36 is: Arg, Glu, or His; R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
Another preferred group of GLP-1 compounds is composed of GLP-1 analogs of formula IV (SEQ ID NO : 5):
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Thr-Xaa-Thr-Ser-Asp-Xaa-Ser- 18 19 20 21 22 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Xaa-Xaa-Ala-Ala-Xaa-Glu-Phe- 29 30 31 32 33 34 35 36 37
Ile-Xaa-Trp-Leu-Val-Lys-Xaa-Arg-R formula IV (SEQ ID NO: 5)
wherein: Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine; Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, Met, or Thr;
Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
Val, Glu, or Ala; Xaa at position 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or Cys;
Xaa at position 23 is: His, Asp, Lys, Glu, or Gin; Xaa at position 26 is: Asp, Lys, Glu, or His; Xaa at position 30 is: Ala, Glu, Asp, Ser, or His; Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His, or Glu;
R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted. Another preferred group of GLP-1 compounds is composed of GLP-1 analogs of formula V (SEQ ID NO: 6) :
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
18 19 20 21 22 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Xaa-Xaa-Xaa-Ala-Lys-Glu-Phe- 29 30 31 32 33 34 35 36 37 Ile-Xaa-Trp-Leu-Val-Lys-Gly-Arg-R formula V (SEQ ID NO: 6) wherein :
Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine;
Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr; Xaa at position 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or
Cys; Xaa at position 23 is: His, Asp, Lys, Glu, or Gin; Xaa at position 24 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys; Xaa at position 30 is: Ala, Glu, Asp, Ser, or His; R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
Preferred GLP-1 compounds of formula I, II, III, IV, and V comprise GLP-1 analogs or fragments of GLP-1 analogs wherein the analogs or fragments contain an amino acid other than alanine at position 8 (position 8 analogs) . It is preferable that these position 8 analogs contain one or more additional changes at positions 9, 11, 12, 16, 18, 22, 23, 24, 26, 27, 30, 31, 33, 34, 35, 36, and 37 compared to the corresponding amino acid of native GLP-1 (7-37) OH. It is also preferable that these analogs have 6 or fewer changes compared to the corresponding amino acids in native GLP-1 (7- 37) OH or GLP-1 (7-36) OH. More preferred analogs have 5 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH or GLP-1 (7-36) OH or have 4 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH or GLP-1 (7-36) OH. It is even more preferable that these analogs have 3 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH or GLP- 1(7-36) OH. It is most preferable that these analogs have 2 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37 ) OH.
It has been found that the compounds of formula II, III, IV, and V have a reduced propensity to aggregate and generate insoluble forms. This is also important in the context of a fusion protein wherein the relatively small GLP-1 peptide must maintain an active conformation despite being fused to a much larger protein. Preferred GLP-1 compounds of formula II, III, IV, and V encompassed by the fusion proteins of the present invention comprise GLP-1 analogs or fragments of GLP-1 analogs in which glycine at position 22 and preferably alanine at position 8 have been replaced with another amino acid.
When position 22 is aspartic acid, glutamic acid, arginine or lysine, position 8 is preferably glycine, valine, leucine, isolecine, serine, threonine or methionine and more preferably valine or glycine. When position 22 is a sulfonic acid such as cysteic acid, position 8 is preferably glycine, valine, leucine, isolecine, serine, threonine or methionine and more preferably valine or glycine. Other preferred GLP-1 compounds include GLP-1 analogs of formula IV (SEQ ID NO: 5) wherein the analogs have the sequence of GLP-1 (7-37) OH except that the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 30 is glutamic acid, aspartic acid, serine, or histidine and more preferably glutamatic acid.
Other preferred GLP-1 compounds include GLP-1 analogs of formula IV (SEQ ID NO: 5) wherein the analogs have the sequence of GLP-1 (7-37) OH except that the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 37 is histidine, lysine, arginine, threonine, serine, glutamic acid, aspartic acid, tryptophan, tyrosine, phenylalanine and more preferably histidine.
Other preferred GLP-1 compounds include GLP-1 analogs of formula IV (SEQ ID NO: 5) wherein the analogs have the sequence of GLP-1 (7-37) OH except that the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 22 is glutamic acid, lysine, aspartic acid, or arginine and more preferably glutamic acid or lysine and position 23 is lysine, arginine, glutamic acid, aspartic acid, and histidine and more preferably lysine or glutamic acid.
Other preferred GLP-1 compounds include GLP-1 analogs of formula V (SEQ ID NO: 6) wherein the analogs have the sequence of GLP-1 (7-37) OH except that the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 22 is glutamic acid, lysine, aspartic acid, or arginine and more preferably glutamine acid or lysine and position 27 is alanine, lysine, arginine, tryptophan, tyrosine, phenylalanine, or histidine and more preferably alanine .
Other preferred GLP-1 compounds include GLP-1 analogs of formula II wherein the analogs have the sequence of GLP- 1(7-37) OH except that the amino acid at position 8 and one, two, or three amino acids selected from the group consisting of position 9, position 11, position 12, position 16, position 18, position 22, position 23, position 24, position 26, position 27, position 30, position 31, position 33, position 34, position 35, position 36, and position 37, differ from the amino acid at the corresponding position of native GLP-1 (7-37 ) OH.
Other preferred GLP-1 compounds of formula II include: Val8-GLP-l(7-37)OH, Gly8-GLP-1 (7-37 ) OH, Glu22-GLP-1 (7-37 ) OH, Asp22-GLP-l(7-37)OH, Arg22_GLP-l (7-37 ) OH, Lys22_GLP-l (7- 37)OH, Cys22_GLP-l(7-37)OH, Val8-Glu22-GLP-1 (7-37 ) OH, Val8- Asp2 -GLP-l(7-37)OH, Val8-Arg22_GLP-l (7-37 ) OH, Val8-Lys22- GLP-l(7-37)OH, Val8-Cys22-GLP-1 (7-37 ) OH, Gly8-Glu22-GLP-1 (7- 37) OH, Gly8-Asp22-GLP-l(7-37)OH, Gly8-Arg22_GLP-l (7-37 ) OH, Gly8-Lys22-GLP-l(7-37)OH, Gly8-Cys22_GLP-l (7-37 ) OH, Glu22- GLP-l(7-36)OH, Asp2 -GLP-1 (7-36.0H, Arg22-GLP-1 (7-36) OH, Lys22-GLP-l(7-36)OH, Cys22-GLP-1 (7-36) OH, Val8-Glu22-GLP- 1(7-36) OH, Val8-Asp22_GLP-l(7-36)OH, Val8-Arg22-GLP-1 (7- 36) OH, Val8-Lys22_GLP-l(7-36)OH, Val8-Cys22_GLP-l (7-36) OH, Gly8-Glu22_GLP-l(7-36)OH, Gly8-Asp22-GLP-1 (7-36) OH, Gly8- Arg22-GLP-l(7-36)OH, Gly8-Lys22-GLP-1 (7-36) OH, Gly8-Cys22- GLP-l(7-36)OH, Lys23-GLP-1 (7-37 ) OH, Val8-Lys23_GLP-l (7- 37) OH, Gly8-Lys23_GLP-l(7-37)OH, His24_GLP-l (7-37) OH, Val8- His2 -GLP-l(7-37)OH, Gly8-His24-GLP-1 (7-37) OH, Lys2 -GLP- 1(7-37) OH, Val8-Lys24-GLP-l(7-37)OH, Gly8-Lys23_GLP-l (7- 37)OH, Glu30_GLP-l(7-37)OH, Val8-Glu30-GLP-1 (7-37) OH, Gly8- G1U30_G P-1(7-37)OH, Asp30_GLP-l (7-37 ) OH, Val8-Asp30_GLP- l(7-37)OH, Gly8-Asp30_GLP-l(7-37)OH, Gln30_GLP-l (7-37 ) OH, Val8-Gln30-GLP-1(7-37)OH, Gly8-Gln30-GLP-1 (7-37 ) OH, Tyr30_ GLP-1 (7-37)OH, Val8-Tyr30-GLP-1 (7-37 ) OH, Gly8-Tyr30-GLP-1 (7- 37)OH, Ser30-GLP-1(7-37)OH, Val8-Ser30_GLP-l (7-37) OH, Gly8- Ser 0-GLP-1(7-37)OH, His30-GLP-1 (7-37) OH, Val8-His30-GLP- 1(7-37) OH, Gly8-His30_GLP-l(7-37)OH, Glu34-GLP-1 (7-37) OH, Val8-Glu3 -GLP-l(7-37)OH, Gly8-Glu34-GLP-1 (7-37 ) OH, Ala34- GLP-l(7-37)OH, Val8-Ala34-GLP-1 (7-37 ) OH, Gly8-Ala34-GLP-1 (7- 37) OH, Gly34-GLP-l(7-37)OH, Val8-Gly34-GLP-1 (7-37 ) OH, Gly8- Gly34-GLP-l(7-37)OH, Ala35-GLP-1 (7-37) OH, Val8-Ala35-GLP- l(7-37)OH, Gly8-Ala35-GLP-l(7-37)OH, Lys35-GLP-1 (7-37) OH, Val8-Lys35_GLP-l(7-37)OH, Gly8-Lys35-GLP-1 (7-37) OH, His35- GLP-1 (7-37)OH Val8-His35-GLP-1 (7-37 ) OH, Gly8-His35-GLP-1 (7- 37) OH, Pro35_GLP-l(7-37)OH, Val8-Pro35_GLP-l (7-37) OH, Gly8- Pro35_G P-l(7-37)OH, Glu35_GLP-l (7-37 ) OH Val8-Glu35-GLP-1 (7- 37)OH, Gly8-Glu35_GLP-l(7-37)OH, Val8-Ala27_GLP-l (7-37) OH, Val8-His37_GLP-l(7-37)OH, Val8-Glu22_Lys23-GLP-l (7-37 ) OH, Val8-Glu22-Glu23_GLP-l(7-37)OH, Val8-Glu22-Ala27_GLP-l (7- 37) OH, Val8-Gly3 -Lys35-GLP-l(7-37)OH, Val8-His37-GLP-1 (7- 37)OH, Gly8-His37-GLP-l(7-37)OH, Val8-Glu22-Ala27-GLP-1 (7- 3-7) OH, Gly8-Glu22-Ala27_GLP-l(7-37)OH, Val8-Lys22_Glu23-GLP- 1(7-37) OH, and Gly8-Lys22-Glu2 -GLP-1 (7-37) OH.
Another preferred group of GLP-1 analogs and derivatives for use in the present invention is composed of molecules of formula VI (SEQ ID NO : 7)
Rl-X-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-
Ser-Tyr-Leu20-Y -Gly-Gln-Ala-Ala25_ ys- Z - Phe-Ile-Ala30-Trp-Leu-Val-Lys-Gly35-Arg-R2 formula VI (SEQ ID NO : 7 ) wherein: Ri is selected from the group consisting of L- histidine, D-histidine, desa ino-histidine, 2-amino- histidine, β-hydroxy-histidine, homohistidine, alpha- fluoromethyl-histidine, and alpha-methyl-histidine; X is selected from the group consisting of Ala, Gly, Val, Thr, lie, and alpha-methyl-Ala; Y is selected from the group consisting of Glu, Gin, Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu, Gin, Ala, Thr, Ser, and Gly; and R2 is Gly-OH. Another preferred group of GLP-1 compounds for use in the present invention is disclosed in WO 91/11457, and consists essentially of GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7- 36), or GLP-l(7-37), or the amide form thereof, and pharmaceutically-acceptable salts thereof, having at least one modification selected from the group consisting of:
(a) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine, or D-lysine for lysine at position 26 and/or position 34; or substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, lysine, or a D-arginine for arginine at position 36;
(b) substitution of an oxidation-resistant amino acid for tryptophan at position 31;
(c) substitution of at least one of: tyrosine for valine at position 16; lysine for serine at position 18; aspartic acid for glutamic acid at position 21; serine for glycine at position 22; arginine for glutamine at position 23; arginine for alanine at position 24; and glutamine for lysine at position 26; and
(d) substitution of at least one of: glycine, serine, or cysteine for alanine at position 8; aspartic acid, glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glutamic acid at position 9; serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glycine at position 10; and glutamic acid for aspartic acid at position 15; and
(e) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine, or the D- or N-acylated or alkylated form of histidine for histidine at position 7; wherein, in the substitutions is (a), (b) ,
(d) , and (e) , the substituted amino acids can optionally be in the D-form and the amino acids substituted at position 7 can optionally be in the N-acylated or N-alkylated form.
Because the enzyme, dipeptidyl-peptidase IV (DPP IV) , may be responsible for the observed rapid in vivo inactivation of administered GLP-1, [see, e.g., Mentlein, R., et al., Eur. J. Biochem. , 214:829-835 (1993)], GLP-1 analogs and derivatives that are protected from the activity of DPP IV in the context of a fusion protein are preferred, and fusion proteins wherein the GLP-1 compound is Gly8-GLP- l(7-37)OH, Val8-GLP-l(7-37)OH, α-methyl-Ala8-GLP-l (7-37 ) OH, or Gly8-Gln l-GLP-1 (7-37) OH are more preferred.
Another preferred group of GLP-1 compounds for use in the present invention consists of the compounds of formula VII (SEQ ID NO: 8) claimed in U.S. Patent No. 5,512,549, which is expressly incorporated herein by reference.
R^-Ala-Glu-Gly10- Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20- Glu-Gly-Gln-Ala-Ala25_χaa-Glu-Phe-Ile-Ala30-
Trp-Leu-Val-Lys-Gly35-Arg-R3
I
R2 formula VII (SEQ ID NO: 8) wherein R! is selected from the group consisting of 4- imidazopropionyl, 4-imidazoacetyl, or 4-imidazo- α, α dimethyl-acetyl ; R2 is selected from the group consisting of C6-C10 unbranched acyl, or is absent; R3 is selected from the group consisting of Gly-OH or NH2 ; and,
Xaa is Lys or Arg.
More preferred compounds of formula IV for use in the present invention are those in which Xaa is Arg and R2 is 6~ ιo unbranched acyl. Even more preferred compounds of formula IV for use in the present invention are those in which Xaa is Arg, R is Cg-Cio unbranched acyl, and R3 is Gly-OH. Other highly-preferred compounds of formula IV for use in the present invention are those in which Xaa is Arg,
R2 is Cg-Cio unbranched acyl, R3 is Gly-OH, and R^ is 4- imidazopropionyl . An especially preferred compound of formula IV for use in the present invention is that in which
Xaa is Arg, R2 is Cs unbranched acyl, R3 is Gly-OH, and R! is 4-imidazopropionyl .
Preferably, the GLP-1 compounds comprise GLP-1 analogs wherein the backbone for such analogs or fragments contains an amino acid other than alanine at position 8 (position 8 analogs) . The backbone may also include L-histidine, D- histidine, or modified forms of histidine such as desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, or α-methyl- histidine at position 7. It is preferable that these position 8 analogs contain one or more additional changes at positions 12, 16, 18, 19, 20, 22, 25, 27, 30, 33, and 37 compared to the corresponding amino acid of native GLP-1 (7- 37) OH. It is more preferable that these position 8 analogs contain one or more additional changes at positions 16, 18, 22, 25 and 33 compared to the corresponding amino acid of native GLP-1 (7-37) OH.
In a preferred embodiment, the GLP-1 analog is GLP-1 (7- 37) OH wherein the amino acid at position 12 is selected from the group consisting of tryptophan or tyrosine. It is more preferred that in addition to the substitution at position 12, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 12 and 8, the amino acid at position 22 is substituted with glutamic acid.
In another preferred embodiment, the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 16 is selected from the group consisting of tryptophan, isoleucine, leucine, phenylalanine, or tyrosine. It is more preferred that in addition to the substitution at position 16, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 16 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 16 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 16 and 8, the amino acid at position 37 is substituted with histidine.
In another preferred embodiment, the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 18 is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, leucine, or isoleucine, preferably tryptophan, tyrosine, and isoleucine. It is more preferred that in addition to the substitution at position 18, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 18 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 18 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 18 and 8, the amino acid at position 37 is substituted with histidine
In another preferred embodiment, the GLP-1 analog is GLP-1 (7-37 ) OH wherein the amino acid at position 19 is selected from the group consisting of tryptophan or phenylalanine, preferably tryptophan. It is more preferred that in addition to the substitution at position 19, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 19 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 19 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 19 and 8, the amino acid at position 37 is substituted with histidine
In another preferred embodiment, the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 20 is phenylalanine, tyrosine, or tryptophan. It is more preferred that in addition to the substitution at position 20, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 20 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 20 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 20 and 8, the amino acid at position 37 is substituted with histidine
In another preferred embodiment, the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 25 is selected from the group consisting of valine, isoleucine, and leucine, preferably valine. It is more preferred that in addition to the substitution at position 25, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 25 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 25 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 25 and 8, the amino acid at position 37 is substituted with histidine.
In another preferred embodiment, the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 27 is selected from the group consisting of isoleucine or alanine. It is more preferred that in addition to the substitution at position 27, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 27 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 27 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 27 and 8, the amino acid at position 37 is substituted with histidine
In another preferred embodiment, the GLP-1 analog is GLP-1 (7-37) OH wherein the amino acid at position 33 is isoleucine. It is more preferred that in addition to the substitution at position 33, the amino acid at position 8 is substituted with glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine. It is even more preferred that in addition to the substitutions at position 33 and 8, the amino acid at position 22 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 33 and 8, the amino acid at position 30 is substituted with glutamic acid. It is also preferred that in addition to the substitutions at positions 33 and 8, the amino acid at position 37 is substituted with histidine
The GLP-1 compounds have modifications at one or more of the following positions: 8, 12, 16, 18, 19, 20, 22, 25, 27, 30, 33, and 37. These GLP-1 compounds show increased potency compared with GLP-1 (7-37 ) OH and comprise the amino acid sequence of formula IX (SEQ ID NO: 12)
Xaa7-Xaa8-Glu-Gly-Thr-Xaa2-Thr-Ser-Asp-Xaaι6-Ser- Xaaχ8-Xaaι9-Xaa2o-Glu-Xaa22-Gln-Ala-Xaa25-Lys-Xaa27- Phe-Ile-Xaa30-Trp-Leu-Xaa33-Lys-Gly-Arg-Xaa37
Formula IX (SEQ ID NO : 12 ) wherein:
Xaa7 is: L-histidine, D-histidine, desamino-histidine, 2- amino-histidine, β-hydroxy-histidine, homohistidine, α-f luoromethyl-histidine, or α- methyl-histidine; Xaa8 is: Ala, Gly, Val, Leu, lie, Ser, or Thr; Xaai2 is: Phe, Trp, or Tyr; Xaai6 is: Val, Trp, lie, Leu, Phe, or Tyr; Xaais is: Ser, Trp, Tyr, Phe, Lys, lie, Leu, Val; Xaai9 is: Tyr, Trp, or Phe; Xaa2o is: Leu, Phe, Tyr, or Trp; Xaa 2 is: Gly, Glu, Asp, or Lys; Xaa25 is: Ala, Val, lie, or Leu; Xaa27 is: Glu, lie, or Ala;
Xaa3o is : Ala or Glu Xaa33 is: Val, or lie; and Xaa37 is: Gly, His, NH2, or is absent.
Some preferred GLP-1 compounds of formula IX include
GLP-1 (7-37) OH, GLP-1 (7-36) -NH2, Gly8-GLP-1 (7-37 ) OH, Gly8-GLP- 1(7-36)NH2, Val8-GLP-l(7-37)OH, Val8-GLP-1 (7-36)NH2, Leu8- GLP-l(7-37)OH, Leu8-GLP-1 (7-36)NH2, Ile8-GLP-1 (7-37 ) OH, Ile8- GLP-1(7-36)NH2, Ser8-GLP-1 (7-37 ) OH, Ser8-GLP-1 (7-36)NH2, Thr8-GLP-l(7-37)OH, Thr8-GLP-1 (7-36) H2, Val8-Tyr12-GLP-1 (7- 37)OH, Val8-Tyr12-GLP-1(7-36)NH2, Val8-Tyr16-GLP-1 (7-37) OH, Val8-Tyrlδ-GLP-1(7-36)NH2, Val8-Glu 2-GLP-1 (7-37 ) OH, Val8- Glu22-GLP-1(7-36)NH2, Gly8-Glu22-GLP-1 (7-37) OH, Gly8-Glu22-GLP- 1(7-36)NH2, Val8-Asp22-GLP-l(7-37)OH, Val8-Asp2 -GLP-1 (7- 36)NH2, Gly8-Asp22-GLP-l(7-37)OH, Gly8-Asp22-GLP-1 (7-36)NH2, Val8-Lys22-GLP-l(7-37)OH, Val8-Lys22-GLP-1 (7-36)NH2, Gly8- Lys2 -GLP-l(7-37)OH, Gly8-Lys22-GLP-1 (7-36) NH2 , Leu8-Glu22-GLP- 1(7-37) OH, Leu8-Glu22-GLP-1(7-36)NH2, Ile8-Glu22-GLP-1 (7- 37)OH, Ile8-Glu22-GLP-1(7-36)NH2, Leu8-Asp22-GLP-1 (7-37 ) OH, Leu8-Asp 2-GLP-1(7-36)NH2, Ile8-Asp22-GLP-1 (7-37 ) OH, Ile8- Asp22-GLP-1(7-36)NH2, Leu8-Lys22-GLP-1 (7-37) OH, Leu8-Lys22-GLP- 1(7-36)NH2, Ile8-Lys22-GLP-l(7-37)OH, Ile8-Lys22-GLP-1 (7- 36)NH2, Ser8-Glu22-GLP-l(J-37)OH, Ser8-Glu22-GLP-1 (7-36)NH2/ Thr8-Glu22-GLP-l(7-37)OH, Thr8-Glu22-GLP-1 (7-36) H2, Ser8- Asp22-GLP-l(7-37)OH, Ser8-Asp22-GLP-1 (7-36)NH2, Thr8-Asp22-GLP- l(7-37)OH, Thr8-Asp22-GLP-1(7-36)NH2, Ser8-Lys22-GLP-1 (7- 37)OH, Ser8-Lys22-GLP-1(7-36)NH2, Thr8-Lys22-GLP-1 (7-37 ) OH, Thr8-Lys22-GLP-1(7-36)NH2/ Glu22-GLP-1 (7-37) OH, Glu22-GLP-1 (7- 36)NH2, Asp22-GLP-l(7-37)OH, Asp22-GLP-1 (7-36)NH2 , Lys22-GLP- l(7-37)OH, Lys22-GLP-1(7-36)NH2, Val8-Ala27-GLP-1 (7-37 ) OH, Val8-Glu22-Ala27-GLP-l(7-37)OH, Val8-Glu30-GLP-1 (7-37) OH, Val8- Glu30-GLP-1(7-36)NH2, Gly8-Glu30-GLP-1 (7-37 ) OH, Gly8-Glu30-GLP- 1(7-36)NH2, Leu8-Glu30-GLP-1(7-37)OH, Leu8-Glu30-GLP-1 (7-
36)NH2, Ile8-Glu30-GLP-1(7-37)OH, Ile8-Glu30-GLP-1 (7-36)NH2 , Ser8-Glu30-GLP-1(7-37)OH, Ser8-Glu30-GLP-1 (7-36)NH2, Thr8- Glu30-GLP-1(7-37)OH, Thr8-Glu30-GLP-1 (7-36)NH2, Val8-His37-GLP- l(7-37)OH, Val8-His37-GLP-1(7-36)NH2, Gly8-His37-GLP-1 (7-37 ) OH, Gly8-His37-GLP-1(7-36)NH2, Leu8-His37-GLP-1 (7-37) OH, Leu8-His37- GLP-1(7-36)NH2, Ile8-His37-GLP-1 (7-37 ) OH, Ile8-His37-GLP-1 (7- 36)NH2, Ser8-His37-GLP-l(7-37)OH, Ser8-His37-GLP-1 (7-36)NH2, Thr8-His37-GLP-l(7-37)OH, Thr8-His37-GLP-1 (7-36)NH2.
Some preferred GLP-1 compounds of formula IX having multiple substitutions include GLP-1 (7-37) OH wherein position 8 is valine or glycine, position 22 is glutamic acid, position 16 is tyrosine, leucine or tryptophan, position 18 is tyrosine, tryptophan, or isoleucine, position 25 is valine and position 33 is isoleucine. Other preferred GLP-1 compounds include the following: Val8-Tyr16-GLP-1 ((7- 37) OH, Val8-Tyr12-Glu22-GLP-l(7-37)OH, Val8-Tyr16-Phe19-GLP- l(7-37)OH. Val8-Tyr16-Glu22-GLP-l(7-37)OH, Val8-Trp15-Glu22- GLP-l(7-37)OH, Val8-Leu16-Glu2 -GLP-1 (7-37 ) OH, Val8-Ile15- Glu22-GLP-l(7-37)OH, Val8-Phe16-Glu22-GLP-1 (7-37 ) OH/ Val8- Trp18-Glu2-GLP-1 (7-37 ) OH, Val8-Tyr18-Glu2 -GLP-1 (7-37 ) OH, Val8-Phe18-Glu22-GLP-l(7-37)OH, and Val8-Ile18-Glu22-GLP-1 (7- 37)OH. The GLP-1 compounds of the present invention also encompass Exendin compounds . Exendin-3 and Exendin-4 are biologically active peptides first isolated from Helodermatidae lizard venoms and have been shown to bind the GLP-1 receptor and stimulate cAMP-dependent H+ production in mammalian parietal cells. Exendin-3 and Exendin-4 are both 39 amino acid peptides which are approximately 53% homologous to GLP-1. They act as potent agonists of GLP-1 activity. Notably, an N-terminally truncated derivative of Exendin, known as Exendin (9-39 amino acids) , is an inhibitor of Exendin-3, Exendin-4 and GLP-1.
An Exendin compound typically comprises a polypeptide having the amino acid sequence of Exendin-3, Exendin-4, or an analog or fragment thereof. Exendin-3 and Exendin-4 are disclosed in U.S. Patent No. 5,424,286.
Exendin-3 has the amino acid sequence of SEQ ID NO : 9:
7 8 9 10 11 12 13 14 15 16 17
His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-
18 19 20 21 22 23 24 25 26 27 28 Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-
29 30 31 32 33 34 35 36 37 38 39 Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser- 40 41 42 43 44 45 Gly-Ala-Pro-Pro-Pro-Ser (SEQ ID NO: 9) Exendin-4 has the amino acid sequence of SEQ ID NO: 10: 7 8 9 10 11 12 13 14 15 16 17 His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- 18 19 20 21 22 23 24 25 26 27 28
Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe- 29 30 31 32 33 34 35 36 37 38 39 Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser- 40 41 42 43 44 45 Gly-Ala-Pro-Pro-Pro-Ser
(SEQ ID NO: 10)
GLP-1 compounds also include Exendin fragments which are polypeptides obtained after truncation of one or more amino acids from the N-terminus and/or C-terminus of Exendin or an Exendin analog. Furthermore, GLP-1 compounds include Exendin polypeptides in which one or more amino acids have been added to the N-terminus and/or C-terminus of Exendin or fragments thereof. Exendin compounds of this type have up to about forty-five amino acids.
GLP-1 compounds also include "Exendin analogs." An Exendin analog has sufficient homology to Exendin-4, Exendin-3, or a fragment thereof such that the analog has insulinotropic activity. The activity of Exendin fragments and/or analogs can be assessed using in vi tro assays such as those described' in EP 619,322 and U.S. Patent No. 5,120,712.
Preferably, an Exendin analog has the amino acid sequence of Exendin-4 or a fragment thereof, modified so that from one, two, three, four or five amino acids differ from the amino acid in corresponding position of Exendin-4 or the fragment of Exendin-4. In the nonmenclature used herein to designate Exendin compounds, the substituting amino acid and its position is indicated prior to the parent o structure. For example, Val -Exendin-4 designates an
Exendin compound in which the glycine normally found at position 8 of Exendin-4 has been replaced with valine.
Another preferred group of GLP-1 compounds is composed of GLP-1/Exendin-4 analogs of formula VIII (SEQ ID NO: 11).
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa-Ser- 18 19 20 21 22 23 24 25 26 27 28 Xaa-Xaa-Xaa-Glu-Xaa-Xaa-Ala-Xaa-Xaa-Xaa-Phe-
29 30 31 32 33 34 35 36 37 Ile-Xaa-Trp-Leu-Xaa-Xaa-Gly-Xaa-R formula VIII (SEQ ID NO: 11)
wherein:
Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine; Xaa at position 8 is: Gly, Ala, or Val;
Xaa at position 16 is: Leu or Val;
Xaa at position 18 is Lys or Ser;
Xaa at position 19 is: Gin or Tyr;
Xaa at position 20 is: Met or Leu; Xaa at position 22 is: Glu or Gin;
Xaa at position 23 is: Glu, or Gin;
Xaa at position 25 is: Val or Ala;
Xaa at position 26 is: Arg or Lys;
Xaa at position 27 is Leu or Glu; Xaa at position 30 is: Glu or Ala;
Xaa at position 33 is: Val or Lys;
Xaa at position 34 is: Asn or Lys;
Xaa at position 36 is: Gly or Arg; and R at position 37 is: Gly, Pro, Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser, or is absent. The activity of 18 different species that fall within this genus is provided in Table 6.
Further Exendin-analogs that are useful for the present invention are described in PCT patent publications WO
99/25728 (Beeley et al . ) , WO 99/25727 Beeley et al . ) , WO 98/05351 (Young et al . ) , WO 99/40788 (Young et al . ) , WO 99/07404 (Beeley et al) , and WO 99/43708 (Knudsen et al) .
The GLP-1 fusion proteins of the present invention can comprise glycosylation sites. Glycosylation is a chemical modification wherein sugar moieties are added to the protein at specific sites. Glycosylation of proteins play a role in ensuring the correct charge, confirmation, and stability of maturing protein and can target the protein to the cell surface and eventual secretion of the protein. Most importantly, glycosylation effects the in vivo clearance rate for many proteins. Sugars can be O-linked or N-linked. Generally, O-linked sugars are added to the hydroxyl-group oxygen of serine and threonine, while N-linked sugars are added to the amide nitrogen of asparagine. The consensus site for N-glycosylation is Asn XI X2 wherein XI is any amino acid except Pro and X2 is Ser or Thr.
GLP-1 compounds are generally not glycosylated in vivo; however, interestingly the GLP-1 fusion proteins of the present invention that comprise a GLP-1 compound with a C terminal extension fused to an Fc sequence is glycosylated at the last serine in the C terminal extension (SSGAPPPS*) and at threonine at position 11 in the N terminal region of Fc (AEPKSCDKTHT*CPPC . . . ) . Heterologous Fc fusion proteins :
The GLP-1 compounds described above can be fused directly or via a peptide linker to the Fc portion of an immunoglobulin.
Immunoglobulins are molecules containing polypeptide chains held together by disulfide bonds, typically having two light chains and two heavy chains. In each chain, one domain (V) has a variable amino acid sequence depending on the antibody specificity of the molecule. The other domains (C) have a rather constant sequence common to molecules of the same class.
As used herein, the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, this term refers to an antibody fragment which is obtained by removing the two antigen • binding regions (the Fab fragments) from the antibody. One way to remove the Fab fragments is to digest the immunoglobulin with papain protease. Thus, the Fc portion is formed from approximately equal sized fragments of the constant region from both heavy chains, which associate through non-covalent interactions and disulfide bonds. The Fc portion can include the hinge regions and extend through the CH2 and CH3 domains to the C-terminus of the antibody. Representative hinge regions for human and mouse immunoglobulins can be found in Antibody Engineering, A Practical Guide, Borrebaeck, C.A.K., ed., W.H. Freeman and Co., 1992, the teachings of which are herein incorporated by reference. The Fc portion can further include one or more glycosylation sites. The amino acid sequence of a representative Fc protein containing a hinge region, CH2 and CH3 domains, and one N-glycosylation site at position 82 is shown in Figure 1. There are five types of human immunoglobulin Fc regions with different effect or and pharmacokinetic properties: IgG, IgA, IgM, IgD, and IgE. IgG is the most abundant immunoglobulin in serum. IgG also has the longest half-life in serum of any immunoglobulin (23 days) . Unlike other immunoglobulins, IgG is efficiently recirculated following binding to an Fc receptor. There are four IgG subclasses GI, G2 , G3 , and G4, each of which have different effect or functions. Gl, G2 , and G3 can bind Clq and fix complement while G4 cannot. Even though G3 is able to bind Clq more efficiently than Gl, Gl is more effective at mediating complement-directed cell lysis. G2 fixes complement very inefficiently. The Clq binding site in IgG is located at the carboxy terminal region of the CH2 domain. All IgG subclasses are capable of binding to Fc receptors (CD16, CD32, CD64) with Gl and G3 being more effective than G2 and G4. The Fc receptor binding region of IgG is formed by residues located in both the hinge and the carboxy terminal regions of the CH2 domain. IgA can exist both in a monomeric and dimeric form held together by a J-chain. IgA is the second most abundant Ig in serum, but it has a half-life of only 6 days. IgA has three effect or functions. It binds to an IgA specific receptor on macrophages and eosinophils, which drives phagocytosis and degranulation, respectively. It can also fix complement via an unknown alternative pathway.
IgM is expressed as either a pentamer or a hexamer, both of which are held together by a J-chain. IgM has a serum half-life of 5 days. It binds weakly to Clq via a binding site located in its CH3 domain. IgD has a half-life of 3 days in serum. It is unclear what effect or functions are attributable to this Ig. IgE is a monomeric Ig and has a serum half-life of 2.5 days. IgE binds to two Fc receptors which drives degranulation and results in the release of proinflammatory agents. Depending on the desired in vivo effect, the heterologous fusion proteins of the present invention may contain any of the isotypes described above or may contain mutated Fc regions wherein the complement and/or Fc receptor binding functions have been altered. Thus, the heterologous fusion proteins of the present invention may contain the entire Fc portion of an immunoglobulin, fragments of the Fc portion of an immunoglobulin, or analogs thereof fused to a GLP-1 compound. The fusion proteins of the present invention can consist of single chain proteins or as multi-chain polypeptides . Two or more Fc fusion proteins can be produced such that they interact through disulfide bonds that naturally form between Fc regions. These multimers can be homogeneous with respect to the GLP-1 compound or they may contain different GLP-1 compounds fused at the N- terminus of the Fc portion of the fusion protein.
Regardless of the final structure of the fusion protein, the Fc or Fc-like region must serve to prolong the in vivo plasma half-life of the GLP-1 compound fused at the N-terminus. Furthermore, the fused, GLP-1 compound must retain some biological activity. An increase in half-life can be demonstrated using the method described in Example 7 wherein the half-life of the fusion protein is compared to the half-life of the GLP-1 compound alone. Biological activity can be determined by in vitro and in vivo methods known in the art . Representative biological assays are described in Examples 6, 8, and 9.
Since the Fc region of IgG produced by proteolysis has the same in vivo half-life as the intact IgG molecule and
Fab fragments are rapidly degraded, it is believed that the relevant sequence for prolonging half-life reside in the CH2 and/or CH3 domains. Further, it has been shown in the literature that the catabolic rates of IgG variants that do not bind the high-affinity Fc receptor or Clq are indistinguishable from the rate of clearance of the parent wild-type antibody, indicating that the catabolic site is distinct from the sites involved in Fc receptor or Clq binding. [Wawrzynczak et al . , (1992) Molecular Immunology 29:221] . Site-directed mutagenesis studies using a murine IgGl Fc region suggested that the site of the IgGl Fc region that controls the catabolic rate is located at the CH2-CH3 domain interface.
Based on these studies, Fc regions can be modified at the catabolic site to optimize the half-life of the fusion proteins. It is preferable that the Fc region used for the heterologous fusion proteins of the present invention be derived from an IgGl or an IgG4 Fc region. It is even more preferable that the Fc region be IgG4 or derived from IgG4. Preferably the IgG Fc region contains both the CH2 and CH3 regions including the hinge region.
Heterologous albumin fusion proteins :
The GLP-1 compounds described above can be fused directly or via a peptide linker to albumin or an analog, fragment, or derivative thereof.
Generally the albumin proteins making up part of the fusion proteins of the present invention can be derived from albumin cloned from any species. However, human albumin and fragments and analogs thereof are preferred to reduce the risk of the fusion protein being immunogenic in humans. Human serum albumin (HSA) consists of a single non- glycosylated polypeptide chain of 585 amino acids with a formula molecular weight of 66,500. The amino acid sequence of human HSA is shown in figure 2. [See Meloun, et al . (1975) FEBS Letters 58:136; Behrens, et al . (1975) Fed. Proc. 34:591; Lawn, et al . (1981) Nucleic Acids Research 9:6102-6114; Minghetti, et al . (1986) J. Biol. Chem. 261:6747]. A variety of polymorphic variants as well as analogs and fragments of albumin have been described. [See Weitkamp, et al . , (1973) Ann. Hum. Genet. 37:219]. For example, in EP 322,094, the inventors disclose various shorter forms of HSA. Some of these fragments include HSA(l-373), HSA(l-388) , HSA(l-389), HSA(l-369), and HS (1- 419) and fragments between 1-369 and 1-419. EP 399,666 discloses albumin fragments that include HS (1-177) and HSA(l-200) and fragments between HSA(1-177) and HSA(l-200) . It is understood that the heterologous fusion proteins of the present invention include GLP-1 compounds that are coupled to any albumin protein including fragments, analogs, and derivatives wherein such fusion protein is biologically active and has a longer plasma half-life than the GLP-1 compound alone. Thus, the albumin portion of the fusion protein need not necessarily have a plasma half-life equal to that of native human albumin. Fragments, analogs, and derivatives are known or can be generated that have longer half-lives or have half-lives intermediate to that of native human albumin and the GLP-1 compound of interest.
The heterologous fusion proteins of the present invention encompass proteins having conservative amino acid substitutions in the GLP-1 compound and/or the Fc or albumin portion of the fusion protein. A "conservative substitution" is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number carbon and heteroatoms in their side chains differs by no more than about four . They have approximately the same shape when the number of branches in their side chains differs by no more than one. Amino acids with phenyl or substituted phenyl groups in their side chains are considered to have about the same size and shape. Except as otherwise specifically provided herein, conservative substitutions are preferably made with naturally occurring amino acids .
However, the term "amino acid" is used herein in its broadest sense, and includes naturally occurring amino acids as well as non-naturally occurring amino acids, including amino acid analogs and derivatives . The latter includes molecules containing an amino acid moiety. One skilled in the art will recognize, in view of this broad definition, that reference herein to an amino acid includes, for example, naturally occurring proteogenic L-amino acids; D- amino acids; chemically modified amino acids such as amino acid analogs and derivatives; naturally occurring non- proteogenic amino acids such as norleucine, β-alanine, ornithine, GABA, etc.; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids. As used herein, the term "proteogenic" indicates that the amino acid can be incorporated into a peptide, polypeptide, or protein in a cell through a metabolic pathway. The incorporation of non-natural amino acids, including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the heterologous fusion proteins of the present invention can be advantageous in a number of different ways. D-amino acid-containing peptides, etc., exhibit increased stability in vi tro or in vivo compared to L-amino acid-containing counterparts. Thus, the construction of peptides, etc., incorporating D-amino acids can be particularly useful when greater intracellular stability is desired or required. More specifically, D- peptides, etc., are resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of the molecule, and prolonged lifetimes in vivo when such properties are desirable. Additionally, D-peptides, etc., cannot be processed efficiently for major histocompatibility complex class Il-restricted presentation to T helper cells, and are therefore, less likely to induce humoral immune responses in the whole organism.
In addition to structure/function analyses of the various polypeptides encompassed by the present invention, there are numerous factors that can be considered when selecting amino acids for substitution. One factor that can be considered in making such changes is the hydropathic index of amino acids. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein has been discussed by Kyte and Doolittle (1982, J. Mol . Biol . , 157: 105-132). It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resultant protein. This, in turn, affects the interaction of the protein with molecules such as enzymes, substrates, receptors, ligands, DNA, antibodies, antigens, etc. Based on its hydrophobicity and charge characteristics, each amino acid has been assigned a hydropathic index as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate/glutamine/aspartate/asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
As is known in the art, certain amino acids in a peptide, polypeptide, or protein can be substituted for other amino acids having a similar hydropathic index or score and produce a resultant peptide, etc., having similar or even improved biological activity. In making such changes, it is preferable that amino acids having hydropathic indices within ±2 are substituted for one another. More preferred substitutions are those wherein the amino acids have hydropathic indices within ±1. Most preferred substitutions are those wherein the amino acids have hydropathic indices within ±0.5.
Like amino acids can also be substituted on the basis of hydrophilicity. U.S. Patent No. 4,554,101 discloses that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0±1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4) . Thus, one amino acid in a peptide, polypeptide, or protein can be substituted by another amino acid having a similar hydrophilicity score and still produce a resultant peptide, etc., having similar biological activity, i.e., still retaining correct biological function. In making such changes, amino acids having hydropathic indices within ±2 are preferably substituted for one another, those within +1 are more preferred, and those within ±0.5 are most preferred.
As outlined above, amino acid substitutions in the fusion proteins of the present invention can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Furthermore, substitutions can be made based on secondary structure propensity. For example, a helical amino acid can be replaced with an amino acid that would preserve the helical structure. Exemplary substitutions that take various of the foregoing characteristics into consideration in order to produce conservative amino acid changes resulting in silent changes within the present peptides, etc., can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids .
General methods for making the heterologous fusion proteins of the present invention .
Although the heterologous fusion proteins of the present invention can be made by a variety of different methods, recombinant methods are preferred. For purposes of the present invention, as disclosed and claimed herein, the following general molecular biology terms and abbreviations are defined below. The terms and abbreviations used in this document have their normal meanings unless otherwise designated. For example, "°c" refers to degrees Celsius; "mmol" refers to millimole or millimoles; "mg" refers to milligrams; "μg" refers to micrograms; "ml or mL" refers to milliliters; and "μl or μL" refers to microliters. Amino acids abbreviations are as. set forth in 37 C.F.R. § 1.822 (b) (2) (1994) .
"Base pair" or "bp" as used herein refers to DNA or RNA. The abbreviations A,C,G, and T correspond to the 5'- monophosphate forms of the deoxyribonucleosides (deoxy) adenosine, (deoxy) cytidine, (deoxy) guanosine, and thymidine, respectively, when they occur in DNA molecules. The abbreviations U,C,G, and A correspond to the 5'- monophosphate forms of the ribonucleosides uridine, cytidine, guanosine, and adenosine, respectively when they occur in RNA molecules. In double stranded DNA, base pair may refer to a partnership of A with T or C with G. In a DNA/RNA, heteroduplex base pair may refer to a partnership of A with U or C with G. (See the definition of "comp1ementary" , infra . )
"Digestion" or "Restriction" of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA ("sequence- specific endonucleases" ) . The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements were used as would be known to one of ordinary skill in the art.
Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can be readily found in the literature.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments. Unless otherwise provided, ligation may be accomplished using known buffers and conditions with a DNA ligase, such as T4 DNA ligase.
"Plasmid" refers to an extrachromosomal (usually) self- replicating genetic element. Plasmids are generally designated by a lower case "p" followed by letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the -art and will be apparent to the ordinarily skilled artisan.
"Recombinant DNA cloning vector" as used herein refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
"Recombinant DNA expression vector" as used herein refers to any recombinant DNA cloning vector in which a promoter to control transcription of the inserted DNA has been incorporated.
"Transcription" refers to the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
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"Complementary" or "Complementarity", as used herein, refers to pairs of bases (purines and pyrimidines) that associate through hydrogen bonding in a double stranded nucleic acid. The following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil .
"Hybridization" as used herein refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing. The conditions employed in the hybridization of two non-identical, but very similar, complementary nucleic acids varies with the degree -of complementarity of the two strands and the length of the strands. Such techniques and conditions are well known to practitioners in this field. "Isolated amino acid sequence" refers to any amino acid sequence, however, constructed or synthesized, which is locationally distinct from the naturally occurring sequence.
"Isolated DNA compound" refers to any DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location in genomic DNA.
"Isolated nucleic acid compound" refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.
"Primer" refers to a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation.
"Promoter" refers to a DNA sequence which directs transcription of DNA to RNA.
"Probe" refers to a nucleic acid compound or a fragment, thereof, which hybridizes with another nucleic acid compound.
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stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5X SSC (750 mM sodium chloride, 75 mM sodium citrate) , 50 mM sodium phosphate at pH 7.6, 5X Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in IX SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc., as necessary to accommodate factors such as probe length and the like.
"PCR" refers to the widely-known polymerase chain reaction employing a thermally-stable DNA polymerase.
"Leader sequence" refers to a sequence' of amino acids which can be enzymatically or chemically removed to produce the desired polypeptide of interest.
"Secretion signal sequence" refers to a sequence of amino acids generally present at the N-terminal region of a larger polypeptide functioning to initiate association of that polypeptide with the cell membrane compartments like endoplasmic reticulum and secretion of that polypeptide through the plasma membrane .
Construction of DNA encoding the heterologous fusion proteins of the present invention : Wild-type albumin and Immunoglobulin proteins can be obtained from a variety of sources. For example, these proteins can be obtained from a cDNA library prepared from tissue or cells which express the mRNA of interest at a detectable level . Libraries can be screened with probes designed using the published DNA or protein sequence for the particular protein of interest. For example, immunoglobulin light or heavy chain constant regions are described in Adams, et al . (1980) Biochemistry 19:2711-2719; Goughet, et al . (1980) Biochemistry 19:2702-2710; Dolby, et al . (1980) Proc. Natl. Acad. Sci. USA 77:6027-6031; Rice et al . (1982) Proc. Natl. Acad. Sci. USA 79:7862-7862; Falkner, et al . (1982) Nature 298:286-288; and Morrison, et al . (1984) Ann. Rev. Immunol. 2:239-256. Some references disclosing albumin protein and DNA sequences include Meloun, et al . (1975) FEBS Letters 58:136; Behrens, et al . (1975) Fed. Proc. 34:591; Lawn, et al . (1981) Nucleic Acids Research 9:6102-6114; and Minghetti, et al . (1986) J. Biol. Chem. 261:6747
Screening a cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al . , Molecular Cloning: A
Laboratory Manual , Cold Spring Harbor Laboratory Press, NY (1989) . An alternative means to isolate a gene encoding an albumin or immunoglobulin protein is to use PCR methodology [Sambrook et al . , supra; Dieffenbach et al . , PCR Primer: A Laboratory Manual , Cold Spring Harbor Laboratory Press, NY (1995)] . PCR primers can be designed based on published sequences .
Generally the full-length wild-type sequences cloned from a particular species can serve as a template to create analogs, fragments, and derivatives that retain the ability to confer a longer plasma half-life on the GLP-1 compound that is part of the fusion protein. It is preferred that the Fc and albumin portions of the heterologous fusion proteins of the present invention be derived from the native human sequence in order to reduce the risk of potential immunogenicity of the fusion protein in humans .
In particular, it is preferred that the immunoglobulin portion of a fusion protein encompassed by the present invention contain only an Fc fragment of the immunoglobulin. Depending on whether particular effect or functions are desired and the structural characteristics of the fusion protein, an Fc fragment may contain the hinge region along with the CH2 and CH3 domains or some other combination thereof . These Fc fragments can be generated using PCR techniques with primers designed to hybridize to sequences corresponding to the desired ends of the fragment. Similarly, if fragments of albumin are desired, PCR primers can be designed which are complementary to internal albumin sequences. PCR primers can also be designed to create 5 restriction enzyme sites to facilitate cloning into expression vectors .
DNA encoding the GLP-1 compounds of the present invention can be made by a variety of different methods including cloning methods like those described above as well
10 as chemically synthesized DNA. Chemical synthesis may be attractive given the short length of the encoded peptide. The amino acid sequence for GLP-1 has been published as well as the sequence of the preproglucagon gene. [Lopez, et al . (1983) Proc. Natl. Acad. Sci., USA 80:5485-5489; Bell, et
15 al . (1983) Nature, 302:716-718; Heinrich, G., et al . (1984) Endocrinol, 115:2176-2181; Ghiglione, M. , et al . 91984) Diabetologia 27:599-600]. Thus, primers can be designed to PCR native GLP-1 compounds and fragments thereof.
The gene encoding a fusion protein can then be
20 constructed by ligating DNA encoding a GLP-1 compound in- frame to DNA encoding an albumin or Fc protein. The gene encoding the GLP-1 compound and the gene encoding the albumin or Fc protein can also be joined in-frame via DNA encoding a linker peptide. 25. The in vivo function and stability of the heterologous fusion proteins of the present invention can be optimized by adding small peptide linkers to prevent potentially unwanted domain interactions . Although these linkers can potentially be any length and consist of any combination of amino acids,
30 it is preferred that the length be no longer than necessary to prevent unwanted domain interactions and/or optimize biological activity and/or stability. Generally, the linkers should not contain amino acids with extremely bulky side chains or amino acids likely to introduce significant 35 secondary structure. It is preferred that the linker be serine-glycine rich and be less than 30 amino acids in length. It is more preferred that the linker be no more than 20 amino acids in length. It is even more preferred that the linker be no more than 15 amino acids in length. A preferred linker contains repeats of the sequence Gly-Gly- Gly-Gly-Ser. It is preferred that there be between 2 and 6 repeats of this sequence. It is even more preferred that there be between 3 and 4 repeats of this sequence.
The DNA encoding wild-type GLP-1, albumin, and Fc polypeptides and fragments thereof can be mutated either before ligation or in the context of a cDNA encoding an entire fusion protein. A variety of mutagenesis techniques are well known in the art. For example, a mutagenic PCR method utilizes strand overlap extension to create specific base mutations for the purposes of changing a specific amino acid sequence in the corresponding protein. This PCR mutagenesis requires the use of four primers, two in the forward orientation (primers A and C) and two in the reverse orientation (primers B and D) . " A mutated gene is amplified from the wild-type template in two different stages. The first reaction amplifies the gene in halves by performing an A to B reaction and a separate C to D reaction wherein the B and C primers target the area of the gene to be mutated. When aligning these primers with the target area, they contain mismatches for the bases that are targeted to be changed. Once the A to B and C to D reactions are complete, the reaction products are isolated and mixed for use as the template for the A to D reaction. This reaction then yields the full, mutated product. Once a gene encoding an entire fusion protein is produced it can be cloned into an appropriate expression vector. Specific strategies that can be employed to make the GLP-1 fusion proteins of the present invention are described in example 1. General methods to recombinantly express the heterologous fusion proteins of the present invention :
Host cells are transfected or transformed with expression or cloning vectors described herein for heterologous fusion protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook, et al . , supra. Methods of transfection are known to the ordinarily skilled artisan, for example, CaP0 and electroporation. General aspects of mammalian cell host system transformations have been described in U.S. Patent No. 4,399,216. Transformations into yeast are typically carried out according to the method of van Solingen et al . , J" Bact . 130(2): 946-7 (1977) and Hsiao et al . , Proc . Natl . Acad. Sci . USA 76(8): 3829-33 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene or polyomithine, may also be used. For various techniques for transforming mammalian cells, see Keown, et al . , Methods in Enzymology 185: 527-37 (1990) and Mansour, et al . , Nature 336(6197): 348-52 (1988).
Suitable host cells for cloning or expressing the nucleic acid (e.g., DNA) in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram- negative or Gram-positive organisms, for example, Enterobacteriacea such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 3 1.446); E. coli XI 776 (ATCC 3 1.537); E. coli strain W3 110 (ATCC 27.325) and K5 772 (ATCC 53.635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g.. E. coli, Enterobacter , Erwinia, Klebisella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigeila, as well as Bacilli such as B. subtilis and B. lichentforrais (e.g., B. licheniformis 4 1 P disclosed in DD266,7 10, published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces . These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3 110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1A2 , which has the complete genotype ronA; E. coli W3 110 strain 9E4, which has the complete genotype ton4 ptr3 ; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA, ptr3 phoA E15 (argF-lac) 169 degP ompT /can'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vivo methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for fusion protein vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe [Beach and Nurse, Nature 290: 140-3 (1981); EP 139,383 published 2 May 1995]; Muyveromyces hosts [U.S. Patent No. 4,943,529; Fleer, et al . , Bio /Technology 9 (10) : 968-75 (1991)] such as, e.g., K lactis (MW98-8C, CBS683, CBS4574) [de Louvencourt et al . , J". Bacteriol . 154(2): 737-42 (1983)]; K. fiagilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K wickeramii (ATCC 24,178), K waltii (ATCC 56,500), K. drosophilarum (ATCC 36.906) [Van den Berg et al . , Bio / 'Technology 8 (2) : 135-9 (1990)]; K. thermotoierans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070) [Sreekrishna et al . , J. Basic Microbiol . 28(4): 265-78 (1988)]; Candid; Trichoderma reesia (EP 244,234); Neurospora crassa [Case, et al . , Proc . Natl . Acad Sci . USA 76(10): 5259-63 (1979)]; Schwanniomyces such as Schwanniomyces occidentulis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991) , and Aspergillus hosts such as A. nidulans [Ballance et al., Biochem . Biophys . Res . Comm. 112(1): 284-9 (1983)]; . Tilburn, et al . , Gene 26(2-3): 205-21 (1983); Yelton, et al . , Proc . Natl . Acad. Sci . USA 81(5): 1470-4 (1984)] and A. niger [Kelly and Hynes, EMBO J. 4(2): 475-9 (1985)]. Methylotropic yeasts are selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotoruia. A list of specific species that are exemplary of this class of yeast may be found in C. Antony, The Biochemistry of Methylotrophs 269 (1982) .
Suitable host cells for the expression of the fusion proteins of the present invention are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sp, Spodoptera high5 as well as plant cells . Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line [293 or 293 cells subcloned for growth in suspension culture, Graham, et al . , J. Gen Virol . , 36(1): 59-74 (1977)]; Chinese hamster ovary cells/-DHFR [CHO, Urlaub and Chasin, Proc . Natl . Acad . Sci . USA, 77(7): 4216-20 (1980)]; mouse sertoli cells [TM4, Mather, Biol . Reprod. 23(l):243-52 (1980)]; human lung cells (W138. ATCC CCL 75); human liver cells (Hep G2 , HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51) . The selection of the appropriate host cell is deemed to be within the skill in the art. The fusion proteins of the present invention may be recombinantly produced directly, or as a protein having a signal sequence or other additional sequences which create a specific cleavage site at the N-terminus of the mature fusion protein. In general, the signal sequence may be a component of the vector, or it may be a part of the fusion protein- • encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpρ, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyvero yces cc-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179), or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species as well as viral secretory leaders . Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells . Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria, the 2u plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells .
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement autotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the fusion protein-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line ,deficient in DHFR activity, prepared and propagated as described [Urlaub and Chasin, Proc . Natl . Acad . Sci . USA, 77(7): 4216-20 (1980) ] . A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 [Stinchcomb, et al . , Nature 282(5734): 39-43 (1979); Kingsman, et al . , Gene 7(2): 141-52 (1979); Tschu per, et al . , Gene 10(2): 157-66 (1980)] . The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEPC1 [Jones, Genetics 85: 23-33 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to the fusion protein-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β- lactamase and lactose promoter systems [Chang, et al . , Nature 275(5681): 617-24 (1978); Goeddel, et al . , Nature 281(5732) : 544-8 (1979)], alkaline phosphatase, a tryptophan (up) promoter system [Goeddel, Nucleic Acids Res . 8(18): 4057-74 (1980); EP 36,776 published 30 September 1981], and hybrid promoters such as the tat promoter [deBoer, et al . , Proc . Natl . Acad. Sci . USA 80(1): 21-5 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the fusion protein. Examples ,of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman, et al . , J. Biol . Chem . 255(24): 12073-80 (1980)] or other glycolytic enzymes [Hess et al . , J. Adv. Enzyme Reg. 7: 149 (1968); Holland, Biochemistry 17(23) : 4900- 7 (1978) ] , such as enolase, glycer ldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase .
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Transcription of fusion protein-encoding mRNA from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis- B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems . Transcription of a polynucleotide encoding a fusion protein by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-ketoprotein, and insulin) . Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the fusion protein coding sequence but is preferably located at a site 5' from the promoter. Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNAs or cDNAs . These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the fusion protein. Various forms of a fusion protein may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells employed in expression of a fusion protein can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
Purification of the heterologous fusion proteins of the present invention :
Once the heterologous fusion proteins of the present invention are expressed in the appropriate host cell, the analogs can be isolated and purified. The following procedures are exemplary of suitable purification procedures: fractionation on carboxymethyl cellulose; gel filtration such as Sephadex G-75; anion exchange resin such as DEAE or Mono-Q; cation exchange such as CM or Mono-S; protein A sepharose to remove contaminants such as IgG; metal chelating columns to bind epitope-tagged forms of the polypeptide; reversed-phase HPLC; chromatofocusing; silica gel; ethanol precipitation; and ammonium sulfate precipitation.
Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-9 (1990) and Scopes, Protein Purification : Principles and Practice,
Springer-Verlag, NY (1982). The purification step(s) selected will depend on the nature of the production process used and the particular fusion protein produced. For example, fusion proteins comprising an Fc fragment can be effectively purified using a Protein A or Protein G affinity matix. Low or high pH buffers can be used to elute the fusion protein from the affinity matrix. Mild elution conditions will aid in preventing irreversible denaturation of the fusion protein. Imidazole-containing buffers can also be used. Example 3 describes some successful purification protocols for the fusion proteins of the present invention.
Characterization of the heterologous fusion proteins of the presen t inven ti on : Numerous methods exist to characterize the fusion proteins of the present invention. Some of these methods include: SDS-PAGE coupled with protein staining methods or immunoblotting using anti-IgG or anti-HSA antibodies. Other methods include matrix assisted laser desporption/ionization- mass spectrometry (MALDI-MS) , liquid chromatography/mass spectrometry, isoelectric focusing, analytical anion exchange, chromatofocussing, and circular dichroism to name a few. A representative number of heterologous fusion proteins were characterized using SDS-PAGE coupled with immunoblotting as well as mass spectrometry (See examples 4 and 5 and figures 3 and 4) .
For example table 3 (see example 5) illustrates the calculated molecular mass for a representative number of fusion proteins as well as the mass as determined by mass spectrometry. In addition, Figures 3 and 4 illustrate molecular weights of a representative number of fusion proteins as determined by SDS PAGE. All heterologous fusion proteins tested were expressed and secreted transiently. In addition, the Igκ signal sequence was cleaved to yield proteins with the correct N-terminus.
Further, table 3 illustrates that in some instances the mass determined by mass spectrometry is greater than expected. This is the result of glycosylation of the Fc portion and the C terminal extension. Enzymatic digestion of the fusion proteins followed by reversed-phase HPLC and mass spectrometry can identify peptide fractions that contain sugar moieties. These fractions can then be N-terminal amino acid sequenced to identify the potential glycosylation site. For example, characterization of Exendin-4-Fc (SEQ ID NO: 29) shows that the serine at position 39 and threonine at position 50 are 0- linked glycosylated and the asaparagine at position 122 is N- linked glycosylated.
A representative number of GLP-1 fusion proteins were also tested for activity. Numerous methods exist to detect
GLP-1 activity in vi tro and in vivo (see examples 6, 7, 8 , and
9) . Table 4 (example 6) illustrates GLP-1 receptor activity associated with several GLP-1 fusions. The numbers are
8 relative to the activity associated with Val -GLP-1 (7-37 ) OH. All fusion proteins tested had GLP-1 receptor activity. A low level of in vi tro activity is not necessarily indicative of a weak effect in vivo . Because of the substantial increase in the half-life of these fusion proteins, weak n vi tro activity is not generally a predictor of weak in vivo activity. Figure 7 and example 7. illustrate the prolonged half-life associated LO LO t to μ> H
LΠ O LΠ o LΠ O Π
Figure imgf000062_0001
formulation. Glycerin is an example of an isotonicity- adjusting excipient. Pharmaceutically acceptable means suitable for administration to a human or other animal and thus, does not contain toxic elements or undesirable contaminants and does not interfere with the activity of the active compounds therein.
A pharmaceutically-acceptable salt form of the heterologous fusion proteins of the present invention may be used in the present invention. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p- bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzole acid, acetic acid, and the like.
Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.
Administration of Compositions :
Administration may be via any route known to be effective by the physician of ordinary skill. Peripheral, parenteral is one such method. Parenteral administration is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump. Peripheral parenteral routes can include intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration.
The heterologous fusion proteins of the present invention may also be amenable to administration by oral, rectal, nasal, or lower respiratory routes, which are non-parenteral routes. Of these non-parenteral routes, the lower respiratory route and the oral route are preferred.
The fusion proteins of the present invention can be used to treat a wide variety of diseases and conditions. The fusion proteins of the present invention primarily exert their biological effects by acting at a receptor, referred to as the "GLP-1 receptor." Subjects with diseases and/or conditions that respond favorably to GLP-1 receptor stimulation or to the administration of GLP-1 compounds can therefore be treated with the GLP-1 fusion proteins of the present invention. These subjects are said to "be in need of treatment with GLP-1 compounds" or "in need of GLP-1 receptor stimulation" . Included are subjects with non-insulin dependent diabetes, insulin dependent diabetes, stroke (see WO 00/16797), myocardial infarction (see WO 98/08531) , obesity (see WO 98/19698), catabolic changes after surgery (see U.S. Patent No. 6,006,753), functional dyspepsia and irritable bowel syndrome (see WO 99/64060). Also included are subjects requiring prophylactic treatment with a GLP-1 compound, e.g., subjects at risk for developing non-insulin dependent diabetes (see WO 00/07617). Subjects with impaired glucose tolerance or impaired fasting glucose, subjects whose body weight is about 25% above normal body weight for the subject's height and body build, subjects with a partial pancreatectomy, subjects having one or more parents with non-insulin dependent diabetes, subjects who have had gestational diabetes and subjects who have, had acute or chronic pancreatitis are at risk for developing non-insulin dependent diabetes . An "effective amount" of a GLP-1 compound is the quantity which results in a desired therapeutic and/or prophylactic effect without causing unacceptable side- effects when administered to a subject in need of GLP-1 receptor stimulation. A "desired therapeutic effect" includes one or more of the following: 1) an amelioration of the symptom(s) associated with the disease or condition; 2) a delay in the onset of symptoms associated with the disease or condition; 3) increased longevity compared with the absence of the treatment; and 4) greater quality of life compared with the absence of the treatment. For example, an "effective amount" of a GLP-1 compound for the treatment of diabetes is the quantity that would result in greater control of blood glucose concentration than in the absence of treatment, thereby resulting in a delay in the onset of diabetic complications such as retinopathy, neuropathy or kidney disease. An "effective amount" of a GLP-1 compound for the prevention of diabetes is the quantity that would delay, compared with the absence of treatment, the onset of elevated blood glucose levels that require treatment with anti-hypoglycaemic drugs such as sulfonyl ureas, thiazolidinediones, insulin and/or bisguanidines .
The dose of fusion protein effective to normalize a patient's blood glucose will depend on a number of factors, among which are included, without limitation, the subject's sex, weight and age, the severity of inability to regulate blood glucose, the route of administration and bioavailability, the pharmacokinetic profile of the fusion protein, the potency, and the formulation.
The present invention comprises GLP-1 compounds that have improved biochemical and biophysical properties by virtue of being fused to an albumin protein, an albumin fragment, an albumin analog, a Fc protein, a Fc fragment, or a Fc analog. These heterologous proteins can be successfully expressed in host cells, retain signaling activities associated with activation of the GLP-1 receptor, and have prolonged half-lives.
The following examples are presented to further describe the present invention. The scope of the present invention is not to be construed as merely consisting of the following examples. Those skilled in the art will recognize that the particular reagents, equipment, and procedures described are merely illustrative and are not intended to limit the present invention in any manner.
Example 1 : Construction of DNA encoding .heterologous fus.ion progteins
Example la Construction of DNA encoding Val -GLP-1 (7-37) -
Fc : A Fc portion of human IgGl was isolated from a cDNA library and contains the full hinge region and the CH2 and CH3 domains. A fragment containing 696 base pairs of this Fc portion of human IgGl was subcloned into the Nhel and Eco47III sites of mammalian expression vector pJB02 to create pJB02/Fc (see Figure 5) . DNA encoding the Igκ
8 secretion signal sequence fused to Val -GLP-1 (7-37) was generated by in vi tro hybridization of four overlapping and complementary oligonucleotides :
5 ' - CTAGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTT CCAGGTTCCACTGGTGACCAGTG - 3' [SEQ ID NO: 12]
5 ' - GAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTGGAGGGCCAGGCCGCCAAGGA GTTCATCGCCTGGCTGGTGAAGGGAAGAGGC - 3' [SEQ ID NO: 13]
5 ' - TGAAGGTGCCCTCCACGTGGTCACCAGTGGAACCTGGAACCCAGAGCAGCAGTA CCCATAGCAGGAGTGTGTCTGTCTCCATGGTGG - 3' [SEQ ID
NO: 14]
5 ' - GCCTCTTCCCTTCACCAGCCAGGCGATGAACTCCTTGGCGGCCTGGCCCTCCAGA TAGGAGGACACGTCGGAGG - 3' [SEQ ID NO: 15]
The hybridization reaction was carried out using equivalent amounts of each oligonucleotide (1 pm/μl final concentration for each oligo) . The mixture of oligonucleotides was heated for 5 min at 100°C in ligation buffer (50 mM Tris-HCl, pH 7.5 , 10 mM MgCl2, 10 mM DTT, ImM ATP, 25 μg/iϊil bovine serum albumin) and then cooled over at least 2 hours to 30°C.
The resulting hybridization product was ligated for 2 hours at room temperature or overnight at 16°C to the pJB02/Fc vector backbone which had been digested with Nhel and Eco47III . The ligation products were used to transform competent XL-1 Blue cells (Stratagene) . Recombinant plasmids were screened for the presence of peptide coding inserts by digesting clones with Ncol (encoding the Kozak sequence and first Met of the signal peptide) and sequenced. The resulting expression plasmid used for transfection assays was denoted pJB02-V8-GLP-l-Fc (Figure 5) .
8 Example lb Construction of DNA encoding Val -GLP-1 (7-37) - HSA: The plasmid HSA/pcDNA3.1GS was purchased from
Invitrogen (Catalog # H-M12523M-pcDNA3.1/GS) and used as a template to isolate the cDNA encoding human serum albumin (HSA) . The HSA cDNA was prepared using PCR wherein the DNA encoding the leader sequence as well as the six amino acid pro-peptide was removed from the 5' end. In addition, stop codons were added directly at the 3 ' end of the HSA coding sequence. Finally, restriction enzyme sites were engineered at the 5' and 3' end to facilitate cloning. The HSA DNA sequence present in the original vector purchased from Invitrogen contained a single base change in the 3' region of the gene (position 667) compared to the native human sequence. This change would result in a codon for Asn instead of Asp. Thus, using the strand overlapping PCR mutagenesis method discussed above, the codon was changed to code for Asp at this position. The resulting HSA encoding DNA was cloned into the Nhel and Hindlll sites of pJB02 to create pJB02-HSA (Figure 6) .
8
The Igκ leader sequence fused to the Val -GLP-1 (7-37) sequence was generated as discussed in Example la. This DNA was ligated into the Nhel and Fspl sites of pJB02-HSA to
8 create pJB02- Val -GLP-1-HSA. o
Example lc Construction of DNA encoding Val -GLP-1 (7-37) - linker-HSA:
The vector pJB02-HSA was prepared as discussed in
Example lb. DNA encoding the linker sequence [GGGGS]3 was ligated in frame to the 5 ' end of the HSA encoding DNA to create pJB02-linker-HSA (Figure 7) . DNA encoding the Igκ
8 leader sequence and fused to the Val -GLP-1 (7-37) sequence and the 5 ' part of the linker sequence was generated as discussed in Example la. This DNA was ligated into the Nhel
8 and BspEI sites of pJB02 to create pJB02- Val -GLP-1-linker-
HSA.
Example Id Construction of DNA encoding Exendin-4-Fc : The plasmid pJB02/Fc was prepared as described in
Example la. DNA encoding the IgK signal sequence fused to
Exending-4 was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides:
5 ' - CTAGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTG GGTTCCAGGTTCCACCGGTCAC - 3' [SEQ ID NO: 16]
5' - GGAGAGGGAACCTTCACCAGCGACCTGAGCAAGCAGATGGAGGAGGAGGCCGT GAGACTG - 3' [SEQ ID NO: 17]
5 ' - TTCATCGAGTGGCTGAAGAACGGAGGACCAAGCAGCGGAGCCCCTCCTCCT
AGC - 3' [SEQ ID NO: 18]
5 ' - GAACCTGGAACCCAGAGCAGCAGTACCCATAGCAGGAGTGTGTCTGTCTCCA
TGGTGG - 3' [SEQ ID NO: 19]
5 ' - CTCCTCCTCCATCTGCTTGCTCAGGTCGCTGGTGAAGGTTCCCTCTCCGTGA CCGGTG - 3' [SEQ ID NO: 20] 5 ' - GCTAGGAGGAGGGGCTCCGCTGCTTGGTCCTCCGTTCTTCAGCCACTCGAT
GAACAGTCTCACGGC - 3' [SEQ ID NO: 21]
The hybridization reaction was carried out as described in Example la. The hybridized product was ligated to the pJB02 vector which had been digested with Nhel and Eco47III as described in Example la to create pJB02-Exendin-4-Fc .
Example le Construction of DNA encoding Exendin-4-HSA: The plasmid pJB02-HSA was prepared as described in
Example lb. DNA encoding the lgκ signal sequence fused to Exending-4 was generated by in vi tro hybridization of the same overlapping and complementary oligonucleotides described in Example Id. Hybridization reactions were also carried out as described above. DNA was cloned into unique Nhel and Fspl sites in pJB02-HSA to create pJB02-Exendin-4- HSA.
Example If Construction of DNA encoding Exendin-4-linker- HSA:
The plasmid pJB02-linker-HSA was constructed as described in Example lc . DNA encoding the Igκ signal sequence fused to Exendin-4 and the 5 ' part of the linker sequence was generated as in Example Id. This DNA was cloned into unique Nhel and BspEI sites in pJB02-linker-HSA to create pJB02-Exendin-4-linker-HSA. o
Example Ig Construction of DNA encoding Val -GLP-1/C-Ex-Fc : The plasmid pJB02-Exendin-4-Fc was prepared as described in Example Id. The Exendin-4 encoding DNA was o excised from the vector with Agel and Eco47III. The Val - GLP-1/C-Ex encoding DNA was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides : 5 ' -CCGGTCACGTGGAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTGGA GGGCCAGGCCGCCA - 3' [SEQ ID NO: 22]
5 ' - AGGAATTCATCGCCTGGCTGGTGAAGGGCCGGGGCAGCAGCGG AGCCCCTCCTCCTAGC - 3' [SEQ ID NO: 23]
5 ' - CTCCAGATAGGAGGACACGTCGGAGGTGAAGGTGCCCTCCAC GTGA - 3' [SEQ ID NO: 24]
5' - GCTAGGAGGAGGGGCTCCGCTGCTGCCCCGGCCCTTCACCAGCCAGGCGA TGAATTCCTTGGCGGCCTGGCC - 3' [SEQ ID NO: 25]
The hybridization reaction was carried out as described in Example la. The hybridized product was ligated in place of Exendin-4 in the pJB02-Exendin-4-Fc expression vector to
8 create pJB02-Val -GLP-1/C-Ex-Fc .
8 22 Example Ih Construction of DNA encoding Val -Glu -GLP-l-Fc:
The plasmid pJB02-Exendin-4-Fc was prepared as described in Example Id. The Exendin-4 encoding DNA was o excised from the vector with Agel and Eco47III . The Val -
22 Glu -GLP-1 encoding DNA was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides :
5 ' -CCGGTCACGTGGAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTCGA GGAGCAGGCCGCCA - 3' [SEQ ID NO: 26]
5' - AGGAGTTCATCGCCTGGCTGGTGAAGGGCCGGGGC - 3' [SEQ ID NO: 27]
5 ' - GCCCCGGCCCTTCACCAGCCAGGCGATGAACTCCTTGGCGGCC TGCTC - 3' [SEQ ID NO:28]
5' - CTCGAGATAGGAGGACACGTCGGAGGTGAAGGTGCCCT CCACGTGA - 3' [SEQ ID NO:29] The hybridization reaction was carried out as described in Example la. The hybridized product was ligated in place of Exendin-4 in the pJB02-Exendin-4-Fc expression vector to
8 22 create pJB02-Val -Glu -GLP-l-Fc.
8 22 Example li Construction of DNA encoding Val -Glu GLP-1/C-
Ex-Fc :
The plasmid pJB02-Exendin-4-Fc was prepared as described in Example Id. The Exendin-4 encoding DNA was
8 excised from the vector with Agel and Eco47III . The Val -
22 Glu GLP-1/C-Ex encoding DNA was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides :
5 ' - CCGGTCACGTGGAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTCGA GGAGCAGGCCGCCA - 3' [SEQ ID NO: 30]
5 ' - AGGAATTCATCGCCTGGCTGGTGAAGGGCCGGGGCAGCAGCGGA GCCCCTCCTCCTAGC - 3' [SEQ ID NO: 31]
5' - CTCGAGATAGGAGGACACGTCGGAGGTGAAGGTGCCC TCCACGTGA - 3' [SEQ ID NO: 32]
5' - GCTAGGAGGAGGGGCTCCGCTGCTGCCCCGGCCCTTCACCAGCCAGGCGA . TGAATTCCTTGGCGGCCTGCTC - 3' [SEQ ID NO: 33]
The hybridization reaction was carried out as described in Example la. The hybridized product was ligated in place of Exendin-4 in the pJB02-Exendin-4-Fc expression vector to o create pJB02-Val -Glu -GLP-1/C-Ex-Fc .
Q
Example lj Construction of DNA encoding Gly -GLP-l-Fc: The plasmid pJB02-Exendin-4-Fc was prepared as described in Example Id. The Exendin-4 encoding DNA was g excised from the vector with Λgrel and Eco47III . The Gly - GLP-1 encoding DNA was generated by in vi tro hybridization of the following overlapping and complementary oligonucleotides :
5 ' - CCGGTCACGGCGAGGGCACCTTCACTAGTGACGTGTCCTCCTATCTGGA GGGCCAGGCCGCCA - 3' [SEQ ID NO: 34]
5' - AGGAGTTCATCGCCTGGCTGGTGAAGGGCCGGGGC - 3' [SEQ ID NO: 35]
5' - CTCCAGATAGGAGGACACGTCACTAGTGAAGGTGCCCTC GCCGTGA - 3' [SEQ ID NO: 36]
5 ' - GCCCCGGCCCTTCACCAGCCAGGCGATGAACTCCTTGGCGGC CTGGCC - 3' [SEQ ID NO: 37]
The hybridization reaction was carried out as described in Example la. The hybridized product was ligated in place of Exendin-4 in the pJB02-Exendin-4-Fc expression vector to
8 create pJB02-Gly -GLP-l-Fc.
Example 2 : Expression of heterologous fusion proteins Expression of the fusion proteins encoded by the DNA constructs of Example 1 was carried out by transiently transfecting HEK 293EBNA cells (both adherent and suspension) . Cells were counted and seeded 24 hours prior to transfection. The transfection cocktail was prepared by mixing FuGene™6 transfection reagent (Roche Molecular Biochemicals, catalog # 1814443) with OptiMEM (Gibco/BRL) and incubating at room temperature for 5 min at which point
DNA was added and the cocktail was incubated for an additional 15 min. Immediately before transfection, fresh growth media was added to the plate. Tables 1 and 2 provide further transfection details. Table 1: Reagents used in transient transfections of 293EBNA cells .
Figure imgf000073_0001
Table 2 : Media composition
Figure imgf000073_0002
For small-scale transfections (35mm - 10mm vessels) , cells were rinsed with PBS and switched to harvesting media 24 hours post- transfection and media was collected and replaced every 24 hours for several days. In the case of large-scale transfections (700 cm2 roller bottles) , the roller bottles were rinsed with PBS 48 hours post- transfection and changed to harvesting media. Media was collected and changed every 24 hours for at least 10 consecutive days. Routinely, only 10 harvests were used for subsequent protein purification. Example 3 : Puri ication of heterologous fusion proteins
8 Example 3a purification of Val -GLP-l-Fc
Approximately 4.5 liters of conditioned medium (fusion protein expression level approximately 20 μg/ml) from large- scale transfections was filtered using a CUNO filter system and concentrated to 250 ml using a ProFlux tangenti Qal flow filtration system with a 10 K filter membrane. Val -GLP-l-Fc was captured with a 5 ml HiTrap protein A column in lx PBS, pH 7.4 at a flow rate of 2 ml/min and eluted with 50 mM citric acid pH 3.3. Fractions (1 ml) were collected in tubes containing 4 ml lx PBS and lOOμl 1 M Tris pH 8.
Fractions containing the fusion protein, as determined by SDS-PAGE and reverse phase-HPLC on Zorbax C8, were pooled and applied to a Superdex 75 60/60 column in lx PBS pH 7.4 at a flow rate of 10 ml/min. Positive fractions (20 mis/tube) were collected and pooled. Pooled fractions were then subjected to C4 Reverse Phase Chromatography in 0.1
8 %TFA water at a flow rate of 3 ml/mm. Val -GLP-l-Fc was eluted using a gradient from 5% B (0.1% TFA in acetonitrile) to 100% B in 70 min. Eluant fractions (3 mis/tube) were collected. Acetonitrile was removed by vacuum drying and 1 ml of H20 was added. The purified sample (approximately 32 mis) was dialyzed twice against 4 liters of lx PBS pH7.4.
The dialyzed sample was then filtered using a MILLEX-GV 0.22 um Filter Unit and concentration was determined using absorption at 280 nm.
8 8
Example 3b purification of Val -GLP-1-HSA or Val -GLP-1-
Linker-HSA Approximately 6.5 liters of conditioned medium (fusion protein expression level approximately lOμg/ml) was filtered using a CUNO filter system and concentrated to 380 is using a ProFlux tangential flow filtration system with a 10 K filter membrane. The fusion protein was captured using a 50 ml Fast Flow Q column (Pharmacia) in 20 mM Tris pH 7.4 at a flow rate of 5ml/min. Protein was eluted using a gradient: from 0% to 50% 20mM Tris pH 7.4, 1M NaCl in 10 CV, then to 100%B in 2 CV. Fractions containing the fusion protein were pooled and subjected to C4 Reverse Phase Chromatography in 0.1% TFA water at a flow rate of 5 ml/min. The fusion protein was eluted using a gradient from 20% B (0.1% TFA in acetonitrile) to 90% B in 120min. Fractions (3.5 ml/tube) were collected. Acetonitrile was removed by vacuum drying.
Approximately 9 mis of pooled sample was diluted with lx PBS pH 7.4 to 40ml and dialyzed against 4 liters of lx PBS pH 7.4 overnight. The sample was filtered and concentration was determined by absorption at 280nm.
Example 3c purification of Exendin-4-Fc :
Approximately 4 liters of conditioned medium (fusion protein expression level approximately 8 μg/ml) was filtered using a CUNO filter system and concentrated to 250 mis using a ProFlux tangential flow filtration system with a 3 OK filter membrane.
Exendin-4-Fc was captured with a 5 ml HiTrap protein A column in lx PBS, pH 7.4 at a flow rate of 2 ml/min and eluted with 50 mM citric acid pH 3.3. Fractions containing the fusion protein were pooled, filtered, and dialyzed against 4 liters of 1 x PBS over night. The dialyzed sample was then applied to a Superdex 75 60/60 column in lx PBS pH7.4, 0.5M NaCI at a flow rate of 10 ml/min. Fractions (20 ml/tube) containing the fusion protein were collected, pooled, and concentrated to about 1 mg/ml . Concentrated samples were then filtered using a MILLEX-GV 0.22 um Filter Unit. Example 3d purification of Exendin-4-HSA and Exendin-4- linker-HSA:
Approximately 1.1 liters of conditioned medium (fusion protein expression level approximately 6μg/ml) was filtered using a CUNO filter system and concentrated to 175 is using a ProFlux tangential flow filtration system with a 3 OK filter membrane .
The fusion protein was captured using a 5 ml HiTrap Q- sepharose column (Pharmacia) in 20 mM Tris pH 7.4 at a flow rate of 2 ml/min. Protein was eluted using a gradient from 0% to 50% 20mM Tris pH 7.4 , 1M NaCI in 12 CV and then to 100%B in 4 CV.
Fractions containing the fusion protein were pooled and subjected to C4 Reverse Phase Chromatography in 0.1% TFA water at a flow rate of 5 ml/min. The fusion protein was eluted using a gradient from 10% B (0.1% TFA in acetonitrile) to 100% B in 70 min. Fractions (10 ml/tube) containing the fusion protein were collected. Acetonitrile was removed using a vacuum dryer.
Approximately 8 mis of pooled sample was dialyzed against 4 liters of lx PBS pH 7.4 overnight. The sample was filtered and concentration was determined by absorption at 280nm. The dialyzed sample was then applied to a Superdex 200 26/60 column in lx PBS pH 7.4 , 0.5 M NaCI at a flow rate of 2 ml/min. Fractions (3 ml/tube) containing the fusion protein were collected, pooled, concentrated, and filtered.
Example 4: Characterization of fusion proteins by SDS PAGE: SDS-PAGE followed by immunoblotting was used to analyze both purified fusion protein as well as conditioned medium from cells transfected with various fusion protein expression vectors. SDS-PAGE was performed on a Novex Powerease 500 system using Novex 16% Tris-Glycine Precast gels (EC6498) , running buffer (lOx, LC2675) and sample buffer (L2676) . Samples were reduced with 50 mM DTT and heated 3-5 min at 95°C prior to loading.
After running the SDS-PAGE gel, water and transfer buffer (IX Tris-Glycine Seprabuff (Owl Scientific Cat. No. ER26-S) with 20% methanol) were used to rinse SDS from the gels. A Novex transfer apparatus was used with PVDF (BioRad, Cat. No. 162-0174) and nitrocellulose membranes (BioRad, Cat. No. 1703965 or 1703932) . Transfer was carried out at room temperature for 90 min at 30-35 V. Membranes were blocked in IX PBS with 0.1% Tween-20 (Sigma, Cat. No. P-
7949) and 5% Milk (BioRad, Cat. No. 170-6404) for 1-12 hours at 4°C. Antibodies are diluted into IX PBS +5% Milk and the blots are incubated in these solutions for 1-2 h at 4°C . Between incubations, the blots are washed four times for 5 min each with IX PBS and 0.2% Tween-20 at room temperature. PBS was made from either GIBCO 10X PBS (Cat No. 70011), to give a final composition of 1 mM monobasic potassium phosphate, 3 mM dibasic sodium phosphate, 153 mM sodium chloride, pH 7.4, or PBS pouches from Sigma (Cat. No. 1000- 3), to give 120 mM NaCI, 2.7 mM KCl and 10 mM phosphate, pH 7.4 at 25°C.
The primary antibodies were either a polycolonal goat anti-IgGl or rabbit anti-HSA. The secondary antibody was either an anti-goat IgG HRP or an anti-rabbit IgG HRP . The secondary antibody was diluted 1:5000. An ECL system
(Amersham Pharmacia Biotech, Cat. No. RN2108 and Cat. No. RPN1674H) was used for developing blots.
Figure 3A compares purified Fc protein to conditioned
8 media from pJB02-Val -GLP-l-Fc and pJB02-Exendin-4-Fc transfected cells. The decrease in mobility is consistent with the increased size due to the GLP-1 portion of the fusion protein. Figure 3B similarly compares purified HSA with conditioned media from cells transfected with pJB02-
Val -GLP-2-HSA, pJB02- Val -GLP-1-Linker-HSA, pJB02-Exendin- 4-HSA, or pJB02-Exendin-4-Linker-HSA. Figure 4 identifies purified fusion protein preparations.
Example 5: Characterization of fusion proteins using mass spectrometry:
All experiments were performed on a Micro ass TofSpec- 2E mass spectrometer equipped with Time Lag Focusing electronics, a Reflectron (used in analysis of the 0-8000 Da peptide range) , a Linear detector (used during high mass / good signal analysis) , and Post Acceleration Detector (or P.A.D., used for high mass / extremely low signal analysis) The effective path length of the instrument in Linear mode is 1.2 meters, in Reflectron mode it is 2.3 meters. Two dual micro-channel plate detectors are fitted for linear and reflectron mode detection. The laser used is a Laser
Science Inc. VSL-337i nitrogen laser operating at 337 nm at 5 laser shots per second. All data were acquired using a 2 GHz, 8 bit internal digitizer and up to 50 laser shots were averaged per spectrum. The instrument was operated in linear mode for the analysis of the GLP-1 fusion proteins in question. The linear detector is a device that detects ions that travel down the flight tube of the MALDI-ToF-MS instrument. It measures the ion abundance over time and sends a signal to the digitizer for conversion. The digitizer is an analog- to-digital converter that allows the signal from the mass spectrometer to be transferred to the computer, where it is reconstructed into a usable m/z spectrum.
A recrystallized saturated sinapinic acid solution (diluted in 50/50 Acn / H20 and 0.1% TFA) was utilized as the ionization matrix. Sinapinic acid is a proper matrix' for proteins above 10 kDa. Mass appropriate reference proteins were used for internal and external calibration files in order to obtain accurate mass determinations for the samples analyzed. Samples were all analyzed using a 1:2 sample to matrix dilution. The instrument was initially set up under the following linear detector conditions:
Source Voltage: 20.0 keV Pulse Voltage : 3.0 keV Extraction Voltage: 20.0 keV Laser Coarse: 50 Focus Voltage: 16.0 keV Laser Fine: 50 Linear detector : 3.7 keV
P.A.D.: (off line)
These settings were modified (as needed) to give the best signal/noise ratio and highest resolution. Table 3 provides a characterization of different GLP-1 fusion proteins .
Table 3
Figure imgf000079_0001
CEx refers to a C-terminal extension and comprises the sequence of Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser .
Linker is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly- Gly-
Gly-Gly-Ser. Example 6: Activity of heterologous fusion proteins:
The ability of the fusion proteins of the present invention to activate the GLP-1 receptor was assessed using in vi tro assays such as those described in EP 619,322 to
Gelfand, et al . , and U.S. Patent No. 5,120,712, respectively. The activity of these compounds relative to
8 the activity of Val -GLP-1 (7-37 ) OH is reported Table 4.
Figure 8 represents in vi tro dose response curves for Val -
GLP-1 and Exendin-4 fusion proteins. In addition, Table 5a and 5b provide the in vi tro activity of a large group of
GLP-1 analogs that can be fused to an Fc or an albumin protein to make biologically active fusion proteins . These activities are compared to GLP-1 (7-37) OH.
Table 4: In vitro activity of GLP-1 fusion proteins
Figure imgf000080_0001
CEx refers to a C-terminal extension and comprises the sequence of Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser .
Linker is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly- Gly-
Gly-Gly-Ser C2 is Ser-Ser-Gly-Ala-Ser-Ser-Gly-Ala.
The amino acid sequences of the fusion proteins described in Tables 3 and 4 are represented in SED ID NO: 13 to SEQ ID NO: 31.
Val8-GLP-1-Human serum albumin amino acid sequence is represented by SEQ ID NO: 13.
1 HVEGTFTSDV SSYLEGQAAK EFIA VKGR GDAHKSEVAH RFKD GΞENF KALVLIAFAQ
61 YLQQCPFEDH VKLVNEVTEF AKTCVADESA ENCDKS HTL FGDKLCTVAT LRETYGEMAD
121 CCAKQEPER ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETF KKYL YEIARRHPYF
181 YAPELLFFAK RY AAFTECC QAADKAACLL PKLDELRDEG KASSAKQR K CASLQKFGER 241 AFKAWAVARL SQRFPKAEFA EVS VTD T KVHTECCHGD LLECADDRAD LAKYICENQD
301 SISSKLKΞCC EKP EKSHC IAEVENDEMP AD PS AADF VESKDVCKNY AEAKDVFLGM 361 F YEYARRHP DYSWLLLRL AKTYETTLEK CCAAADPHEC YAKVFDEFKP VEEPQNLIK 421 QNCELFEQ G EYKFQNAL V RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA 481 EDYLSW NQ CV HEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP KEFNAETFTF 541 HADICT SEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD DFAAFVEKCC KADDKETCFA
601 EEGKKLVAAS QAALGL [ SEQ ID NO : 13 ]
8
Val -GLP-1-Linker-Human serum albumin ammo acid sequence is represented by SEQ ID NO: 14. 1 HVEGTFTSDV SSYLEGQAAK EFIAWLVKGR GGGGGSGGGG SGGGGSDAHK SEVAHRFKDL
61 GEENFKALVL I FAQYLQQC PFEDHVKLVN EVTEFAKTCV ADΞSAENCDK SLHTLFGDKL 121 CTVATLRETY GEMADCCAKQ EPERNECFLQ HKDDNPNLPR LVRPEVDVMC TAFHDNEETF 181 LKKYLYEIAR RHPYFYAPEL LFFAKRYKAA FTECCQAADK AACLLPKLDE LRDEGKASSA 241 KQRLKCASLQ KFGERAFKAW AVARLSQRFP KAEFAEVSKL VTDLTKVHTE CCHGDLLECA 301 DDRADLAKYI CENQDSISSK LKECCEKPLL EKSHCIAEVE NDEMPADLPS LAADFVESKD
361 VCKNYAEAKD VFLGMFLYEY ARRHPDYSλ V LLLRLAKTYΞ TTLEKCCAAA DPHECYAKVF 421 DEFKPLVEEP QNLIKQNCEL FEQLGΞYKFQ NALLVRYTKK VPQVSTPTLV EVSRNLGKVG 481 SKCCKHPEAK RMPCAEDYLS WLNQLCVLH EKTPVSDRVT KCCTESLVNR RPCFSALEVD 541 ETYVPKEFNA ETFTFHADIC TLSEKERQIK KQTALVELVK HKPKATKΞQL KAVMDDFAAF 601 VEKCCKADDK ETCFAEEGKK LVAASQAALG L [ SEQ ID NO : 14 ] o Gly -Glu -GLP-1-CEx-Linker-Human serum albumin amino acid sequence is represented by SEQ ID NO: 15.
1 HGEGTFTSDV SSYLΞEQAAK EFIAWLVKGR GSSGAPPPSG GGGGSGGGGS GGGGSDAHKS
61 EVAHRFKDLG ΞENFKALVLI AFAQYLQQCP FEDHVKLVNE VTEFAKTCVA DESAENCDKS 121 LHTLFGDKLC TVATLRETYG EMADCCAKQE PERNECFLQH KDDNPNLPRL VRPEVDVMCT
181 AFHDNEETFL KKYLYEIARR HPYFYAPELL FFAKRYKAAF TECCQAADKA ACLLPKLDEL
241 RDEGKASSAK QRLKCASLQK FGERAFKAWA VARLSQRFPK AEFAEVSKLV TDLTKVHTΞC
301 CHGDLLECAD DRADLAKYIC ENQDSISSKL KECCEKPLLE KSHCIAEVEN DEMPADLPSL
361 AADFVESKDV CKNYAEAKDV FLGMFLYΞYA RRHPDYSWL LLRLAKTYET TLEKCCAAAD 421 PHECYAKVFD EFKPLVEΞPQ NLIKQNCELF EQLGEYKFQN ALLVRYTKKV PQVSTPTLVE
481 VSRNLGKVGS KCCKHPEAKR MPCAΞDYLSV VLNQLCVLHE KTPVSDRVTK CCTESLVNRR
541 PCFSALEVDE TYVPKEFNAE TFTFHADICT LSEKERQIKK QTALVELVKH KPKATKEQLK
601 AVMDDFAAFV EKCCKADDKE TCFAEEGKKL VAASQAALGL [ SEQ ID NO : 15 ]
Exendin-4-Human serum albumin amino acid sequence is represented by SEQ ID NO: 16.
1 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPSD AHKSEVAHRF KDLGEENFKA
61 LVLIAFAQYL QQCPFEDHVK LVNEVTEFAK TCVADESAEN CDKSLHTLFG DKLCTVATLR
121 ETYGEMADCC AKQEPERNEC FLQHKDDNPN LPRLVRPEVD VMCTAFHDNE ETFLKKYLYΞ 181 IARRHPYFYA PELLFFAKRY KAAFTECCQA ADKAACLLPK LDELRDEGKA SSAKQRLKCA
241 SLQKFGERAF KAWAVARLSQ RFPKAEFAEV SKLVTDLTKV HTECCHGDLL ΞCADDRADLA
301 KYICΞNQDSI SSKLKECCEK PLLEKSHCIA EVENDEMPAD LPSLAADFVE SKDVCKNYAΞ
361 AKDVFLGMFL YEYARRHPDY SWLLLRLAK TYETTLEKCC AAADPHECYA KVFDEFKPLV
421 EEPQNLIKQN CELFEQLGEY KFQNALLVRY TKKVPQVSTP TLVEVSRNLG KVGSKCCKHP 481 EAKRMPCAED YLSWLNQLC VLHEKTPVSD RVTKCCTESL VNRRPCFSAL EVDETYVPKΞ
541 FNAETFTFHA DICTLSEKER QIKKQTALVE LVKHKPKATK EQLKAVMDDF AAFVΞKCCKA
601 DDKETCFAEΞ GKKLVAASQA ALGL [ SEQ ID NO : 16 ]
Exendin-4-Linker-Human serum albumin amino acid sequence is represented by SEQ ID NO: 17.
1 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPSG GGGGSGGGGS GGGGSDAHKS
61 EVAHRFKDLG EENFKALVLI AFAQYLQQCP FEDHVKLVNE VTEFAKTCVA DESAENCDKS
121 LHTLFGDKLC TVATLRETYG EMADCCAKQE PERNECFLQH KDDNPNLPRL VRPEVDVMCT
181 AFHDNEETFL KKYLYEIARR HPYFYAPELL FFAKRYKAAF TECCQAADKA ACLLPKLDEL 241 RDEGKASSAK QRLKCASLQK FGERAFKAWA VARLSQRFPK AEFAEVSKLV TDLTKVHTEC 301 CHGDLLECAD DRADLAKYIC ENQDSISSKL KECCEKPLLE KSHCIAEVEN DEMPADLPSL 361 AADFVESKDV CKNYAEAKDV FLGMFLYEYA RRHPDYSWL LLRLAKTYET TLEKCCAAAD 421 PHECYAKVFD EFKPLVEEPQ NLIKQNCELF EQLGEYKFQN ALLVRYTKKV PQVSTPTLVE 481 VSRNLGKVGS KCCKHPΞAKR MPCAΞDYLSV VLNQLCVLHE KTPVSDRVTK CCTESLVNRR 541 PCFSALEVDE TYVPKEFNAE TFTFHADICT LSEKERQIKK QTALVELVKH KPKATKEQLK 601 AVMDDFAAFV ΞKCCKADDKΞ TCFAEEGKKL VAASQAALGL [SEQ ID NO: 17]
Q Val -GLP-1-IgGl amino acid sequences represented by SEQ ID
NO: 18.
1 HVEGTFTSDV SSYLEGQAAK EFIAWLVKGR GAEPKSCDKT HTCPPCPAPE LLGGPSVFLF
61 PPKPKDTLMI SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW
121 SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP RΞPQVYTLPP SRΞEMTKNQV 181 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF
241 SCSVMHEALH NHYTQKSLSL SPGK [ SEQ ID NO : 18 ]
8
Val -GLP-1-Cex-IgGl ammo acid sequence is represented by
SEQ ID NO : 19 . 1 HVEGTFTSDV SSYLEGQAAK EFIAWLVKGR GSSGAPPPSA EPKSCDKTHT CPPCPAPELL
61 GGPSVFLFPP KPKDTLMISR TPEVTC WD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ
121 YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
181 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS
241 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK [ SEQ ID NO : 19 ]
8 22 Val -Glu GLP-1-IgGl ammo acid sequence is represented by
SEQ ID NO: 20.
1 HVEGTFTSDV SSYLEEQAAK EFIAWLVKGR GAEPKSCDKT HTCPPCPAPE LLGGPSVFLF
61 PPKPKDTLMI SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW 121 SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV
181 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF
241 SCSVMHEALH NHYTQKSLSL SPGK [ SEQ ID NO : 20 ]
8 22
Val -Glu GLP-1-CEx-IgGl ammo acid sequence is represented by SEQ ID NO : 21 .
1 HVEGTFTSDV SSYLEEQAAK EFIAWLVKGR GSSGAPPPSA EPKSCDKTHT CPPCPAPELL
61 GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ
121 YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
181 EEMTKNQVSL TCLVKGFYPS • DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS 241 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK [ SEQ ID NO : 21 ] 8 22 Gly -Glu GLP-l-C2-IgGl amino acid sequence is represented by SEQ ID NO: 22.
1 HGEGTFTSDV SSYLEEQAAK EFIAWLVKGR GSSGASSGAA EPKSCDKTHT CPPCPAPELL
61 GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 121 YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
181 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS
241 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK [ SEQ ID NO : 22 ]
8 22
Gly -Glu GLP- 1-CEx-Linker- IgGl amino acid sequence is represented by SEQ ID NO : 23 .
1 HGEGTFTSDV SSYLEEQAAK EFIAWLVKGR GSSGAPPPSG GGGSGGGGSG GGGSAEPKSC
61 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD
121 GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
181 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 241 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK [ SEQ ID NO : 23 ;
8 22
Gly -Glu GLP-l-CEx-Linker-IgG4 amino acid sequence is represented by SEQ ID NO : 24 .
1 HGEGTFTSDV SSYLEEQAAK EFIAWLVKGR GSSGAPPPSG GGGSGGGGSG GGGSAESKYG 61 PPCPSCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSQEDPEV QFNWYVDGVE
121 VHNAKTKPRE EQFNSTYRW SVLTVLHQDW LNGKEYKCKV SNKGLPSSIΞ KTISKAKGQP 181 REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS 241 FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK [ SEQ ID NO : 24 ]
8 22 . . Gly -Glu GLP-l-CEx-2Lmker-IgGl ammo acid sequence s represented by SEQ ID NO : 25 .
1 HGEGTFTSDV SSYLEEQAAK EFIAWLVKGR GSSGAPPPSG GGGSGGGGSG GGGSGGGGSG
61 GGGSGGGGSA EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD
121 VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKEYKCKVSN 181 KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG
241 QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP 301 GK [ SEQ ID NO : 25 ]
8 22
Gly -Glu GLP-l-2Linker-IgGl amino acid sequence is represented by SEQ ID NO: 26. 1 HGEGTFTSDV SSYLEEQAAK EFIAWLVKGR GGGGGSGGGG SGGGGSGGGG SGGGGSGGGG
61 SAEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSHEDPEV
121 KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE
181 KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 241 TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK
[ SEQ ID NO : 26 ]
8 22
Gly -Glu GLP-l-2CEx-IgGl ammo acid sequence is represented by SEQ ID NO : 27 . 1 HGEGTFTSDV SSYLEEQAAK EFIAWLVKGR GSSGAPPPSS SGAPPPSAEP KSCDKTHTCP
61 PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA 121 KTKPREEQYN STYRWSVLT VLHQDWLNGK ΞYKCKVSNKA LPAPIEKTIS KAKGQPREPQ 181 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ΞNNYKTTPPV LDSDGSFFLY 241 SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK [ SEQ ID NO : 27 ]
Gly -Glu -Val -lie GLP-1-CEx-Linker-IgGl amino acid sequence is represented by SEQ ID NO : 28 .
1 HGEGTFTSDV SSYLEEQAVK EFIAWLIKGR GSSGAPPPSG GGGSGGGGSG GGGSAEPKSC
61 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD 121 GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
181 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS
241 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK [ SEQ ID NO : 28 ]
Exendin-4-IgGl amino acid sequence is represented by SEQ ID NO : 29 .
1 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPSA EPKSCDKTHT CPPCPAPELL
61 GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ
121 YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
181 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS 241 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK [ SEQ ID NO : 29 ] Exendin-4-C2-IgGl amino acid sequence is represented by SEQ ID NO : 30 .
1 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGASSGAA EPKSCDKTHT CPPCPAPELL 61 GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ
121 YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
181 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS
241 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK [ SEQ ID NO : 30 ]
Exendin-4-Linker-IgGl amino acid sequence is represent by SEQ ID NO : 31 .
1 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPSG GGGSGGGGSG GGGSAEPKSC
61 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD
121 GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 181 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS
241 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK [ SEQ ID NO : 31 ] Table 5a : In vi tro GLP-1 analog activity
GLP-1 Receptor
GLP-1 Compound Activation
GLP-l(7-37)OH 1.0
Val8-GLP-l(7-37)OH 0.47
(n = 6)
Gly -His ,11-L-GLP-l(7-37)OH 0.282
Val -Ala .11-GLP-1 (7-37) OH 0.021
Val8-Lys -GLP-l(7-37)OH 0.001
Val8-Tyr12-GLP-l(7-37)OH 0.81
Val8-Glu16-GLP-l(7-37)OH 0.047 Var -Ala16-GLP- l(7-37)OH 0.112
Val -Tyr .16b-GLP-l(7-37)OH 1.175
Valβ-Lys .20 -GLP-1 (7-37) OH 0.33
Gln22-GLP- l(7-37)OH 0.42
Val8-Ala22-GLP-l(7-37)OH 0.56
Val8-Ser22-GLP-l(7-37)OH 0.50
Val8-Asp22-GLP-l(7-37)OH 0.40
Val8-Glu22-GLP-l(7-37)OH 1.29
Val8-Lys22-GLP-l(7-37)OH 0.5S
Val8-Pro22-GLP-1(7-37)OH 0.01
Val8-His22-GLP-l(7-37)OH 0.14
Val8-Lys2 -GLP-1(7-36)NH2 0.53
Val8-Glu22-GLP-1(7-36)NH2 1.0
Gly8-Glu22-GLP-l(7-37)OH 1.07
Val8-Lys23-GLP-l(7-37)OH 0.18
Val8-His24-GLP-l(7-37)OH 0.007 Val -Lys 2 -GLP-1 7-37)OH 0.02
Val -His 2D6-GLP-l 7-37)OH 1.6
Val -Glu 26 -GLP-1 7-37)OH 1.5
Val°-His 27 -GLP-1 7-37)OH 0.37
Val8-Ala2AQLP-l 7-37)OH 0.47
Gly -Glu 3J0U-GLP-l 7-37)OH 0.29
Val -GluJ 3U0-GLP-l 7-37)OH 0.29
Val -Asp 30 -GLP-1 7-37)OH 0.15
Val -SeA 30 -GLP-1 7-37)OH 0.19
Val -His 30 -GLP-1 7-37)OH 0.19
Val -GluJ 3J3-GLP-l 7-37)OH 0.039
Val -Ala 33 -GLP-1 7-37)OH 0.1
Val°-Gly 33 -GLP-1 7-37)OH 0.01
34
Valβ-Glu -GLP-1 7-37)OH 0.17
Val°-ProJ 3D5-GLP-l 7-37)OH 0.094
Val°-His 3J5 -GLP-1 7-37)OH 0.41
Val°-G1A 35-GLP-1 7-37)OH 0.15 Val 8 - rG-1t-A,36 -GLP-1 (7-37)OH 0.11
Val' -His ,36 -GLP- l(7-37)OH 0.22
Val8-His3AQLP-l(7-37)OH 0.33
Figure imgf000089_0001
Val 8 -L τ„ys„2""2 -Gl-.u 30 -GLP-1 (7-37) OH 0.37
Figure imgf000089_0002
(7-37) OH 0.35
Val8-Glu22-Ala AGLP-l(7-37)OH 1.02
Val8-Glu22-Lys2AGLP-l(7-37)OH 1.43
Val8-Lys3Aval3AGLP-l(7-37)OH 0.05
Val8-Lys33-Asn3AGLP-l(7-37)OH 0.09
Val8-Gly3 -Lys35-GLP-l(7-37)OH 0.34
Val8-Gly35-Pro3AGLP-l(7-37)NH2 0.53
Table 5b. In vitro GLP-1 analog activity
Figure imgf000090_0001
Figure imgf000091_0001
Table 6 : In vitro activity of GLP/Exendin analogs
Figure imgf000091_0002
Figure imgf000092_0001
Example 7 : In vivo pharmacokinetics of Val -GLP-1-IgGl
8 and Val -GLP-1-HSA:
8 8
A pharmacok etic study of Val -GLP-1-IgGl and Val -
GLP-1-HSA was performed in cynomologus monkeys. Monkeys
8 were dosed at 5.6 nmoles/kg with either purified Val -GLP-1-
8 IgGl' or Val -GLP-1-HSA . The compounds were administered as an intravenous bolus administration. Blood was collected pre-dose and at 0.083, 0.25, 0.5, 1, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 216 hours post-dose into tubes containing EDTA. Plasma concentrations of immunoreactive
8 Val -GLP-1 were determined using a radioimmunoassay that utilizes a polyclonal antiserum whose primary specificity is
8 for the N-terminal (7-16) region of Val -GLP-1 (7-37) .
8 Figure 9 depicts the plasma concentration of Val -GLP-l-Fc o and Val -GLP-1-Linker-HSA following a single intravenous dose to two cynomologus monkeys. The Fc fusion protein had a half-life of approximately 45 hours and the albumin fusion had a half-life of approximately 87 hours. Example 8: In vivo pharmacodynamics of Exendin-4-IgGl : Two chronically cannulated normal male beagle dogs were studied after an overnight fast. Arterial and venous vascular access ports were accessed, and a catheter was inserted percutaneously into a cephalic vein and secured. Animals were placed in cages, and their catheters were attached to a swivel/tether system. A solution containing the fusion protein Exendin-4-IgGl (11.8 μM) was injected intravenously (1.0 nmol/kg) through the cephalic vein catheter. The catheter was then cleared with 10 ml of saline. Two hours later, a hyperglycemic (150 mg/dl) clamp was initiated and continued for three hours. Arterial blood samples were drawn throughout this 5-hour period for determination of plasma concentrations of the fusion protein, glucose, and insulin.
The results of this study were compared to those from a similar, previous study in which both of the animals had received a bolus of saline, s.c, and three hours later were studied using a 3-hour hyperglycemic (150 mg/dl) clamp. In both sets of studies, plasma glucose concentrations were determined using a Beckman glucose analyzer. Plasma insulin concentrations were determined by employees of Linco
Research, Inc. using an RIA kit developed in their laboratories. The data is illustrated in Figures 10 and 11.
Example 9 : In vivo pharmacokinetics of
Gly8-Glu22-GLP-l-CEx-Linker-IgGl:
Two groups of three normal male beagle dogs received
0.1 mg/kg of Gly8-Glu22-GLP-l-CEx-Linker-IgGl by subcutaneous (SC) or intravenous (IV) administration. Plasma concentrations of Gly8-Glu2-GLP-l-CEx-Linker-IgGl immunoreactivity were determined by radioimmunoassay in samples collected from 30 minutes predose to 216 hours postdose for both the IV and SC groups . These concentrations were subsequently used to determine the reported pharmacokinetic parameters . The mean elimination half-life of IV administered Gly8-Glu22-GLP-l-CEx-Linker-IgGl was approximately 55 hours and the total body clearance was 1.5 mL/h/kg. The mean elimination half-life of SC administered Gly8-Glu22-GLP-l-CEx-Linker-IgGl was approximately 38 hours.

Claims

WE CLAIM :
1. A heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) human albumin; b) human albumin analogs; and c) fragments of human albumin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide.
2. A heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) human albumin; b) human albumin analogs; and c) fragments of human albumin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide via a peptide linker.
3. The heterologous fusion protein of the Claim 2 wherein the peptide linker is selected from the group consisting of: a) a glycine rich peptide; b) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]n where n is 1, 2, 3, 4, 5 or 6; and c) a peptide having the sequence [Gly-Gly-Gly-Gly- Ser]3.
4. The heterologous fusion protein of Claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula 1 [SEQ ID NO: 2]
7 8 9 10 11 12 13 14 15 16 17
His-Xaa-Xaa-Gly-Xaa-Phe-Thr-Xaa-Asp-Xaa-Xaa- 18 19 20 21 22 23 24 25 26 27 28 Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe- 29 30 31 32 33 34 35 36 37 38 39 Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-
40 41 42 43 44 45 Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
Formula I (SEQ ID NO: 2)
wherein:
Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, lie, Val,
Glu, Asp, or Lys; Xaa at position 9 is Glu, Asp, or Lys; Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, lie, Val, Glu, Asp, or Lys;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, lie,
Val, Glu, Asp, or Lys; Xaa at position 16 is Val, Ala, Gly, Ser, Thr,. Leu, lie, Tyr, Glu, Asp, Trp, or Lys; Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, lie,
Val, Glu, Asp, or Lys; Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, lie,
Val, Glu, Asp, Trp, Tyr, or Lys; Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, or Lys ;
Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, lie,
Val, Glu, Asp, Met, Trp, Tyr, or Lys; Xaa at position 21 is Glu, Asp, or Lys; Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys; Xaa at position 23 is Gin, Asn, Arg Glu, Asp, or Lys; Xaa at position 24 is Ala, Gly, Ser Thr, Leu, lie,
Val, Arg, Glu, Asp, or Lys; Xaa at position 25 is Ala, Gly, Ser Thr, Leu, lie,
Val, Glu, Asp, or Lys; Xaa at position 26 is Lys, Arg, Gin Glu, Asp, or His; Xaa at position 27 is Leu, Glu, Asp or Lys ; Xaa at position 30 is Ala, Gly, Ser Thr, Leu, lie,
Val, Glu, Asp, or Lys; Xaa at position 31 is Trp, Phe, Tyr Glu, Asp, or Lys; Xaa at position 32 is Leu, Gly, Ala Ser, Thr, lie,
Val, Glu, Asp, or Lys; Xaa at position 33 is Val, Gly, Ala Ser, Thr, Leu, lie, Glu, Asp, or Lys; Xaa at position 34 is Asn, Lys, Arg Glu, Asp, or His; Xaa at position 35 is Gly, Ala, Ser Thr, Leu, lie,
Val, Glu, Asp, or Lys; Xaa at position 36 is Gly, Arg, Lys Glu, Asp, or His; Xaa at position 37 is Pro, Gly, Ala Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys, or is deleted; Xaa at position 38 is Ser, Arg, Lys Glu, Asp, or His, or is deleted; Xaa at position 39 is Ser, Arg, Lys Glu, Asp, or His, qr is deleted; Xaa at position 40 is Gly, Asp, Glu or Lys, or is deleted; Xaa at position 41 is Ala, Phe, Trp Tyr, Glu, Asp, or
Lys, or is deleted; Xaa at position 42 is Ser, Pro, Lys Glu, or Asp, or is deleted; Xaa at position 43 is Ser, Pro, Glu Asp, or Lys, or is deleted; Xaa at position 44 is Gly, Pro, Glu Asp, or Lys, or is deleted; and Xaa at position 45 is Ala, Ser, Val, Glu, Asp, or Lys, or is deleted; provided that when the amino acid at position 37, 38, 39, 40, 41, 42, 43, or 44 is deleted, then each amino acid downstream of that amino acid is also deleted.
5. The heterologous fusion protein of Claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula II (SEQ ID NO: 3) :
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Thr-Ser-Asp-Xaa-Ser- 18 19 20 21 22 23 24 25 26 27 28 Xaa-Tyr-Leu-Glu-Xaa-Xaa-Xaa-Ala-Xaa-Xaa-Phe-
29 30 31 32 33 34 35 36 37 Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Xaa-Xaa Formula II (SEQ ID NO : 3)
wherein:
Xaa at position 7 is: L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy- histidine, homohistidine, α-fluoromethyl-histidine or α-methyl-histidine; Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr;
Xaa at position 9 is: Thr, Ser, Arg, Lys, Trp, Phe, Tyr, Glu, or His;
Xaa at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His; Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
Val, Tyr, Glu, or Ala; Xaa at position 18 is: His, Pro, Asp, Glu, Arg, Ser, Ala, or
Lys; Xaa at position 19 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or Cys;
Xaa at position 23 is: His, Asp, Lys, Glu, Gin, or Arg;
Xaa at position 24 is: Glu, Arg, Ala, or Lys;
Xaa at position 26 is: Trp, Tyr, Phe, Asp, Lys, Glu, or His; Xaa at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or
Lys;
Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
Xaa at position 31 is: Asp, Glu, Ser, Thr, Arg, Trp, or Lys;
Xaa at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu; Xaa at position 34 is: Glu, Lys, or Asp;
Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
Asp, Gly, Pro, His, or Glu;
Xaa at position 36 is: Thr, Ser, Asp, Trp, Tyr, Phe, Arg,
Glu, or His; Xaa at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp,
Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
6. The heterologous fusion protein of Claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula III (SEQ ID NO: 4) :
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Xaa-Xaa-Thr-Ser-Asp-Xaa-Ser- 18 19 20 21 22 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Xaa-Xaa-Xaa-Ala-Xaa-Xaa-Phe- 29 30 31 32 33 34 35 36 37
Ile-Ala-Xaa-Leu-Xaa-Xaa-Xaa-Xaa-Xaa formula III (SEQ ID NO : 4)
wherein : Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine; Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr; Xaa at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His; Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
Val, Glu, or Ala; Xaa at position 22: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or Cys ;
Xaa at position 23 is: His, Asp, Lys, Glu, or Gin; Xaa at position 24 is: Glu, His, Ala, or Lys; Xaa at position 25 is: Asp, Lys, Glu, or His; Xaa at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys;
Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
Xaa at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or
Glu; Xaa at position 34 is: Glu, Lys, or Asp; Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His, or Glu; Xaa at position 36 is: Arg, Glu, or His;
Xaa at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
7. The heterologous fusion protein of Claim 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula IV (SEQ ID NO: 5) :
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Thr-Xaa-Thr-Ser-Asp-Xaa-Ser-
18 19 20 21 22 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Xaa-Xaa-Ala-Ala-Xaa-Glu-Phe- 29 30 31 32 33 34 35 36 37 Ile-Xaa-Trp-Leu-Val-Lys-Xaa-Arg-Xaa formula IV (SEQ ID NO : 5)
wherein :
Xaa at position 7 is : L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine;
Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr;
Xaa at position 12 is: His, Trp, Phe, or Tyr; Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu, or Ala;
Xaa at position 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or Cys;
Xaa at position 23 is: His, Asp, Lys, Glu, or Gin; Xaa at position 26 is: Asp, Lys, Glu, or His;
Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His, or Glu;
Xaa at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH2, Gly, Gly-Pro, or Gly-Pro-NH2, or is deleted.
8. The heterologous fusion protein of Claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula V (SEQ ID NO: 6)
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser- 18 19 20 21 22 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Xaa-Xaa-Ala-Ala-Lys-Xaa-Phe-
29 30 31 32 33 34 35 36 37 Ile-Xaa-Trp-Leu-Val-Lys-Gly-Arg-Xaa formula V (SEQ ID NO : 6)
wherein :
Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine; Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr; Xaa at position 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or
Cys;
Xaa at position 23 is: His, Asp, Lys, Glu, or Gin; Xaa at position 24 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys
Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
Xaa at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp,
Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
9. The heterologous fusion protein of Claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula VIII (SEQ ID NO: 11) .
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa-Ser-
18 19 20 21 22 23 24 25 26 27 28 Xaa-Xaa-Xaa-Glu-Xaa-Xaa-Ala-Xaa-Xaa-Xaa-Phe- 29 30 31 32 33 34 35 36 37 Ile-Xaa-Trp-Leu-Xaa-Xaa-Gly-Xaa-Xaa formula VIII (SEQ ID NO: 11)
wherein :
Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine;
Xaa at position 8 is: Gly, Ala, or Val;
Xaa at position 16 is: Leu or Val;
Xaa at position 18 is Lys or Ser; Xaa at position 19 is: Gin or Tyr;
Xaa at position 20 is: Met or Leu; Xaa at position 22 is: Glu or Gin; Xaa at position 23 is: Glu, or Gin; Xaa at position 25 is: Val or Ala; Xaa at position 26 is: Arg or Lys; Xaa at position 27 is Leu or Glu; Xaa at position 30 is: Glu or Ala; Xaa at position 33 is: Val or Lys; Xaa at position 34 is: Asn or Lys; Xaa at position 36 is: Gly or Arg; and
Xaa at position 37 is: Gly, Pro, Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser, or is absent.
10. The heterologous fusion protein of claims 1 through 9 wherein the GLP-1 compound has no more than 6 amino acids that are different from the corresponding amino acid in GLP- l(7-37)OH, GLP-1 (7-36.0H, or Exendin-4.
11. The heterologous fusion protein of Claim 10 wherein the GLP-1 compound has no more than 5 amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH, GLP-1 (7- 36) OH, or Exendin-4.
12. The heterologous fusion protein of Claim 11 wherein the GLP-1 compound has no more than 4 amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH, GLP-1 (7- 36) OH, or Exendin-4.
13. The heterologous fusion protein of Claim 12 wherein the GLP-1 compound has no more than 3 amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH, GLP-1 (7- 36) OH, or Exendin-4.
14. The heterologous fusion protein of Claim 13 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH, GLP-1 (7- 36) OH, or Exendin-4.
15. The heterologous fusion protein of any one of Claims 1 through 14 wherein Xaa at position 8 is glycine or valine.
16. The heterologous fusion protein of Claim 8 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37) OH or GLP- 1(7-36) OH and Xaa at position 8 is glycine or valine and Xaa at position 30 is alanine, glutamic acid, aspartic acid, serine, or histidine.
17. The heterologous fusion protein of Claim 16 wherein Xaa at position 30 is Glutamic acid.
18. The heterologous fusion protein of Claim 8 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH or GLP- 1(7-36) OH and Xaa at position 8 is glycine or valine and Xaa at position 37 is histidine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, serine, threonine, arginine, or lysine.
19. The heterologous fusion protein of Claim 18 wherein Xaa at position 37 is histidine.
20. The heterologous fusion protein of Claim 8 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH or GLP-
1(7-36) OH and Xaa at position 8 is glycine, valine, leucine, isoleucine, serine, threonine, or methionine and Xaa at position 22 is aspartic acid, glutamic acid, lysine, arginine, asparagine, glutamine or histidine.
21. The heterologous fusion protein of Claim 20 wherein Xaa at position 22 is lysine or glutamic acid.
22. The heterologous fusion protein of Claim 8 wherein the GLP-1 compound is Val8-GLP-1 (7-37 ) .
23. The heterologous fusion protein of Claim 8 wherein the GLP-1 compound is Gly8-GLP-1 (7-37 ) .
24. The heterologous fusion protein of any one of Claims 1 through 23 wherein the second polypeptide is human albumin.
25. The heterologous fusion protein of Claim 24 wherein the second polypeptide has the sequence of SEQ ID NO: 34.
26. The heterologous fusion protein of any one of Claims 1 through 23 wherein the second polypeptide is an N-terminal fragment of albumin.
27. ■ A heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) the Fc portion of an immunoglobulin; b) an analog of the Fc portion of an immunoglobulin; and c) fragments of the Fc portion of an immunoglobulin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide.
28. A heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of a) the Fc portion of an immunoglobulin; b) an analog of the Fc portion of an immunoglobulin; and c) fragments of the Fc portion of an immunoglobulin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide via a peptide linker.
29. The heterologous fusion protein of the Claim 28 wherein the peptide linker is selected from the group consisting of: a) a glycine rich peptide; b) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]n where n is 1, 2, 3, 4, 5, or 6; and c) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser] 3.
30. The heterologous fusion protein of any one of Claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula 1 [SEQ ID NO : 2] .
7 8 9 10 11 12 13 14 15 16 17 His-Xaa-Xaa-Gly-Xaa-Phe-Thr-Xaa-Asp-Xaa-Xaa- 18 19 20 21 22 23 24 25 26 27 28
Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe- 29 30 31 32 33 34 35 36 37 38 39 Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa- 40 41 42 43 44 45 Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
Formula I (SEQ ID NO : 2)
wherein: Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys;
Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, lie, Val,
Glu, Asp, or Lys; Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys; Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, lie,
Tyr, Glu, Asp, Trp, or Lys; Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, or Lys; Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu, Asp, Trp, Tyr, or Lys; Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, or Lys; Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, lie, Val, Glu, Asp, Met, Trp, Tyr, or Lys; Xaa at position 21 is Glu, Asp, or Lys;
Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, lie, Val,
Glu, Asp, or Lys; Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, or Lys; Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, lie, Val, Arg, Glu, Asp, or Lys;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, lie, Val,
Glu, Asp, or Lys; Xaa at position 26 is Lys, Arg, Gin, Glu, Asp, or His; Xaa at position 27 is Leu, Glu, Asp, or Lys; Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys; Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys; Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, lie, Val, Glu, Asp, or Lys; Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, lie, Glu, Asp, or Lys; Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His; Xaa at position 35 is Gly, Ala, Ser,- Thr, Leu, lie, Val, Glu, Asp, or Lys; Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His;
Xaa at position 37 is Pro, Gly, Ala, Ser, Thr, Leu, lie,
Val, Glu, Asp, or Lys, or is deleted; Xaa at position 38 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted; Xaa at position 39 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted; Xaa at position 40 is Gly, Asp, Glu, or Lys, or is deleted; Xaa at position 41 is Ala, Phe, Trp, Tyr, Glu, Asp, or Lys, or is deleted; Xaa at position 42 is Ser, Pro, Lys, Glu, or Asp, or is deleted;
Xaa at position 43 is Ser, Pro, Glu, Asp, or Lys, or is deleted;
Xaa at position 44 is Gly, Pro, Glu, Asp, or Lys, or is deleted; and Xaa at position 45 is Ala, Ser, Val, Glu, Asp, or Lys, or is deleted; provided that when the amino acid at position 37, 38, 39, 40, 41, 42, 43, or 44 is deleted, then each amino acid downstream of that amino acid is also deleted.
31. The heterologous fusion protein of Claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula II (SEQ ID NO: 3) :
7 8 .9 10 11 12 13 14 15 16 17 Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Thr-Ser-Asp-Xaa-Ser- 18 19 20 21 22 23 24 25 26 27 28 Xaa-Tyr-Leu-Glu-Xaa-Xaa-Xaa-Ala-Xaa-Xaa-Phe-
29 30 31 32 33 34 35 36 37 Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Xaa-Xaa Formula II (SEQ ID NO : 3)
wherein :
Xaa at position 7 is : L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-f luoromethyl-histidine or α-methyl- histidine ; Xaa at position 8 is : Gly, Ala, Val , Leu, lie, Ser, or Thr; Xaa at position 9 is: Thr, Ser, Arg, Lys, Trp, Phe, Tyr,
Glu, or His; Xaa at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His; Xaa at position 12 is: His, Trp, Phe, or Tyr; Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
Val, Tyr, Glu, or Ala; Xaa at position 18 is: His, Pro, Asp, Glu, Arg, Ser, Ala, or
Lys; Xaa at position 19 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or Cys;
Xaa at position 23 is: His, Asp, Lys, Glu, Gin, or Arg; Xaa at position 24 is: Glu, Arg, Ala, or Lys; Xaa at position 26 is: Trp, Tyr, Phe, Asp, Lys, Glu, or His; Xaa at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys;
Xaa at position 30 is: Ala, Glu, Asp, Ser, or His; Xaa at position 31 is: Asp, Glu, Ser, Thr, Arg, Trp, or Lys; Xaa at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu; Xaa at position 34 is: Glu, Lys, or Asp; Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
Asp, Gly, Pro, His, or Glu; Xaa at position 36 is: Thr, Ser, Asp, Trp, Tyr, Phe, Arg,
Glu, or His; Xaa at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
32. The heterologous fusion protein of Claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula III (SEQ ID NO: 4) : 7 8 9 10 11 12 13 14 15 16 17
Xaa-Xaa-Glu-Gly-Xaa-Xaa-Thr-Ser-Asp-Xaa-Ser- 18 19 20 21 22 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Xaa-Xaa-Xaa-Ala-Xaa-Xaa-Phe- 29 30 31 32 33 34 35 36 37 Ile-Ala-Xaa-Leu-Xaa-Xaa-Xaa-Xaa-R formula III (SEQ ID NO : 4)
wherein :
Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine; Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr; Xaa at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His; Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
Val, Glu, or Ala; Xaa at position 22: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or Cys; Xaa at position 23 is: His, Asp, Lys, Glu, or Gin; Xaa at position 24 is: Glu, His, Ala, or Lys; Xaa at position 25 is: Asp, Lys, Glu, or His; Xaa at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys; Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
Xaa at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or
Glu; Xaa at position 34 is: Glu, Lys, or Asp;
Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His, or Glu;
Xaa at position 36 is: Arg, Glu, or His;
Xaa at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
33. The heterologous fusion protein of Claim 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula IV (SEQ ID NO: 5) :
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Thr-Xaa-Thr-Ser-Asp-Xaa-Ser- ' 18 19 20 21 22 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Xaa-Xaa-Ala-Ala-Xaa-Glu-Phe- 29 30 31 32 33 34 35 36 37 Ile-Xaa-Trp-Leu-Val-Lys-Xaa-Arg-Xaa formula IV (SEQ ID NO: 5)
wherein :
Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine;
Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr; Xaa at position 12 is: His, Trp, Phe, or Tyr; Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu, or Ala; Xaa at position 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or Cys; Xaa at position 23 is: His, Asp, Lys, Glu, or Gin; Xaa at position 26 is: Asp, Lys, Glu, or His; Xaa at position 30 is: Ala, Glu, Asp, Ser, or His; Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His, or Glu; Xaa at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
34. The heterologous fusion protein of Claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula V (SEQ ID NO: 6)
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
18 19 20 21 22' 23 24 25 26 27 28 Ser-Tyr-Leu-Glu-Xaa-Xaa-Ala-Ala-Lys-Xaa-Phe- 29 30 31 32 33 34 35 36 37 Ile-Xaa-Trp-Leu-Val-Lys-Gly-Arg-Xaa formula V (SEQ ID NO : 6) wherein :
Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine; Xaa at position 8 is: Gly, Ala, Val, Leu, lie, Ser, or Thr; Xaa at position 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or Cys; Xaa at position 23 is: His, Asp, Lys, Glu, or Gin;
Xaa at position 24 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys Xaa at position 30 is: Ala, Glu, Asp, Ser, or His; Xaa at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, Gly, Gly-Pro, or is deleted.
35. The heterologous fusion protein of Claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula VIII (SEQ ID NO: 11) .
7 8 9 10 11 12 13 14 15 16 17 Xaa-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa-Ser- 18 19 20 21 22 23 24 25 26 27 28 Xaa-Xaa-Xaa-Glu-Xaa-Xaa-Ala-Xaa-Xaa-Xaa-Phe- 29 30 31 32 33 34 35 36 37
Ile-Xaa-Trp-Leu-Xaa-Xaa-Gly-Xaa-R formula VIII (SEQ ID NO: 11)
wherein : Xaa at position 7 is: L-histidine, D-histidine, desamino- histidine, 2-amino~histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- histidine,- Xaa at position 8 is: Gly, Ala, or Val; Xaa at position 16 is: Leu or Val; Xaa at position 18 is Lys or Ser; Xaa at position 19 is: Gin or Tyr; Xaa at position 20 is: Met or Leu; Xaa at position 22 is: Glu or Gin; Xaa at position 23 is: Glu, or Gin; Xaa at position 25 is: Val or Ala; Xaa at position 26 is: Arg or Lys; Xaa at position 27 is Leu or Glu; Xaa at position 30 is: Glu or Alaj Xaa at position 33 is: Val or Lys, Xaa at position 34 is: Asn or Lys; Xaa at position 36 is: Gly or Arg; and Xaa at position 37 is: Gly, Pro, Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser, or is absent.
36. The heterologous fusion protein of claims 27 through 35 wherein the GLP-1 compound has no more than 6 amino acids that are different from the corresponding amino acid in GLP- l(7-37)OH, GLP-l(7-36)OH, or Exendin-4.
37. The heterologous fusion protein of Claim 36 wherein the GLP-1 compound has no more than 5 amino acids that differ from the corresponding amino acid in GLP-1 (7-37) OH, GLP-1 (7- 36) OH, or Exendin-4.
38. The heterologous fusion protein of Claim 37 wherein the GLP-1 compound has no more than 4 amino acids that differ from the .corresponding amino acid in GLP-1 (7-37 ) OH, GLP-1 (7- 36) OH, or Exendin-4.
39. The heterologous fusion protein of Claim 38 wherein the GLP-1 compound has no more than 3 amino acids that differ from the corresponding amino acid in GLP-1 (7-37 ) OH, GLP-1 (7- 36) OH, or Exendin-4.
40. The heterologous fusion protein of Claim 39 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37) OH, GLP-1 (7- 36) OH, or Exendin-4.
41. The heterologous fusion protein of any one of Claims 27 through 40 wherein Xaa at position 8 is glycine or valine.
42. The heterologous fusion protein of Claim 34 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37) OH or GLP- 1(7-36) OH and Xaa at position 8 is glycine or valine and Xaa at position 30 is alanine, glutamic acid, aspartic acid, serine, or histidine.
43. The heterologous fusion protein of Claim 42 wherein Xaa at position 30 is Glutamic acid.
44. The heterologous fusion protein of Claim 34 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37) OH or GLP-1 (7-36) OH and Xaa at position 8 is glycine or valine and Xaa at position 37 is histidine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, serine, threonine, arginine, or lysine.
45. The heterologous fusion protein of Claim 44 wherein Xaa at position 37 is histidine.
46. The heterologous fusion protein of Claim 34 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1 (7-37) OH or GLP- 1(7-36) OH and Xaa at position 8 is glycine, valine, leucine, isoleucine, serine, threonine, or methionine and Xaa at position 22 is aspartic acid, glutamic acid, lysine, arginine, asparagine, glutamine or histidine.
47. The heterologous fusion protein of Claim 46 wherein Xaa at position 22 is lysine or glutamic acid.
48. The heterologous fusion protein of Claim 34 wherein the GLP-1 compound is Val-8-GLP-l (7-37 ) .
49. The heterologous fusion protein of Claim 34 wherein the GLP-1 compound is Gly-8-GLP-l (7-37 ) .
50. The heterologous fusion protein of any one of Claims 27 through 49 wherein the second polypeptide is, the Fc portion of an Ig selected from the group consisting of: IgGl, IgG2 , IgG3, IgG4, IgE, IgA, IgD, or IgM.
51. The heterologous fusion protein of any one of Claims 27 through 50 wherein the second polypeptide is the Fc portion of an Ig selected from the group consisting of: IgGl, IgG2 , IgG3, and IgG4.
52. The heterologous fusion protein of Claim 51 wherein the second polypeptide is the Fc portion of an IgGl immunoglobulin .
53. The heterologous fusion protein of Claim 51 wherein the second polypeptide is the Fc portion of an IgG4 immunoglobulin.
54. The heterologous fusion protein of Claims 50 through 53 wherein the IgG is human.
55. The heterologous fusion protein of any one of Claims 27 through 54 wherein the Fc portion comprises the hinge, CH2 , and CH3 domains .
56. The heterologous fusion protein of Claim 52 wherein the second polypeptide has the sequence of SEQ ID NO: 32.
57. A polynucleotide encoding a heterologous fusion protein of any one of Claims 1 through 56.
58. A vector comprising the polynucleotide of Claim 57.
59. A host cell comprising the vector of Claim 58.
60. A host cell expressing at least one heterologous fusion protein of any one of Claims 1 through 56.
61. The host cell of Claim 60 wherein said host cell is a CHO cell.
62. A process for producing a heterologous fusion protein comprising the steps of transcribing and translating a polynucleotide of Claim 57 under conditions wherein the heterologous fusion protein is expressed in detectable amounts .
63. A method for normalizing blood glucose levels in a mammal in need thereof comprising the administration of a therapeutically effective amount of the heterologous fusion protein of any one of Claims 1 through 56.
64. A method of treating a patient with non-insulin dependent diabetes mellitus comprising the administration of a therapeutically effective amount of the heterologous fusion protein of any one of Claims 1 through 56.
65. A method of treating obesity comprising the administration of a therapeutically effective amount of the heterologous fusion protein of any one of Claims 1 through 56.
66. The use of a heterologous fusion protein as claimed in any one of Claims 1 through 56 for the manufacture of a medicament for the treatment of patients with non-insulin dependent diabetes mellitus.
67. The use of a heterologous fusion protein as claimed in any one of Claims 1 through 56 for the manufacture of a medicament for the treatment of patients with obesity.
68. A pharmaceutical formulation adapted for the treatment of patients with non-insulin dependent diabetes comprising a heterologous fusion protein of any one of Claims 1 through 56.
69. The heterologous fusion protein of Claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula IX [SEQ ID NO: 12]
Xaa7-Xaa8-Glu-Gly-Thr-Xaa12-Thr-Ser-Asp-Xaaιs-Ser- Xaaι8-Xaaι9-Xaa20-Glu-Xaa22-Gln-Ala-Xaa25-Lys-Xaa27-
Phe-Ile-Xaa3o-Trp-Leu-Xaa33-Lys-Gly-Arg-Xaa37 Formula IX (SEQ ID NO: 12)
wherein : Xaa is: L-histidine, D-histidine, desamino-histidine, 2- amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, or α- methyl-histidine;
Xaa8 is: Ala, Gly, Val, Leu, lie, Ser, or Thr; Xaaχ2 is: Phe, Trp, or Tyr;
Xaa16 is: Val, Trp, lie, Leu, Phe, or Tyr;
Xaais is: Ser, Trp, Tyr, Phe, Lys, lie, Leu, Val;
Xaai9 is: Tyr, Trp, or Phe;
Xaa2o is: Leu, Phe, Tyr, or Trp; Xaa22 is: Gly, Glu, Asp, or Lys;
Xaa25 is: Ala, Val, lie, or Leu;
Xaa2 is: Glu, lie, or Ala;
Xaa30 is: Ala or Glu
Xaa33 is: Val, or .lie; and Xaa37 is: Gly, His, NH , or is absent.
70. The heterologous fusion protein of Claim 69 wherein the second polypeptide is human albumin.
71. The heterologous fusion protein of Claim 70 wherein the second polypeptide has the sequence of SEQ ID NO: 34.
72. The heterologous fusion protein of Claim 69 wherein the second polypeptide is an N-terminal fragment of albumin.
73. The heterologous fusion protein of any one of Claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula IX [SEQ ID NO: 12]
Xaa7-Xaa8-Glu-Gly-Thr-Xaai2-Thr-Ser-Asp-Xaaι5-Ser-
Xaai8-Xaai9-Xaa2o-G-lu-Xaa22-Gln-Ala-Xaa25-Lys-Xaa27- Phe-Ile-Xaa3o-Trp-Leu-Xaa3 ~Lys-Gly-Arg-Xaa37 Formula IX ( SEQ ID NO : 12 ) wherein : Xaa7 is L-histidine, D-histidine, desamino-histidine, 2- amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, or α- methyl-histidine;
Xaa8 is Ala, Gly, Val, Leu, lie, Ser, or Thr;
Xaa12 is : Phe, Trp, or Tyr;
Xaais is : Val, Trp, lie, Leu, Phe, or Tyr;
Xaais is : Ser, Trp, Tyr, Phe, Lys, lie, Leu, Val;
Xaai9 is : Tyr, Trp, or Phe;
Xaa2o is : Leu, Phe, Tyr, or Trp;
Xaa22 is : Gly, Glu, Asp, or Lys;
Xaa25 is : Ala, Val, lie, or Leu;
Xaa2 is : Glu, lie, or Ala;
Xaa30 is : Ala or Glu
Xaa33 is : Val, or lie; and
Xaa37 is: Gly, His, NH,, or is absent.
74. The heterologous fusion protein of Claim 73 wherein the second polypeptide is the Fc portion of an Ig selected from the group consisting of: IgGl, IgG2 , IgG3 , IgG4, IgE, IgA, IgD, or IgM.
75. The heterologous fusion protein of Claim 73 or 74 wherein the second polypeptide is the Fc portion of an Ig selected from the group consisting of: IgGl, IgG2 , IgG3 , and IgG4.
76. The heterologous fusion protein of Claim 75 wherein the second polypeptide is the Fc portion of an IgGl immunoglobulin .
77. The heterologous fusion protein of Claim 75 wherein the second polypeptide is the Fc portion of an IgG4 immunoglobulin .
78. The heterologous fusion protein of Claims 73 through 78 wherein the IgG is human.
79. The heterologous fusion protein of Claim 75 wherein the second polypeptide has the sequence of SEQ ID NO: 32.
80. The heterologous fusion protein of any one of Claims 69 through 79 wherein the GLP-1 compound is selected from the group consisting of Gly8-GLP-1 (7-37) , Val8- Tyr12-GLP-l(7-37) , Val8-Tyr12-GLP-1 (7-36) , Val8-Trp12-
GLP-K7-37), Val8-Leu 6-GLP-l(7-37) , Val8-Val16-GLP-1 (7- 37) , Val8-Tyr16-GLP-l(7-37) , Gly8-Glu22-GLP-1 (7-37) , Val8-Glu22-GLP-l(7-37) , Val8-Leu25-GLP-1 (7-37 ) , Val8- Tyr 2-Tyr16-GLP-l(7-37) , Val8-Trp12-Glu22-GLP-1 (7-37 ) , Val8-Tyr12-Glu22-GLP-l(7-37) , Val8-Tyr16-Phe19-GLP-1 (7-
37), Val8-Tyr16-Glu22-GLP-l(7-37) , Val8-Trp16-Glu22-GLP- 1(7-37) , Val8-Leux6-Glu22-GLP-l(7-37) , . Val8-Ile16-Glu22- GLP-KJ-37), Val8-Phe16-Glu22-GLP-l(7-37) , Val8-Trp18- Glu22-GLP-l(7-37) , Val8-Tyr18-Glu22-GLP-1 (7-37 ) , Val8- Phe18-Glu22-GLP-l(7-37) , Val8-Ile18-Glu22-GLP-1 (7-37 ) ,
Val8-Lys18-Glu22-GLP-l(7-37) , Val8-Trp19-Glu22-GLP-1 (7- 37), Val8-Phe19-Glu22-GLP-l(7-37) , Val8-Phe20-Glu22-GLP- 1(7-37) , Val8-Glu22-Leu25-GLP-l(7-37) , Val8-Glu22-Ile2A GLP-K7-37), Val8-Glu22-Val25-GLP-l(7-37) , Val8-Glu22- Ile27-GLP-l(7-37) , Val8-Glu22-Ala27-GLP-1 (7-37 ) , Val8-
Glu22-Ile33-GLP-l(7-37) , Val8-Asp9-Ilexl-Tyr16-Glu22-GLP- 1(7-37) , Val8-Tyr16-Trp19-Glu22-GLP-l(7-37) , Val8-Trp15- Glu22-Val25-Ile33-GLP-l(7-37) , Val8-Trp16-Glu22-Ile33-GLP- 1(7-37) , Val8-Glu22-Val25-Ile33-GLP-l(7-37) , Val8-Trp16- Glu .22z-Val 25D- GLP-1 (7-37), and Val -Cys 16-Lys ,26 -GLP-1 (7-
37)
PCT/US2001/043165 2000-12-07 2001-11-29 Glp-1 fusion proteins WO2002046227A2 (en)

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UA2003065280A UA81897C2 (en) 2000-12-07 2001-11-29 Normal;heading 1;heading 2;GLUCAGON-LIKE PEPTIDE 1 HETEROLOGOUS FUSION PROTEIN FOR THE PREPARATION OF A MEDICINE FOR THE TREATMENT OF NON-INSULIN DEPENDENT DIABETES MELLITUS
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Cited By (136)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003086444A1 (en) 2002-04-04 2003-10-23 Amgen Inc. Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins
EP1411968A2 (en) * 2001-07-31 2004-04-28 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Glp-1 exendin-4 peptide analogs and uses thereof
WO2004050115A2 (en) * 2002-12-03 2004-06-17 Novo Nordisk A/S Combination treatment using exendin-4 and thiazolidinediones
EP1432730A2 (en) * 2001-08-23 2004-06-30 Eli Lilly And Company Glucagon-like peptide-1 analogs
WO2004089985A1 (en) * 2003-04-11 2004-10-21 Novo Nordisk A/S Stable pharmaceutical compositions
WO2004110472A2 (en) * 2003-06-12 2004-12-23 Eli Lilly And Company Fusion proteins
WO2005000892A2 (en) 2003-06-12 2005-01-06 Eli Lilly And Company Glp-1 analog fusion plroteins
EP1545608A2 (en) * 2002-06-28 2005-06-29 Centocor, Inc. Mammalian ch1 deleted mimetibodies, compositions, methods and uses
EP1585959A2 (en) * 2002-01-08 2005-10-19 Eli Lilly And Company Extended glucagon-like peptide-1 analogs
WO2005058958A3 (en) * 2003-12-18 2005-11-24 Novo Nordisk As Novel glp-1 analogues linked to albumin-like agents
WO2005113606A2 (en) 2004-05-13 2005-12-01 Eli Lilly And Company Fgf-21 fusion proteins
EP1605897A2 (en) * 2003-03-19 2005-12-21 Eli Lilly And Company Polyethelene glycol link glp-1 compounds
EP1611093A2 (en) * 2002-08-30 2006-01-04 Biorexis Pharmaceutical Corporation Modified transferrin fusion proteins
JP2006503588A (en) * 2002-08-30 2006-02-02 バイオレクシス ファーマシューティカル コーポレイション Modified transferrin fusion protein
WO2006059106A2 (en) * 2004-12-02 2006-06-08 Domantis Limited Bispecific domain antibodies targeting serum albumin and glp-1 or pyy
EP1682581A1 (en) * 2003-11-13 2006-07-26 Hanmi Pharmaceutical. Co., Ltd. Igg fc fragment for a drug carrier and method for the preparation thereof
EP1688148A1 (en) * 2002-12-03 2006-08-09 Novo Nordisk A/S Combination treatment using exendin-4 and thiazolidinediones
EP1695983A2 (en) * 2000-06-16 2006-08-30 Eli Lilly &amp; Company Glucagon-like peptide-1 analogs
WO2006097537A2 (en) 2005-03-18 2006-09-21 Novo Nordisk A/S Acylated glp-1 compounds
EP1718665A2 (en) * 2004-02-11 2006-11-08 Amylin Pharmaceuticals, Inc. Hybrid polypeptides with selectable properties
WO2005077042A3 (en) * 2004-02-09 2006-11-30 Human Genome Sciences Inc Albumin fusion proteins
WO2007012188A1 (en) * 2005-07-27 2007-02-01 Qinghua Wang GLP/1/EXENDM 4 IgG Fc FUSION CONSTRUCTS FOR TREATMENT OF DIABETES
EP1750754A2 (en) * 2004-03-31 2007-02-14 Centocor, Inc. Human glp-1 mimetibodies, compositions, methods and uses
US7189690B2 (en) 2001-12-21 2007-03-13 Human Genome Sciences, Inc. Albumin fusion proteins
EP1767545A1 (en) 2005-09-22 2007-03-28 Biocompatibles UK Limited GLP-1 (Glucagon-like peptide-1) fusion polypeptides with increased peptidase resistance
WO2007049695A1 (en) * 2005-10-26 2007-05-03 Chugai Seiyaku Kabushiki Kaisha Agglutinable glp-1 analogue and sustained-release pharmaceutical composition
WO2007063907A1 (en) * 2005-11-30 2007-06-07 Shionogi & Co., Ltd. Sugar chain adduct of peptide and pharmaceutical comprising the same as active ingredient
US7259234B2 (en) 2003-05-15 2007-08-21 Trustees Of Tufts College Stable analogs of peptide and polypeptide therapeutics
CN100346834C (en) * 2003-01-10 2007-11-07 株式会社新潟Tlo Vector for gene therapy and method of quantifying target protein in mammal or cultured cells with the administration of the vector for gene theraphy
US7314859B2 (en) 2002-12-27 2008-01-01 Diobex, Inc. Compositions and methods for the prevention and control of insulin-induced hypoglycemia
EP1871811A2 (en) * 2005-03-28 2008-01-02 Centocor, Inc. Human glp-1 mimetibodies, compositions, methods and uses
WO2008081418A1 (en) * 2007-01-05 2008-07-10 Covx Technologies Ireland Limited Glucagon-like protein-1 receptor (glp-1r) agonist compounds
JP2008525477A (en) * 2004-12-22 2008-07-17 セントカー・インコーポレーテツド GLP-1 agonists, compositions, methods and uses
EP1972349A1 (en) * 2007-03-21 2008-09-24 Biocompatibles UK Limited GLP-1 fusion peptides conjugated to polymer(s), their production and use
WO2008131242A1 (en) 2007-04-18 2008-10-30 Zymogenetics, Inc. Single chain fc, methods of making and methods of treatment
JP2009504681A (en) * 2005-08-11 2009-02-05 アミリン・ファーマシューティカルズ，インコーポレイテッド Hybrid polypeptides with selectable properties
EP2059606A2 (en) * 2006-09-06 2009-05-20 Phasebio Pharmaceuticals, Inc. Fusion peptide therapeutic compositions
WO2009121804A1 (en) 2008-03-31 2009-10-08 Glaxo Group Limited Drug fusions and conjugates
WO2010011096A2 (en) 2008-07-23 2010-01-28 Hanmi Pharmaceutical Co., Ltd. A polypeptide complex comprising non-peptidyl polymer having three functional ends
US7655618B2 (en) 2002-12-27 2010-02-02 Diobex, Inc. Compositions and methods for the prevention and control of insulin-induced hypoglycemia
US7723471B2 (en) 2004-02-11 2010-05-25 Amylin Pharmaceuticals, Inc. Pancreatic polypeptide family motifs, polypeptides and methods comprising the same
US7785599B2 (en) 2000-04-12 2010-08-31 Human Genome Sciences, Inc. Albumin fusion proteins
US7799759B2 (en) 2001-12-21 2010-09-21 Human Genome Sciences, Inc. Albumin fusion proteins
WO2010108937A2 (en) 2009-03-27 2010-09-30 Glaxo Group Limited Drug fusions and conjugates
US7867972B2 (en) 2006-07-24 2011-01-11 Pharmacia & Upjohn Company, Llc Fusion protein of exendin-4 to a transferrin (Tf) polypeptide
US7893017B2 (en) 2004-10-07 2011-02-22 Novo Nordisk A/S Protracted GLP-1 compounds
US7897560B2 (en) 2003-09-19 2011-03-01 Novo Nordisk A/S Plasma protein affinity tags
EP2298337A2 (en) 2003-12-09 2011-03-23 Novo Nordisk A/S Regulation of food preference using GLP-1 agonists
WO2011039096A1 (en) 2009-09-30 2011-04-07 Glaxo Group Limited Drug fusions and conjugates with extended half life
EP2316446A1 (en) 2004-06-11 2011-05-04 Novo Nordisk A/S Counteracting drug-induced obesity using GLP-1 agonists
EP2330126A1 (en) 2004-10-07 2011-06-08 Novo Nordisk A/S Protracted exendin-4 compounds
WO2011122921A2 (en) 2010-04-02 2011-10-06 Hanmi Holdings Co., Ltd. An insulin conjugate using an immunoglobulin fragment
WO2011123943A1 (en) 2010-04-09 2011-10-13 Mount Sinai Hospital Methods for treating disorders of the gastrointestinal tract using a glp-1 agonist
WO2011136361A1 (en) 2010-04-30 2011-11-03 株式会社 三和化学研究所 Peptide for improving in vivo stability of physiologically active substance or the like and physiologically active substance with improved in vivo stability
US8067554B2 (en) 1999-03-15 2011-11-29 Novo Nordisk A/S Ion exchange chromatography of GLP-1, analogs and derivatives thereof
EP2394656A2 (en) 2003-11-20 2011-12-14 Novo Nordisk A/S Propylene Glycol-containing peptide formulations which are optimal for production and for use in injection devices
WO2011153965A1 (en) 2010-06-11 2011-12-15 北京精益泰翔技术发展有限公司 Fusion protein of exendin-4 and its analog, preparation method and use thereof
US8110665B2 (en) 2003-11-13 2012-02-07 Hanmi Holdings Co., Ltd. Pharmaceutical composition comprising an immunoglobulin FC region as a carrier
US8129504B2 (en) 2001-08-30 2012-03-06 Biorexis Technology, Inc. Oral delivery of modified transferrin fusion proteins
AU2011254001B2 (en) * 2007-01-05 2012-08-02 Covx Technologies Ireland Limited Glucagon-like protein-1 receptor (GLP-1R) agonist compounds
WO2012118042A1 (en) 2011-02-28 2012-09-07 独立行政法人国立循環器病研究センター Medicinal agent for inhibiting metastasis of malignant tumor
WO2012136792A2 (en) 2011-04-07 2012-10-11 Glaxo Group Limited Cck compositions
WO2012136790A1 (en) 2011-04-07 2012-10-11 Glaxo Group Limited Compositions comprising fusion proteins or conjugates with an improved half -life
US8288339B2 (en) 2006-04-20 2012-10-16 Amgen Inc. GLP-1 compounds
US8293869B2 (en) 2005-12-16 2012-10-23 Nektar Therapeutics Polymer conjugates of GLP-1
AU2010203063B2 (en) * 2003-09-19 2012-10-25 Novo Nordisk A/S Albumin-binding derivatives of therapeutic peptides
WO2013027680A1 (en) 2011-08-19 2013-02-28 独立行政法人国立循環器病研究センター Drug for preventing exacerbation of malignant tumor, comprising combination of natriuretic peptide receptor gc-a agonist and gc-b agonist
WO2013066106A1 (en) 2011-11-04 2013-05-10 Hanmi Science Co., Ltd. Method for preparing physiologically active polypeptide complex
WO2013083826A2 (en) 2011-12-09 2013-06-13 Novo Nordisk A/S Glp-1 agonists
US8476230B2 (en) 2007-01-05 2013-07-02 Hanmi Science Co., Ltd Insulinotropic complex using an immunoglobulin fragment
WO2013100704A1 (en) 2011-12-30 2013-07-04 Hanmi Science Co., Ltd. A site-specific glp-2 conjugate using an immunoglobulin fragment
TWI403331B (en) * 2007-11-29 2013-08-01 Hanmi Science Co Ltd A pharmaceutical composition for treating obesity-related disease comprising insulinotropic peptide conjugate
US8513192B2 (en) 2009-01-22 2013-08-20 Novo Nordisk A/S Stable growth hormone compounds resistant to proteolytic degradation
EP2636680A2 (en) 2006-05-26 2013-09-11 Amylin Pharmaceuticals, LLC Composition and methods for treatment of congestive heart failure
US8541373B2 (en) 2009-12-08 2013-09-24 Teva Pharmaceutical Industries Ltd. BChE albumin fusions for the treatment of cocaine abuse
US8557769B2 (en) 2007-08-03 2013-10-15 Eli Lilly And Company Co-administration of FGF-21 and GLP-1 to treat diabetes and lower blood glucose
WO2013171570A1 (en) 2012-05-16 2013-11-21 Glaxo Group Limited Polypeptide loaded poca nanoparticles for oral administration
US8603972B2 (en) 2005-03-18 2013-12-10 Novo Nordisk A/S Extended GLP-1 compounds
US8697647B2 (en) 2004-02-11 2014-04-15 Odile Esther Levy Hybrid polypeptides with selectable properties
US8709415B2 (en) 2006-01-12 2014-04-29 Alexion Pharmaceuticals, Inc. Antibodies to OX-2/CD200 and uses thereof
KR20140066793A (en) * 2006-02-06 2014-06-02 체에스엘 베링 게엠베하 Modified coagulation factor viia with extended half-life
US8779109B2 (en) 2010-01-22 2014-07-15 Novo Nordisk Health Care Ag Growth hormones with prolonged in-vivo efficacy
US8790705B2 (en) 2006-05-10 2014-07-29 Biocompatibles Uk Ltd. Spherical microcapsules comprising GLP-1 peptides, their production and use
US8841249B2 (en) 2009-08-06 2014-09-23 Novo Nordisk A/S Growth hormones with prolonged in-vivo efficacy
US8840885B2 (en) 2000-12-08 2014-09-23 Alexion Pharmaceuticals, Inc. Methods for treating chronic lymphocytic leukemia
US8865868B2 (en) 2008-08-06 2014-10-21 Novo Nordisk Healthcare Ag Conjugated proteins with prolonged in vivo efficacy
US8895694B2 (en) 2007-09-05 2014-11-25 Novo Nordisk A/S Glucagon-Like Peptide-1 derivatives and their pharmaceutical use
WO2015022420A1 (en) * 2013-08-16 2015-02-19 Medimmune Limited Gip and glp-1 receptor dual-agonists for the treatment of diabetes
US8969538B2 (en) 2006-06-07 2015-03-03 Human Genome Sciences, Inc. Albumin fusion proteins
US8986684B2 (en) 2007-07-25 2015-03-24 Alexion Pharmaceuticals, Inc. Methods and compositions for treating autoimmune disease
US8999328B2 (en) 2000-12-08 2015-04-07 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic Leukemia cells and uses thereof
US9067977B2 (en) 2007-09-05 2015-06-30 Novo Nordisk A/S Peptides derivatized with A-B-C-D- and their therapeutical use
US9150661B2 (en) 2000-12-08 2015-10-06 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
WO2015165480A1 (en) 2014-04-30 2015-11-05 Institute For Research In Biomedicine Human cytomegalovirus vaccine compositions and method of producing the same
US9211342B2 (en) 2010-01-22 2015-12-15 Novo Nordisk Healthcare Ag Stable growth hormone compounds resistant to proteolytic degradation
WO2016055610A1 (en) * 2014-10-10 2016-04-14 Novo Nordisk A/S Stable glp-1 based glp-1/glucagon receptor co-agonists
WO2016073704A1 (en) * 2014-11-06 2016-05-12 Children's Research Institute, Children's National Medical Center Immunotherapeutics for cancer and autoimmune diseases
EP1965823B1 (en) * 2005-11-04 2016-05-18 Glaxosmithkline LLC Methods for administering hypoglycemic agents
US9364772B2 (en) 2003-04-08 2016-06-14 Novo Nordisk A/S Regeneration of chromatographic stationary phases
US9409662B2 (en) 2013-01-15 2016-08-09 Teva Pharmaceutical Industries, Ltd. Formulations of albu-BChE, preparation and uses thereof
US20160280762A1 (en) * 2013-08-01 2016-09-29 Jiangsu T-Mab Biopharma Co., Ltd Glp-1 analog fusion protein and preparation method and use thereof
US9534033B2 (en) 2007-09-21 2017-01-03 The Regents Of The University Of California Targeted interferons demonstrating potent apoptotic and anti-tumor activities
US9616109B2 (en) 2014-10-22 2017-04-11 Extend Biosciences, Inc. Insulin vitamin D conjugates
US9657079B2 (en) 2007-09-05 2017-05-23 Novo Nordisk A/S Truncated GLP-1 derivatives and their therapeutical use
CN107033234A (en) * 2017-01-03 2017-08-11 北京凯因科技股份有限公司 The acylated derivatives of GLP 1
US9789197B2 (en) 2014-10-22 2017-10-17 Extend Biosciences, Inc. RNAi vitamin D conjugates
US9803021B2 (en) 2012-12-07 2017-10-31 The Regents Of The University Of California CD138-targeted interferon demonstrates potent apoptotic and anti-tumor activities
EP3248611A1 (en) 2010-07-21 2017-11-29 Hanmi Science Co., Ltd. Novel long-acting glucagon conjugate and pharmaceutical composition comprising the same for the prevention and treatment of obesity
US9884124B2 (en) 2012-05-17 2018-02-06 Extend Biosciences, Inc. Carriers for improved drug delivery
US9981017B2 (en) 2010-04-02 2018-05-29 Hanmi Science Co., Ltd. Insulin conjugate using an immunoglobulin fragment
US10059773B2 (en) 2013-08-13 2018-08-28 Gmax Biopharm Llc. Antibody specifically binding to GLP-1 R and fusion protein thereof with GLP-1
US10093745B2 (en) 2013-05-29 2018-10-09 The Regents Of The University Of California Anti-CSPG4 fusions with interferon for the treatment of malignancy
WO2018220123A1 (en) 2017-05-31 2018-12-06 University Of Copenhagen Long-acting gip peptide analogues
US10302655B2 (en) 2008-12-05 2019-05-28 Glaxo Group Limited Methods for selecting protease resistant polypeptides
US10406202B2 (en) 2014-10-22 2019-09-10 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
WO2019201328A1 (en) 2018-04-19 2019-10-24 杭州先为达生物科技有限公司 Acylated glp-1 derivative
US10485870B2 (en) 2015-02-11 2019-11-26 Gmax Biopharm Llc. Stable pharmaceutical solution formulation of GLP-1R antibody fusion protein
WO2020115048A1 (en) 2018-12-03 2020-06-11 Antag Therapeutics Aps Modified gip peptide analogues
WO2020125744A1 (en) 2018-12-21 2020-06-25 江苏恒瑞医药股份有限公司 Bispecific protein
US10851144B2 (en) 2015-04-10 2020-12-01 Amgen Inc. Interleukin-2 muteins for the expansion of T-regulatory cells
US10933120B2 (en) 2012-03-22 2021-03-02 Novo Nordisk A/S Compositions of GLP-1 peptides and preparation thereof
US10960052B2 (en) 2010-12-16 2021-03-30 Novo Nordisk A/S Solid compositions comprising a GLP-1 agonist and a salt of N-(8-(2-hydroxybenzoyl) amino) caprylic acid
US10968266B2 (en) 2014-09-05 2021-04-06 University Of Copenhagen GIP peptide analogues
US10987424B2 (en) 2012-07-25 2021-04-27 Hanmi Pharm. Co., Ltd. Liquid formulation of long-acting insulin conjugate
US11034746B2 (en) 2011-04-12 2021-06-15 Novo Nordisk A/S Double-acylated GLP-1 derivatives
US11033499B2 (en) 2012-06-20 2021-06-15 Novo Nordisk A/S Tablet formulation comprising a GLP-1 peptide and a delivery agent
US11045523B2 (en) 2013-04-05 2021-06-29 Novo Nordisk Healthcare Ag Formulation of growth hormone albumin-binder conjugate
US11123296B2 (en) 2012-03-22 2021-09-21 Novo Nordisk A/S Compositions comprising a delivery agent and preparation thereof
US11168109B2 (en) 2012-03-08 2021-11-09 Hanmi Science Co., Ltd. Process for preparation of physiologically active polypeptide complex
EP3889185A3 (en) * 2014-03-31 2022-01-26 Hanmi Pharm. Co., Ltd. Composition for improving the solubility of a protein or peptide by using immunoglobulin fc fragment linkage
US20220089669A1 (en) * 2018-10-22 2022-03-24 Janssen Pharmaceutica Nv Glucagon like peptide 1 (glp1)-growth differentiation factor 15 (gdf15) fusion proteins and uses thereof
US11318191B2 (en) 2020-02-18 2022-05-03 Novo Nordisk A/S GLP-1 compositions and uses thereof
US11421033B2 (en) 2015-09-04 2022-08-23 The Scripps Research Institute Insulin immunoglobulin fusion proteins
US11752198B2 (en) 2017-08-24 2023-09-12 Novo Nordisk A/S GLP-1 compositions and uses thereof
US11761963B2 (en) 2017-09-27 2023-09-19 Alexion Pharmaceuticals, Inc. Biomarker signature for predicting tumor response to anti-CD200 therapy
US11833248B2 (en) 2018-02-02 2023-12-05 Novo Nordisk A/S Solid compositions comprising a GLP-1 agonist and a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid

Families Citing this family (183)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6924264B1 (en) * 1999-04-30 2005-08-02 Amylin Pharmaceuticals, Inc. Modified exendins and exendin agonists
US20050100991A1 (en) * 2001-04-12 2005-05-12 Human Genome Sciences, Inc. Albumin fusion proteins
WO2002048192A2 (en) * 2000-12-13 2002-06-20 Eli Lilly And Company Amidated glucagon-like peptide-1
WO2003033671A2 (en) * 2001-10-18 2003-04-24 Bristol-Myers Squibb Company Human glucagon-like-peptide-1 mimics and their use in the treatment of diabetes and related conditions
US20080194481A1 (en) * 2001-12-21 2008-08-14 Human Genome Sciences, Inc. Albumin Fusion Proteins
WO2003100021A2 (en) 2002-05-24 2003-12-04 Restoragen, Inc. Methods and dna constructs for high yield production of polypeptides
US9321832B2 (en) 2002-06-28 2016-04-26 Domantis Limited Ligand
US20070059376A1 (en) * 2003-02-06 2007-03-15 Shinji Takeoka Peptide conjugate
US8263084B2 (en) * 2003-11-13 2012-09-11 Hanmi Science Co., Ltd Pharmaceutical composition for treating obesity-related disease comprising insulinotropic peptide conjugate
US20090238838A1 (en) * 2003-11-13 2009-09-24 Hanmi Pharm. Ind. Co. Ltd. Insulinotropic peptide conjugate using an immunoglobulin fc
US20060252693A1 (en) * 2004-01-29 2006-11-09 Wolfgang Glaesner Glucagon-like peptide-1 analogs
CN1980687B (en) * 2004-02-09 2015-05-13 人类基因科学公司 Albumin fusion proteins
DK2348114T3 (en) 2004-04-21 2018-09-03 Alexion Pharma Inc BONE DELIVERY CONJUGATES AND PROCEDURE TO USE IT FOR TARGETING PROTEINS AGAINST BONE
EA011166B1 (en) * 2004-12-22 2009-02-27 Эли Лилли Энд Компани Glp-1 analog fusion protein formulations
WO2007022123A2 (en) * 2005-08-11 2007-02-22 Amylin Pharmaceuticals, Inc. Hybrid polypeptides with selectable properties
US20090016959A1 (en) * 2005-02-18 2009-01-15 Richard Beliveau Delivery of antibodies to the central nervous system
PL2650020T3 (en) * 2005-05-06 2017-07-31 Providence Health & Services - Oregon Trimeric OX40-immunoglobulin fusion protein and methods of use
CA2608311C (en) * 2005-05-13 2012-11-27 Eli Lilly And Company Glp-1 pegylated compounds
AU2006279276A1 (en) * 2005-07-29 2007-02-15 Amprotein Corporation Chimeric therapeutic agents
CN101277722A (en) * 2005-08-06 2008-10-01 王庆华 Composition and method for prevention and treatment of type I diabetes
CN1962695B (en) * 2005-11-09 2011-08-31 浙江德清安平生物制药有限公司 GLP-1 infusion proteins, their preparation and use
US8039432B2 (en) 2005-11-09 2011-10-18 Conjuchem, Llc Method of treatment of diabetes and/or obesity with reduced nausea side effect
US20080280328A1 (en) * 2005-11-18 2008-11-13 Novozymes A/S Glucoamylase Variants
US8841255B2 (en) 2005-12-20 2014-09-23 Duke University Therapeutic agents comprising fusions of vasoactive intestinal peptide and elastic peptides
ES2779992T3 (en) 2005-12-20 2020-08-21 Univ Duke Methods and compositions for delivering active agents with enhanced pharmacological properties
US20130172274A1 (en) 2005-12-20 2013-07-04 Duke University Methods and compositions for delivering active agents with enhanced pharmacological properties
US8759292B2 (en) 2006-02-03 2014-06-24 Prolor Biotech, Llc Long-acting coagulation factors and methods of producing same
US8048849B2 (en) 2006-02-03 2011-11-01 Modigene, Inc. Long-acting polypeptides and methods of producing same
US20140113860A1 (en) 2006-02-03 2014-04-24 Prolor Biotech Ltd. Long-acting polypeptides and methods of producing and administering same
US7553941B2 (en) * 2006-02-03 2009-06-30 Modigene Inc Long-acting polypeptides and methods of producing same
US9458444B2 (en) 2006-02-03 2016-10-04 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US8476234B2 (en) * 2006-02-03 2013-07-02 Prolor Biotech Inc. Long-acting coagulation factors and methods of producing same
US10221228B2 (en) 2006-02-03 2019-03-05 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US20150038413A1 (en) 2006-02-03 2015-02-05 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US8450269B2 (en) 2006-02-03 2013-05-28 Prolor Biotech Ltd. Long-acting growth hormone and methods of producing same
US9249407B2 (en) 2006-02-03 2016-02-02 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US8946155B2 (en) 2006-02-03 2015-02-03 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US10351615B2 (en) 2006-02-03 2019-07-16 Opko Biologics Ltd. Methods of treatment with long-acting growth hormone
CN101003574B (en) * 2006-02-21 2010-12-15 大连帝恩生物工程有限公司 Recombined expression of peptide for lowering blood sugar in long acting, and application in medication for treating diabetes
KR20090039787A (en) * 2006-07-18 2009-04-22 센토코 인코포레이티드 Human glp-1 mimetibodies, compositions, methods and uses
BRPI0715754A2 (en) 2006-08-31 2013-07-09 Hoffmann La Roche Method for the production of insulin-like growth factor
CL2007002502A1 (en) 2006-08-31 2008-05-30 Hoffmann La Roche VARIANTS OF THE SIMILAR GROWTH FACTOR TO HUMAN INSULIN-1 (IGF-1) PEGILATED IN LISIN; METHOD OF PRODUCTION; FUSION PROTEIN THAT UNDERSTANDS IT; AND ITS USE TO TREAT ALZHEIMER'S DISEASE.
CL2007002634A1 (en) * 2006-09-13 2008-05-16 Smithkline Beecham Corp USE OF A COMPOSITION THAT INCLUDES AT LEAST A POLYPEPTIDE THAT HAS A PEPTIDE-1 ACTIVITY LIKE GLUCAGON (GLP-1) AS A LONG-TERM HYPOGLUCEMIAN AGENT.
TWI428346B (en) * 2006-12-13 2014-03-01 Imp Innovations Ltd Novel compounds and their effects on feeding behaviour
EP1975176A1 (en) * 2007-03-27 2008-10-01 Biocompatibles UK Limited Novel glp-1 fusion peptides, their production and use
RU2440097C2 (en) 2007-04-23 2012-01-20 Интарсия Терапьютикс, Инк. Method of treating insulin-independent diabetes and obesity, osmotic delivery system and method for making it
JP2009019027A (en) * 2007-07-16 2009-01-29 Hanmi Pharmaceutical Co Ltd Insulin secretion peptide derivative in which amino acid of amino terminal is varied
WO2009059278A1 (en) * 2007-11-02 2009-05-07 Centocor, Inc. Semi-synthetic glp-1 peptide-fc fusion constructs, methods and uses
ATE533060T1 (en) * 2008-04-03 2011-11-15 Hoffmann La Roche ASSAY WITH PEGYLATED INSULIN-LIKE GROWTH FACTORS
CN101328221B (en) * 2008-04-14 2011-03-23 中国药科大学 Hypoglycemic polypeptide fused protein, structure and use of derivate thereof
EP2926825A1 (en) 2008-06-27 2015-10-07 Duke University Therapeutic agents comprising elastin-like peptides
US20100075897A1 (en) * 2008-09-23 2010-03-25 Jinan University Method for sustainedly releasing bioactive peptides and application thereof
RU2011118056A (en) 2008-10-15 2012-11-27 Ангиокем Инк. GLP-1 AGONIC CONJUGATES AND THEIR APPLICATION
WO2010063122A1 (en) 2008-12-05 2010-06-10 Angiochem Inc. Conjugates of neurotensin or neurotensin analogs and uses thereof
EP2373681B1 (en) 2008-12-10 2017-01-18 Glaxosmithkline LLC Pharmaceutical compositions of albiglutide
SG2014012918A (en) 2009-02-11 2014-04-28 Novozymes Biopharma Dk As Albumin variants and conjugates
WO2010096394A2 (en) 2009-02-17 2010-08-26 Redwood Biosciences, Inc. Aldehyde-tagged protein-based drug carriers and methods of use
EP2421562B1 (en) 2009-04-20 2019-03-13 Angiochem Inc. Treatment of ovarian cancer using an anticancer agent conjugated to an angiopep-2 analog
RU2012103240A (en) 2009-07-02 2013-08-10 Ангиокем Инк. MULTI-DIMENSIONAL PEPTIDE CONJUGATES AND THEIR APPLICATION
US9663778B2 (en) 2009-07-09 2017-05-30 OPKO Biologies Ltd. Long-acting coagulation factors and methods of producing same
EP2464370B1 (en) * 2009-08-14 2017-03-29 Phasebio Pharmaceuticals, Inc. Modified vasoactive intestinal peptides
CN101993496B (en) * 2009-08-20 2013-06-05 重庆富进生物医药有限公司 Dual blood sugar and blood fat adjusting fusion protein and preparation method and application thereof
CN101993485B (en) * 2009-08-20 2013-04-17 重庆富进生物医药有限公司 Peptide analog homologous dimer capable of accelerating insulin secretion and application thereof
KR101229610B1 (en) * 2009-10-09 2013-02-05 한남대학교 산학협력단 Fusion molecule of GLP-1 analogue, and composition comprising the same for preventing or treating diabetes mellitus
US8697648B2 (en) 2009-10-20 2014-04-15 Georgia State University Research Foundation, Inc. Protein agent for diabetes treatment and β cell imaging
RU2607374C2 (en) 2009-10-30 2017-01-10 Новозаймс Байофарма Дк А/С Versions of albumin
CN102070717B (en) * 2009-11-19 2013-04-10 东莞太力生物工程有限公司 Fusion protein, preparation method thereof, DNA sequence for coding protein, expression vector, host cell and protein-containing medicinal compoisition
CN101875700B (en) * 2010-04-09 2012-09-26 无锡和邦生物科技有限公司 Method for improving bioactivity of exendin fusion protein
CN102939304B (en) 2010-04-09 2017-04-19 阿尔布麦狄克斯公司 albumin derivatives and variants
JP2013525491A (en) 2010-05-04 2013-06-20 グラクソスミスクライン・リミテッド・ライアビリティ・カンパニー Methods for treating or preventing cardiovascular disorders and providing cardiovascular protection
WO2011153642A1 (en) * 2010-06-10 2011-12-15 Angiochem Inc. Leptin and leptin analog conjugates and fusion proteins and uses thereof
US9234023B2 (en) * 2010-06-24 2016-01-12 Biousian Biosystems, Inc. Glucagon-like peptide-1 glycopeptides
CN103080125A (en) 2010-07-02 2013-05-01 安吉奥开米公司 Short and D-amino acid-containing polypeptides for therapeutic conjugates and uses thereof
CN102311501A (en) * 2010-07-08 2012-01-11 天津药物研究院 Fusion protein comprising GLP-1 (Glucagon-Like Peptide) or analog thereof and preparation method as well as application thereof
CN101906158B (en) * 2010-07-14 2013-10-23 中国药科大学 Pegylation hypoglycemic polypeptide and preparation method and application thereof
CN102532323B (en) * 2010-12-09 2014-07-23 天津药物研究院 Polypeptide complex, pharmaceutical composition, as well as preparation method and application of polypeptide complex
CN102533655A (en) * 2010-12-21 2012-07-04 青岛黄海制药有限责任公司 CHO-S cell strain for efficient expression type human recombinant protein GLP1/Fc and establishment method thereof
US9266939B2 (en) 2010-12-27 2016-02-23 Alexion Pharmaceuticals, Inc. Compositions comprising natriuretic peptides and methods of use thereof
CA2824143C (en) 2011-01-14 2018-12-18 Redwood Bioscience, Inc. Aldehyde-tagged immunoglobulin polypeptides and method of use thereof
JP6101638B2 (en) 2011-03-03 2017-03-22 ザイムワークス，インコーポレイテッド Multivalent heteromultimer scaffold design and construction
ES2669190T3 (en) 2011-06-06 2018-05-24 Phasebio Pharmaceuticals, Inc. Use of modified vasoactive intestinal peptides in the treatment of hypertension
EP2726093A4 (en) 2011-06-28 2015-08-19 Inhibrx Llc Wap domain fusion polypeptides and methods of use thereof
US10400029B2 (en) 2011-06-28 2019-09-03 Inhibrx, Lp Serpin fusion polypeptides and methods of use thereof
US8980266B2 (en) 2011-06-28 2015-03-17 Inhibrx, Llc Serpin fusion polypeptides and methods of use thereof
AU2012280474A1 (en) 2011-07-01 2014-01-16 Bayer Intellectual Property Gmbh Relaxin fusion polypeptides and uses thereof
WO2013007563A1 (en) 2011-07-08 2013-01-17 Bayer Intellectual Property Gmbh Fusion proteins releasing relaxin and uses thereof
JP6118251B2 (en) * 2011-08-25 2017-04-19 株式会社Ｌｓｉメディエンス Method for measuring glucagon-like peptide-1 and kit used therefor
JP6126097B2 (en) 2011-09-06 2017-05-10 ノヴォ ノルディスク アー／エス GLP-1 derivative
WO2013075066A2 (en) 2011-11-18 2013-05-23 Eleven Biotherapeutics, Inc. Proteins with improved half-life and other properties
MX2014009129A (en) 2012-01-26 2014-11-21 Amgen Inc Growth differentiation factor 15 (gdf-15) polypeptides.
ES2664328T3 (en) 2012-03-16 2018-04-19 Albumedix A/S Albumin variants
MY167814A (en) 2012-04-19 2018-09-26 Opko Biologics Ltd Long-acting oxyntomodulin variants and methods of producing same
CN104302772B (en) * 2012-05-18 2017-11-10 爱德迪安（北京）生物技术有限公司 Albumen, protein conjugate and its application for treating diabetes
US10052366B2 (en) 2012-05-21 2018-08-21 Alexion Pharmaceuticsl, Inc. Compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof
SI2859015T1 (en) * 2012-06-08 2018-12-31 Alkermes Pharma Ireland Limited Ligands modified by circular permutation as agonists and antagonists
AU2013289881B2 (en) 2012-07-13 2018-01-18 Zymeworks Bc Inc. Multivalent heteromultimer scaffold design and constructs
AR094821A1 (en) * 2012-07-25 2015-09-02 Hanmi Pharm Ind Co Ltd LIQUID FORMULATION OF AN INSULINOTROPIC PEPTIDE CONJUGATE OF PROLONGED ACTION
AR092862A1 (en) * 2012-07-25 2015-05-06 Hanmi Pharm Ind Co Ltd LIQUID FORMULATION OF PROLONGED ACTION INSULIN AND AN INSULINOTROPIC PEPTIDE AND PREPARATION METHOD
UA116217C2 (en) 2012-10-09 2018-02-26 Санофі Exendin-4 derivatives as dual glp1/glucagon agonists
AU2013343503B2 (en) 2012-11-08 2017-12-14 Albumedix Ltd. Albumin variants
MX363326B (en) 2012-11-20 2019-03-20 Opko Biologics Ltd Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides.
PL2934567T3 (en) 2012-12-21 2018-10-31 Sanofi Exendin-4 derivatives as dual glp1/gip- or trigonal glp1/gip/glucagon agonists
US9580486B2 (en) * 2013-03-14 2017-02-28 Amgen Inc. Interleukin-2 muteins for the expansion of T-regulatory cells
CN104277112B (en) 2013-07-04 2018-01-26 嘉和生物药业有限公司 Long-acting hypoglycemic fusion protein
JP6509852B2 (en) * 2013-07-31 2019-05-08 アムジエン・インコーポレーテツド Constructs of growth differentiation factor 15 (GDF-15)
JP6534654B2 (en) 2013-10-10 2019-06-26 ベス イスラエル デアコネス メディカル センター インコーポレイティッド TM4SF1 binding protein and method of using the same
US20150158926A1 (en) 2013-10-21 2015-06-11 Opko Biologics, Ltd. Long-acting polypeptides and methods of producing and administering same
EP3080156A1 (en) * 2013-12-10 2016-10-19 F. Hoffmann-La Roche AG Use of the binding domain of a subunit of a multi-subunit structure for targeted delivery of pharmaceutically active entities to the multi-subunit structure
WO2015086730A1 (en) 2013-12-13 2015-06-18 Sanofi Non-acylated exendin-4 peptide analogues
TW201609799A (en) 2013-12-13 2016-03-16 賽諾菲公司 Dual GLP-1/GIP receptor agonists
WO2015086728A1 (en) 2013-12-13 2015-06-18 Sanofi Exendin-4 peptide analogues as dual glp-1/gip receptor agonists
EP3080149A1 (en) 2013-12-13 2016-10-19 Sanofi Dual glp-1/glucagon receptor agonists
TW201625669A (en) 2014-04-07 2016-07-16 賽諾菲公司 Peptidic dual GLP-1/glucagon receptor agonists derived from Exendin-4
TW201625670A (en) 2014-04-07 2016-07-16 賽諾菲公司 Dual GLP-1/glucagon receptor agonists derived from EXENDIN-4
TW201625668A (en) 2014-04-07 2016-07-16 賽諾菲公司 Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists
NO2776305T3 (en) * 2014-04-23 2018-01-27
US11052132B2 (en) 2014-05-08 2021-07-06 Phasebio Pharmaceuticals, Inc. Methods and compositions for treating cystic fibrosis
US9932381B2 (en) 2014-06-18 2018-04-03 Sanofi Exendin-4 derivatives as selective glucagon receptor agonists
WO2016007873A1 (en) 2014-07-11 2016-01-14 The Regents Of The University Of Michigan Compositions and methods for treating craniosynostosis
CN104558198A (en) * 2014-07-25 2015-04-29 成都贝爱特生物科技有限公司 Preparation method and application of fusion protein of GLP-1 analogue and amylin analogue
CN104257696B (en) * 2014-09-04 2017-08-25 西安国誉生物科技有限公司 A kind of steady sugar yeast bacterium powder of hypoglycemic and its preparation method and application
CN104327187B (en) * 2014-10-11 2018-06-08 上海兴迪金生物技术有限公司 A kind of recombined human GLP-1-Fc fusion proteins
WO2016090251A1 (en) 2014-12-05 2016-06-09 Alexion Pharmaceuticals, Inc. Treating seizure with recombinant alkaline phosphatase
WO2016094456A1 (en) * 2014-12-08 2016-06-16 1Globe Health Institute Llc Soluble universal adcc-enhancing synthetic fusion gene and peptide technology and its use thereof
WO2016118577A1 (en) * 2015-01-22 2016-07-28 Medimmune, Llc Thymosin-beta-four fusion proteins
CA2973883A1 (en) 2015-01-28 2016-08-04 Alexion Pharmaceuticals, Inc. Methods of treating a subject with an alkaline phosphatase deficiency
CN114652817A (en) 2015-02-09 2022-06-24 费斯生物制药公司 Methods and compositions for treating muscle diseases and disorders
EP3277302A4 (en) * 2015-04-01 2018-12-05 The Scripps Research Institute Methods and compositions related to gpcr agonist polypeptides
AR105616A1 (en) 2015-05-07 2017-10-25 Lilly Co Eli FUSION PROTEINS
AR105319A1 (en) 2015-06-05 2017-09-27 Sanofi Sa PROPHARMS THAT INCLUDE A DUAL AGONIST GLU-1 / GLUCAGON CONJUGATE HIALURONIC ACID CONNECTOR
EP3307326B9 (en) 2015-06-15 2021-02-24 Angiochem Inc. Methods for the treatment of leptomeningeal carcinomatosis
KR102172937B1 (en) 2015-06-19 2020-11-03 옵코 바이오로직스 리미티드 Long-acting coagulation factor and its preparation method
AR105284A1 (en) 2015-07-10 2017-09-20 Sanofi Sa DERIVATIVES OF EXENDINA-4 AS SPECIFIC DUAL PEPTIDE AGONISTS OF GLP-1 / GLUCAGÓN RECEPTORS
RU2745528C2 (en) 2015-08-17 2021-03-26 Алексион Фармасьютикалз, Инк. Production of alkaline phosphatases
CN108137674B (en) 2015-08-20 2022-12-06 阿尔布梅迪克斯医疗有限公司 Albumin variants and conjugates
US11229686B2 (en) 2015-09-28 2022-01-25 Alexion Pharmaceuticals, Inc. Reduced frequency dosage regimens for tissue non-specific alkaline phosphatase (TNSALP)-enzyme replacement therapy of hypophosphatasia
WO2017074466A1 (en) 2015-10-30 2017-05-04 Alexion Pharmaceuticals, Inc. Methods for treating craniosynostosis in a patient
CN105367664B (en) * 2015-11-04 2019-09-20 成都贝爱特生物科技有限公司 Activate GLP-1 receptor and the preparation of the fusion protein of the difunctional effect of Amylin receptor and application thereof
CN114835794A (en) * 2015-11-16 2022-08-02 Ubi蛋白公司 Method for extending the half-life of a protein
ES2882634T3 (en) 2015-11-24 2021-12-02 Transfert Plus Sec Peptide compounds and peptide conjugates for the treatment of cancer by receptor-mediated chemotherapy
EP3426286A4 (en) 2016-03-08 2019-12-04 Alexion Pharmaceuticals, Inc. Methods for treating hypophosphatasia in children
WO2017173395A1 (en) 2016-04-01 2017-10-05 Alexion Pharmaceuticals, Inc. Methods for treating hypophosphatasia in adolescents and adults
CA3019726A1 (en) 2016-04-01 2017-10-05 Alexion Pharmaceuticals, Inc. Treating muscle weakness with alkaline phosphatases
US11208632B2 (en) 2016-04-26 2021-12-28 R.P. Scherer Technologies, Llc Antibody conjugates and methods of making and using the same
WO2017214130A1 (en) 2016-06-06 2017-12-14 Alexion Pharmaceuticals, Inc. Metal impact on manufacturing of alkaline phosphatases
CN106046176B (en) 2016-08-16 2019-09-10 中国药科大学 A kind of high active long-acting hypoglycemic fusion protein and preparation method thereof and medical usage
JP7018933B2 (en) 2016-08-18 2022-02-14 アレクシオン ファーマシューティカルズ， インコーポレイテッド How to treat bronchomalacia
WO2018045872A1 (en) * 2016-09-06 2018-03-15 中国药科大学 Polypeptide and uses thereof
CN109200273B (en) * 2017-07-04 2021-02-19 中国药科大学 Application of polypeptide in preparation of medicine for preventing or treating fatty liver disease
CN106279400A (en) * 2016-09-06 2017-01-04 中国药科大学 Design of P8 incretin peptide and application thereof
KR102641844B1 (en) * 2016-12-14 2024-02-28 리간달 인코포레이티드 Methods and compositions for delivery of nucleic acid and protein payloads
CN106519016A (en) * 2016-12-20 2017-03-22 中国药科大学 Design and application of blood glucose reducing and lipid regulating peptide Progly
EP3574004A1 (en) * 2017-01-25 2019-12-04 Medimmune, LLC Relaxin fusion polypeptides and uses thereof
AU2018243320A1 (en) 2017-03-31 2019-10-10 Alexion Pharmaceuticals, Inc. Methods for treating hypophosphatasia (HPP) in adults and adolescents
CN108727486A (en) * 2017-04-24 2018-11-02 舒泰神（北京）生物制药股份有限公司 Long-acting nerve growth factor, preparation method and combinations thereof
AU2018273406A1 (en) 2017-05-24 2020-01-16 Transfert Plus, S.E.C. Peptide compounds, conjugate compounds and uses thereof for treating inflammatory diseases
MX2020005231A (en) 2017-11-21 2020-08-24 Lilly Co Eli Methods of using and compositions containing dulaglutide.
CN109836503B (en) * 2017-11-24 2022-09-16 浙江道尔生物科技有限公司 Multiple active protein for treating metabolic diseases
CN109836504B (en) * 2017-11-24 2022-08-02 浙江道尔生物科技有限公司 Multi-domain active protein for treating metabolic diseases
CN109929806B (en) 2017-12-19 2020-05-08 北京吉源生物科技有限公司 Stem cell expressing GLP1 and FGF21 and application thereof
CN110028587B (en) * 2018-01-11 2021-10-08 安源医药科技（上海）有限公司 Synergistic bifunctional proteins for regulating blood glucose and lipids
WO2019140021A1 (en) 2018-01-12 2019-07-18 Eli Lilly And Company Combination therapy
CN110092835A (en) * 2018-01-30 2019-08-06 上海惠盾生物技术有限公司 A kind of GLP-1 analog-COL3A1 fusion protein
US11913039B2 (en) 2018-03-30 2024-02-27 Alexion Pharmaceuticals, Inc. Method for producing recombinant alkaline phosphatase
HRP20221054T1 (en) 2018-04-05 2022-11-11 Sun Pharmaceutical Industries Limited Novel glp-1 analogues
AU2019253462A1 (en) 2018-04-09 2020-11-26 Amgen Inc. Growth differentiation factor 15 fusion proteins
CN110964116A (en) * 2018-09-26 2020-04-07 北京辅仁瑞辉生物医药研究院有限公司 GLP1-Fc fusion proteins and conjugates thereof
WO2020118843A1 (en) * 2018-12-12 2020-06-18 四川利通科创生物医药科技有限公司 Glp-1 mutant, preparation method and application thereof
CA3177693A1 (en) 2019-04-05 2020-10-05 Eli Lilly And Company Therapeutic uses of dulaglutide
CN110151980B (en) * 2019-06-30 2022-12-09 中国药科大学 Application of GLP-1 receptor agonist fusion protein in preparation of medicine for preventing or treating hyperlipidemia
TW202140513A (en) 2020-02-22 2021-11-01 日商Ｊｃｒ製藥股份有限公司 Human transferrin receptor binding peptide
EP4222176A4 (en) * 2020-09-30 2024-02-28 Beijing Ql Biopharmaceutical Co Ltd Polypeptide conjugates and methods of uses
KR20230170918A (en) 2021-03-22 2023-12-19 펩티에이드 가부시키가이샤 Peptides and compositions comprising peptides
TW202305012A (en) 2021-03-22 2023-02-01 日商肽夢想股份有限公司 c-Met protein-binding peptide complex
CN113150172B (en) * 2021-04-28 2023-09-22 中国药科大学 GLP-1R/GIPR double-target agonist fusion protein and preparation method and application thereof
WO2022265109A1 (en) 2021-06-18 2022-12-22 Peptidream Inc. Ghr-binding pending peptide and composition comprising same
AU2022329794A1 (en) 2021-08-19 2024-02-22 Jcr Pharmaceuticals Co., Ltd. Human transferrin receptor–binding peptide
WO2023026994A1 (en) 2021-08-21 2023-03-02 武田薬品工業株式会社 Human transferrin receptor binding peptide-drug conjugate
CA3229962A1 (en) 2021-08-24 2023-03-02 Peptidream Inc. Human transferrin receptor-binding antibody-peptide conjugate
CN113801853B (en) * 2021-11-19 2022-03-15 山东兴瑞生物科技有限公司 Exendin-4 fusion gene modified MSC and application thereof
WO2023179796A1 (en) * 2022-03-25 2023-09-28 Beijing Ql Biopharmaceutical Co., Ltd. Pharmaceutical compositions of polypeptide conjugates and methods of uses thereof
EP4323413A1 (en) * 2022-03-30 2024-02-21 Beijing QL Biopharmaceutical Co., Ltd. Liquid pharmaceutical compositions of polypeptide conjugates and methods of uses thereof
CN114774496B (en) * 2022-06-21 2022-10-04 北京惠之衡生物科技有限公司 Method for preparing GLP-1 analogue through high-density fermentation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999043706A1 (en) 1998-02-27 1999-09-02 Novo Nordisk A/S Derivatives of glp-1 analogs

Family Cites Families (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8725529D0 (en) 1987-10-30 1987-12-02 Delta Biotechnology Ltd Polypeptides
ATE92107T1 (en) 1989-04-29 1993-08-15 Delta Biotechnology Ltd N-TERMINAL FRAGMENTS OF HUMAN SERUM ALBUMIN-CONTAINING FUSION PROTEINS.
US5766883A (en) 1989-04-29 1998-06-16 Delta Biotechnology Limited Polypeptides
FR2650598B1 (en) * 1989-08-03 1994-06-03 Rhone Poulenc Sante DERIVATIVES OF ALBUMIN WITH THERAPEUTIC FUNCTION
FR2686899B1 (en) 1992-01-31 1995-09-01 Rhone Poulenc Rorer Sa NOVEL BIOLOGICALLY ACTIVE POLYPEPTIDES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM.
FR2686900B1 (en) 1992-01-31 1995-07-21 Rhone Poulenc Rorer Sa NOVEL POLYPEPTIDES HAVING GRANULOCYTE COLONY STIMULATION ACTIVITY, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM.
EP0804581B1 (en) * 1994-04-22 2001-09-26 Corixa Corporation Compounds and methods for the stimulation and enhancement of protective immune responses and il-12 production
US5990077A (en) * 1995-04-14 1999-11-23 1149336 Ontario Inc. Glucagon-like peptide-2 and its therapeutic use
US5925351A (en) * 1995-07-21 1999-07-20 Biogen, Inc. Soluble lymphotoxin-β receptors and anti-lymphotoxin receptor and ligand antibodies as therapeutic agents for the treatment of immunological disease
GB9526733D0 (en) 1995-12-30 1996-02-28 Delta Biotechnology Ltd Fusion proteins
US6750334B1 (en) * 1996-02-02 2004-06-15 Repligen Corporation CTLA4-immunoglobulin fusion proteins having modified effector functions and uses therefor
AR008077A1 (en) 1996-07-26 1999-12-09 Talarico Salinas Laura Beatriz A FUSION POLYPEPTIDE OR A SALT OF THE SAME, ITS USE, A PROCESS TO PREPARE THEM, A PHARMACEUTICAL COMPOSITION THAT UNDERSTANDS THEM, AND A VECTOR.
EP0966297B2 (en) 1996-08-08 2013-02-27 Amylin Pharmaceuticals, Inc. Regulation of gastrointestinal motility
CA2264243C (en) * 1996-08-30 2004-10-05 Novo Nordisk A/S Glp-1 derivatives
UA65549C2 (en) * 1996-11-05 2004-04-15 Елі Ліллі Енд Компані Use of glucagon-like peptides such as glp-1, glp-1 analog, or glp-1 derivative in methods and compositions for reducing body weight
US6190909B1 (en) * 1997-04-17 2001-02-20 Millennium Pharmaceuticals, Inc. TH2-specific gene
EP0887061A1 (en) * 1997-06-28 1998-12-30 The Procter & Gamble Company Faecal collector
DK1019077T4 (en) 1997-08-08 2011-03-07 Amylin Pharmaceuticals Inc New exendin agonist compounds
NZ504258A (en) 1997-11-14 2002-12-20 Amylin Pharmaceuticals Inc Exendin 3 and 4 agonist compounds for the treatment of diabetes
DK1032587T4 (en) 1997-11-14 2013-04-08 Amylin Pharmaceuticals Llc New exendin agonist compounds
DE69936446T2 (en) 1998-02-13 2008-03-06 Amylin Pharmaceuticals, Inc., San Diego INOTROPIC AND DIETIC EFFECTS OF EXENDIN AND GLP-1
ATE466028T1 (en) * 1998-02-27 2010-05-15 Novo Nordisk As N-TERMINALLY MODIFIED GLP-1 DERIVATIVES
AU3247799A (en) 1998-02-27 1999-09-15 Novo Nordisk A/S Glp-1 derivatives of glp-1 and exendin with protracted profile of action
ATE265224T1 (en) * 1998-02-27 2004-05-15 Novo Nordisk As GLP-1 DERIVATIVES WITH A HELIX CONTENT OVER 25 THAT FORM PARTIALLY STRUCTURED MICELLAR-LIKE AGGREGATES
AU775422B2 (en) * 1998-06-15 2004-07-29 Gtc Biotherapeutics, Inc. Erythropoietin analog-human serum albumin fusion
WO2000034784A1 (en) * 1998-12-10 2000-06-15 Phylos, Inc. Protein scaffolds for antibody mimics and other binding proteins
US6514500B1 (en) * 1999-10-15 2003-02-04 Conjuchem, Inc. Long lasting synthetic glucagon like peptide {GLP-!}
WO2000069911A1 (en) * 1999-05-17 2000-11-23 Conjuchem, Inc. Long lasting insulinotropic peptides
EP1274720A4 (en) 2000-04-12 2004-08-18 Human Genome Sciences Inc Albumin fusion proteins
EP2277888A3 (en) 2001-12-21 2011-04-27 Human Genome Sciences, Inc. Fusion proteins of albumin and erythropoietin
EP1463752A4 (en) 2001-12-21 2005-07-13 Human Genome Sciences Inc Albumin fusion proteins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999043706A1 (en) 1998-02-27 1999-09-02 Novo Nordisk A/S Derivatives of glp-1 analogs

Cited By (273)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8067554B2 (en) 1999-03-15 2011-11-29 Novo Nordisk A/S Ion exchange chromatography of GLP-1, analogs and derivatives thereof
US7785599B2 (en) 2000-04-12 2010-08-31 Human Genome Sciences, Inc. Albumin fusion proteins
EP1695983A2 (en) * 2000-06-16 2006-08-30 Eli Lilly &amp; Company Glucagon-like peptide-1 analogs
EP1695983A3 (en) * 2000-06-16 2007-02-21 Eli Lilly &amp; Company Glucagon-like peptide-1 analogs
US8999328B2 (en) 2000-12-08 2015-04-07 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic Leukemia cells and uses thereof
US9150661B2 (en) 2000-12-08 2015-10-06 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
US8840885B2 (en) 2000-12-08 2014-09-23 Alexion Pharmaceuticals, Inc. Methods for treating chronic lymphocytic leukemia
EP2022505A3 (en) * 2001-07-31 2009-05-27 The Government of the United States of America, as represented by the Secretary of the Department of Health and Human Services GLP-1, exendin-4, peptide analogs and uses thereof
US10941187B2 (en) 2001-07-31 2021-03-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services GLP-1, exendin-4, peptide analogs and uses thereof
EP1411968A4 (en) * 2001-07-31 2005-06-15 Us Gov Health & Human Serv Glp-1 exendin-4 peptide analogs and uses thereof
US8853160B2 (en) 2001-07-31 2014-10-07 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services GLP-1, exendin-4, peptide analogs and uses thereof
US8278272B2 (en) 2001-07-31 2012-10-02 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services GLP-1, exendin-4, peptide analogs and uses thereof
US7576050B2 (en) 2001-07-31 2009-08-18 The United States Of America As Represented By The Department Of Health And Human Services GLP-1 exendin-4 peptide analogs and uses thereof
EP1411968A2 (en) * 2001-07-31 2004-04-28 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Glp-1 exendin-4 peptide analogs and uses thereof
EP1432730A2 (en) * 2001-08-23 2004-06-30 Eli Lilly And Company Glucagon-like peptide-1 analogs
EP1432730A4 (en) * 2001-08-23 2006-10-11 Lilly Co Eli Glucagon-like peptide-1 analogs
US8129504B2 (en) 2001-08-30 2012-03-06 Biorexis Technology, Inc. Oral delivery of modified transferrin fusion proteins
US7176278B2 (en) 2001-08-30 2007-02-13 Biorexis Technology, Inc. Modified transferrin fusion proteins
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
US8513189B2 (en) 2001-12-21 2013-08-20 Human Genome Sciences, Inc. Albumin fusion proteins
US7977306B2 (en) 2001-12-21 2011-07-12 Human Genome Sciences, Inc. Albumin fusion proteins
US8252739B2 (en) 2001-12-21 2012-08-28 Human Genome Sciences, Inc. Albumin fusion proteins
US7238660B2 (en) 2001-12-21 2007-07-03 Human Genome Sciences, Inc. Albumin fusion proteins
US8211439B2 (en) 2001-12-21 2012-07-03 Human Genome Sciences, Inc. Albumin fusion proteins comprising insulin polypeptides
US7189690B2 (en) 2001-12-21 2007-03-13 Human Genome Sciences, Inc. Albumin fusion proteins
US9221896B2 (en) 2001-12-21 2015-12-29 Human Genome Sciences, Inc. Albumin fusion proteins
US7847079B2 (en) 2001-12-21 2010-12-07 Human Genome Sciences, Inc. Albumin fusion proteins
US9296809B2 (en) 2001-12-21 2016-03-29 Human Genome Sciences, Inc. Albumin fusion proteins
US7799759B2 (en) 2001-12-21 2010-09-21 Human Genome Sciences, Inc. Albumin fusion proteins
US8071539B2 (en) 2001-12-21 2011-12-06 Human Genome Sciences, Inc. Albumin fusion proteins
AU2003200839B2 (en) * 2002-01-08 2008-12-11 Eli Lilly And Company Extended glucagon-like peptide-1 analogs
EP1585959A4 (en) * 2002-01-08 2007-11-14 Lilly Co Eli Extended glucagon-like peptide-1 analogs
EP1585959A2 (en) * 2002-01-08 2005-10-19 Eli Lilly And Company Extended glucagon-like peptide-1 analogs
EP2298331A2 (en) 2002-04-04 2011-03-23 Amgen, Inc Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins
WO2003086444A1 (en) 2002-04-04 2003-10-23 Amgen Inc. Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins
EP1545608A2 (en) * 2002-06-28 2005-06-29 Centocor, Inc. Mammalian ch1 deleted mimetibodies, compositions, methods and uses
EP1545608A4 (en) * 2002-06-28 2006-09-13 Centocor Inc Mammalian ch1 deleted mimetibodies, compositions, methods and uses
JP4873860B2 (en) * 2002-08-30 2012-02-08 バイオレクシス ファーマシューティカル コーポレーション Modified transferrin fusion protein
EP1611093A2 (en) * 2002-08-30 2006-01-04 Biorexis Pharmaceutical Corporation Modified transferrin fusion proteins
JP2006503588A (en) * 2002-08-30 2006-02-02 バイオレクシス ファーマシューティカル コーポレイション Modified transferrin fusion protein
EP1611093B1 (en) * 2002-08-30 2015-07-15 Biorexis Pharmaceutical Corporation Modified transferrin fusion proteins
WO2004050115A2 (en) * 2002-12-03 2004-06-17 Novo Nordisk A/S Combination treatment using exendin-4 and thiazolidinediones
WO2004050115A3 (en) * 2002-12-03 2004-07-22 Novo Nordisk As Combination treatment using exendin-4 and thiazolidinediones
EP1688148A1 (en) * 2002-12-03 2006-08-09 Novo Nordisk A/S Combination treatment using exendin-4 and thiazolidinediones
US7678763B2 (en) 2002-12-27 2010-03-16 Diobex, Inc. Compositions and methods for the prevention and control of insulin-induced hypoglycemia
US7642232B2 (en) 2002-12-27 2010-01-05 Diobex, Inc. Compositions and methods for the prevention and control of insulin-induced hypoglycemia
US7314859B2 (en) 2002-12-27 2008-01-01 Diobex, Inc. Compositions and methods for the prevention and control of insulin-induced hypoglycemia
US7655618B2 (en) 2002-12-27 2010-02-02 Diobex, Inc. Compositions and methods for the prevention and control of insulin-induced hypoglycemia
US7678762B2 (en) 2002-12-27 2010-03-16 Diobex, Inc. Methods for reducing the risk of hypoglycemia
US7683027B2 (en) 2002-12-27 2010-03-23 Diobex, Inc. Methods relating to hypoglycemic unawareness
CN100346834C (en) * 2003-01-10 2007-11-07 株式会社新潟Tlo Vector for gene therapy and method of quantifying target protein in mammal or cultured cells with the administration of the vector for gene theraphy
EP1605897A4 (en) * 2003-03-19 2010-08-04 Lilly Co Eli Polyethelene glycol link glp-1 compounds
EP1605897A2 (en) * 2003-03-19 2005-12-21 Eli Lilly And Company Polyethelene glycol link glp-1 compounds
US9364772B2 (en) 2003-04-08 2016-06-14 Novo Nordisk A/S Regeneration of chromatographic stationary phases
WO2004089985A1 (en) * 2003-04-11 2004-10-21 Novo Nordisk A/S Stable pharmaceutical compositions
US7595293B2 (en) 2003-04-11 2009-09-29 Novo Nordisk A/S Stable pharmaceutical compositions
US9102756B2 (en) 2003-05-15 2015-08-11 Trustees Of Tufts College Stable analogs of peptide and polypeptide therapeutics
US7994121B2 (en) 2003-05-15 2011-08-09 Trustees Of Tufts College Stable analogs of peptide and polypeptide therapeutics
US7259234B2 (en) 2003-05-15 2007-08-21 Trustees Of Tufts College Stable analogs of peptide and polypeptide therapeutics
US8273854B2 (en) 2003-06-12 2012-09-25 Eli Lilly And Company GLP-1 analog fusion proteins
WO2005000892A2 (en) 2003-06-12 2005-01-06 Eli Lilly And Company Glp-1 analog fusion plroteins
US7452966B2 (en) 2003-06-12 2008-11-18 Eli Lilly And Company GLP-1 analog fusion proteins
EA008831B1 (en) * 2003-06-12 2007-08-31 Эли Лилли Энд Компани Glp-1 analog fusion proteins
WO2004110472A3 (en) * 2003-06-12 2005-02-03 Lilly Co Eli Fusion proteins
WO2004110472A2 (en) * 2003-06-12 2004-12-23 Eli Lilly And Company Fusion proteins
KR100758755B1 (en) * 2003-06-12 2007-09-14 일라이 릴리 앤드 캄파니 -1 glp-1 analog fusion proteins
CN101974090A (en) * 2003-06-12 2011-02-16 伊莱利利公司 GLP-1 analog fusion proteins
WO2005000892A3 (en) * 2003-06-12 2005-03-03 Lilly Co Eli Glp-1 analog fusion plroteins
JP2007505643A (en) * 2003-06-12 2007-03-15 イーライ リリー アンド カンパニー Fusion protein
JP2007536902A (en) * 2003-06-12 2007-12-20 イーライ リリー アンド カンパニー GLP-1 analog complex protein
CN1802386B (en) * 2003-06-12 2010-12-15 伊莱利利公司 GLP-1 analog fusion proteins
EP2368909A1 (en) 2003-06-12 2011-09-28 Eli Lilly and Company GLP-1 analog fusion proteins
AU2010203063B2 (en) * 2003-09-19 2012-10-25 Novo Nordisk A/S Albumin-binding derivatives of therapeutic peptides
US7897560B2 (en) 2003-09-19 2011-03-01 Novo Nordisk A/S Plasma protein affinity tags
US7737260B2 (en) 2003-11-13 2010-06-15 Hanmi Pharm. Co., Ltd Protein complex using an immunoglobulin fragment and method for the preparation thereof
US7736653B2 (en) 2003-11-13 2010-06-15 Hanmi Pharm. Co., Ltd Pharmaceutical composition comprising an immunoglobulin Fc region as a carrier
JP2007531513A (en) * 2003-11-13 2007-11-08 ハンミ ファーム．インダストリー カンパニー リミテッド IgG Fc fragment useful as drug carrier and method for producing the same
EP1682581A4 (en) * 2003-11-13 2008-11-05 Hanmi Pharmaceutical Co Ltd Igg fc fragment for a drug carrier and method for the preparation thereof
US9750820B2 (en) 2003-11-13 2017-09-05 Hanmi Science Co., Ltd. IgG Fc fragment for a drug carrier and method for the preparation thereof
US8846874B2 (en) 2003-11-13 2014-09-30 Hanmi Science Co., Ltd IgG Fc fragment for a drug carrier and method for the preparation thereof
US10071166B2 (en) 2003-11-13 2018-09-11 Hanmi Science Co., Ltd. Protein complex using an immunoglobulin fragment and method for the preparation thereof
US10272159B2 (en) 2003-11-13 2019-04-30 Hanmi Science Co., Ltd. IgG Fc fragment for a drug carrier and method for the preparation thereof
EP1682581A1 (en) * 2003-11-13 2006-07-26 Hanmi Pharmaceutical. Co., Ltd. Igg fc fragment for a drug carrier and method for the preparation thereof
US11058776B2 (en) 2003-11-13 2021-07-13 Hanmi Science Co., Ltd. IgG Fc fragment for a drug carrier and method for the preparation thereof
US8110665B2 (en) 2003-11-13 2012-02-07 Hanmi Holdings Co., Ltd. Pharmaceutical composition comprising an immunoglobulin FC region as a carrier
EP2394656A2 (en) 2003-11-20 2011-12-14 Novo Nordisk A/S Propylene Glycol-containing peptide formulations which are optimal for production and for use in injection devices
EP3300721A1 (en) 2003-11-20 2018-04-04 Novo Nordisk A/S Propylene glycol-containing peptide formulations which are optimal for production and for use in injection devices
EP2298337A2 (en) 2003-12-09 2011-03-23 Novo Nordisk A/S Regulation of food preference using GLP-1 agonists
JP2007537142A (en) * 2003-12-18 2007-12-20 ノボ　ノルディスク　アクティーゼルスカブ Novel GLP-1 analogues bound to albumin-like substances
WO2005058958A3 (en) * 2003-12-18 2005-11-24 Novo Nordisk As Novel glp-1 analogues linked to albumin-like agents
CN102816241A (en) * 2004-02-09 2012-12-12 人类基因科学公司 Albumin fusion proteins
AU2005211725B2 (en) * 2004-02-09 2010-07-15 Human Genome Sciences, Inc. Albumin fusion proteins
US8143026B2 (en) 2004-02-09 2012-03-27 Human Genome Sciences, Inc. Albumin fusion proteins
US7569384B2 (en) 2004-02-09 2009-08-04 Human Genome Sciences, Inc. Albumin fusion proteins
WO2005077042A3 (en) * 2004-02-09 2006-11-30 Human Genome Sciences Inc Albumin fusion proteins
JP2007522806A (en) * 2004-02-09 2007-08-16 ヒューマン　ジノーム　サイエンシーズ，　インコーポレイテッド Albumin fusion protein
EP1718665A2 (en) * 2004-02-11 2006-11-08 Amylin Pharmaceuticals, Inc. Hybrid polypeptides with selectable properties
US8426361B2 (en) 2004-02-11 2013-04-23 Amylin Pharmaceuticals, Llc Pancreatic polypeptide family motifs, polypeptides and methods comprising the same
US8697647B2 (en) 2004-02-11 2014-04-15 Odile Esther Levy Hybrid polypeptides with selectable properties
EP1718665A4 (en) * 2004-02-11 2008-05-14 Amylin Pharmaceuticals Inc Hybrid polypeptides with selectable properties
EP2422807A3 (en) * 2004-02-11 2012-05-30 Amylin Pharmaceuticals Inc. Hybrid polypeptides with selectable properties
EP2422806A3 (en) * 2004-02-11 2012-06-13 Amylin Pharmaceuticals Inc. Hybrid polypeptides with selectable properties
EP2417980A1 (en) * 2004-02-11 2012-02-15 Amylin Pharmaceuticals Inc. Hybrid polypeptides with selectable properties
JP2007525495A (en) * 2004-02-11 2007-09-06 アミリン・ファーマシューティカルズ，インコーポレイテッド Hybrid polypeptides with selectable properties
US9453063B2 (en) 2004-02-11 2016-09-27 Amylin Pharmaceuticals, Llc. Hybrid polypeptides with selectable properties
US8906849B2 (en) 2004-02-11 2014-12-09 Amylin Pharmaceuticals, Llc Pancreatic polypeptide family motifs, polypeptides and methods comprising the same
US8603969B2 (en) 2004-02-11 2013-12-10 Amylin Pharmaceuticals, Llc Pancreatic polypeptide family motifs and polypeptides comprising the same
US7723471B2 (en) 2004-02-11 2010-05-25 Amylin Pharmaceuticals, Inc. Pancreatic polypeptide family motifs, polypeptides and methods comprising the same
EA011583B1 (en) * 2004-03-31 2009-04-28 Сентокор, Инк. Human glp-1 mimetibodies, compositions, methods and uses
EP1750754A2 (en) * 2004-03-31 2007-02-14 Centocor, Inc. Human glp-1 mimetibodies, compositions, methods and uses
JP2008537873A (en) * 2004-03-31 2008-10-02 セントカー・インコーポレーテツド Human GLP-1 mimetibody, compositions, methods and uses
EP1750754A4 (en) * 2004-03-31 2010-09-22 Centocor Ortho Biotech Inc Human glp-1 mimetibodies, compositions, methods and uses
WO2005113606A2 (en) 2004-05-13 2005-12-01 Eli Lilly And Company Fgf-21 fusion proteins
WO2005113606A3 (en) * 2004-05-13 2006-05-04 Lilly Co Eli Fgf-21 fusion proteins
US7576190B2 (en) 2004-05-13 2009-08-18 Eli Lilly And Company FGF-21 fusion proteins
EP2316446A1 (en) 2004-06-11 2011-05-04 Novo Nordisk A/S Counteracting drug-induced obesity using GLP-1 agonists
US8030273B2 (en) 2004-10-07 2011-10-04 Novo Nordisk A/S Protracted exendin-4 compounds
US7893017B2 (en) 2004-10-07 2011-02-22 Novo Nordisk A/S Protracted GLP-1 compounds
EP2330126A1 (en) 2004-10-07 2011-06-08 Novo Nordisk A/S Protracted exendin-4 compounds
US8772232B2 (en) 2004-10-07 2014-07-08 Novo Nordisk A/S Protracted exendin-4 compounds
WO2006059106A3 (en) * 2004-12-02 2007-01-04 Domantis Ltd Bispecific domain antibodies targeting serum albumin and glp-1 or pyy
AU2005311099B2 (en) * 2004-12-02 2012-02-02 Domantis Limited Bispecific domain antibodies targeting serum albumin and GLP-1 or PYY
CN101128487B (en) * 2004-12-02 2012-10-10 杜门蒂斯有限公司 Bispecific domain antibodies targeting serum albumin and GLP-1 or PYY
WO2006059106A2 (en) * 2004-12-02 2006-06-08 Domantis Limited Bispecific domain antibodies targeting serum albumin and glp-1 or pyy
EP2769990A2 (en) 2004-12-02 2014-08-27 Domantis Limited Bispecific domain antibodies targeting serum albumin and GLP-1 or PYY
JP2008525477A (en) * 2004-12-22 2008-07-17 セントカー・インコーポレーテツド GLP-1 agonists, compositions, methods and uses
EP2322546A1 (en) 2005-03-18 2011-05-18 Novo Nordisk A/S Acylated GLP-1 compounds
US8129343B2 (en) 2005-03-18 2012-03-06 Novo Nordisk A/S Acylated GLP-1 compounds
US8536122B2 (en) 2005-03-18 2013-09-17 Novo Nordisk A/S Acylated GLP-1 compounds
US8603972B2 (en) 2005-03-18 2013-12-10 Novo Nordisk A/S Extended GLP-1 compounds
WO2006097537A2 (en) 2005-03-18 2006-09-21 Novo Nordisk A/S Acylated glp-1 compounds
EP1871811A2 (en) * 2005-03-28 2008-01-02 Centocor, Inc. Human glp-1 mimetibodies, compositions, methods and uses
EP1871811A4 (en) * 2005-03-28 2009-07-22 Centocor Inc Human glp-1 mimetibodies, compositions, methods and uses
JP2008546373A (en) * 2005-03-28 2008-12-25 セントカー・インコーポレーテツド Human GLP-1 mimetibody, compositions, methods and uses
EP1920061A4 (en) * 2005-07-27 2009-05-13 Wang Qinghua GLP/1/EXENDIN 4 IgG Fc FUSION CONSTRUCTS FOR TREATMENT OF DIABETES
US8658174B2 (en) 2005-07-27 2014-02-25 Qinghua Wang GLP/1/exendin 4 IgG Fc fusion constructs for treatment of diabetes
WO2007012188A1 (en) * 2005-07-27 2007-02-01 Qinghua Wang GLP/1/EXENDM 4 IgG Fc FUSION CONSTRUCTS FOR TREATMENT OF DIABETES
EP1920061A1 (en) * 2005-07-27 2008-05-14 Wang, Qinghua GLP/1/EXENDIN 4 IgG Fc FUSION CONSTRUCTS FOR TREATMENT OF DIABETES
JP2009504681A (en) * 2005-08-11 2009-02-05 アミリン・ファーマシューティカルズ，インコーポレイテッド Hybrid polypeptides with selectable properties
AU2006299134B2 (en) * 2005-09-22 2012-02-23 Biocompatibles Uk Ltd. GLP-1 ( glucagon-like peptide-1 ) fusion polypeptides with increased peptidase resistance
US8853159B2 (en) 2005-09-22 2014-10-07 Biocompatibles Uk Ltd GLP-1 Fusion Peptides
EP2045265A1 (en) * 2005-09-22 2009-04-08 Biocompatibles Uk Ltd. GLP-1 fusion peptides, their production and use
EP2261245A1 (en) * 2005-09-22 2010-12-15 Biocompatibles Uk Ltd. GLP-1 (glucagon-like peptide-1) fusion polypeptides with increased peptidase resistance
EP2174952A3 (en) * 2005-09-22 2010-11-17 Biocompatibles Uk Ltd. glp-1 (glucagon-like peptide-1) fusion polypeptides with increased peptidase resistance
EA013796B1 (en) * 2005-09-22 2010-06-30 Байокомпатиблз Юк Лтд. Glp-1 (glucagon-like peptide-1) fusion polypeptides with increased peptidase resistance
EP1767545A1 (en) 2005-09-22 2007-03-28 Biocompatibles UK Limited GLP-1 (Glucagon-like peptide-1) fusion polypeptides with increased peptidase resistance
US8431533B2 (en) 2005-09-22 2013-04-30 Biocompatibles Uk Ltd. GLP-1 fusion peptides, their production and use
WO2007039140A1 (en) * 2005-09-22 2007-04-12 Biocompatibles Uk Ltd. Glp-1 ( glucagon-like peptide-1 ) fusion polypeptides with increased peptidase resistance
WO2007049695A1 (en) * 2005-10-26 2007-05-03 Chugai Seiyaku Kabushiki Kaisha Agglutinable glp-1 analogue and sustained-release pharmaceutical composition
EP1965823B1 (en) * 2005-11-04 2016-05-18 Glaxosmithkline LLC Methods for administering hypoglycemic agents
EP3095456A1 (en) * 2005-11-04 2016-11-23 Glaxosmithkline LLC Methods for administering hypoglycemic agents
WO2007063907A1 (en) * 2005-11-30 2007-06-07 Shionogi & Co., Ltd. Sugar chain adduct of peptide and pharmaceutical comprising the same as active ingredient
US8580919B2 (en) 2005-12-16 2013-11-12 Nektar Therapeutics Polymer conjugates of GLP-1
US8293869B2 (en) 2005-12-16 2012-10-23 Nektar Therapeutics Polymer conjugates of GLP-1
US8709415B2 (en) 2006-01-12 2014-04-29 Alexion Pharmaceuticals, Inc. Antibodies to OX-2/CD200 and uses thereof
US9000133B2 (en) 2006-01-12 2015-04-07 Alexion Pharmaceuticals, Inc. Antibodies to OX-2/CD200 and uses thereof
KR20140066793A (en) * 2006-02-06 2014-06-02 체에스엘 베링 게엠베하 Modified coagulation factor viia with extended half-life
US8765915B2 (en) 2006-02-06 2014-07-01 Csl Behring Gmbh Modified coagulation factor VIIa with extended half-life
KR101439817B1 (en) 2006-02-06 2014-10-02 체에스엘 베링 게엠베하 Modified coagulation factor VIIa with extended half-life
KR101579083B1 (en) 2006-02-06 2015-12-21 체에스엘 베링 게엠베하 Modified coagulation factor VIIa with extended half-life
US8288339B2 (en) 2006-04-20 2012-10-16 Amgen Inc. GLP-1 compounds
US8790705B2 (en) 2006-05-10 2014-07-29 Biocompatibles Uk Ltd. Spherical microcapsules comprising GLP-1 peptides, their production and use
EP2636680A2 (en) 2006-05-26 2013-09-11 Amylin Pharmaceuticals, LLC Composition and methods for treatment of congestive heart failure
US8969538B2 (en) 2006-06-07 2015-03-03 Human Genome Sciences, Inc. Albumin fusion proteins
US7867972B2 (en) 2006-07-24 2011-01-11 Pharmacia & Upjohn Company, Llc Fusion protein of exendin-4 to a transferrin (Tf) polypeptide
US8158579B2 (en) 2006-07-24 2012-04-17 Biorexis Pharmaceutical Corporation Fusion protein of an exendin to modified transferrin
EP2059606A2 (en) * 2006-09-06 2009-05-20 Phasebio Pharmaceuticals, Inc. Fusion peptide therapeutic compositions
EP2059606A4 (en) * 2006-09-06 2010-04-07 Phasebio Pharmaceuticals Inc Fusion peptide therapeutic compositions
US8476230B2 (en) 2007-01-05 2013-07-02 Hanmi Science Co., Ltd Insulinotropic complex using an immunoglobulin fragment
AU2011254001B2 (en) * 2007-01-05 2012-08-02 Covx Technologies Ireland Limited Glucagon-like protein-1 receptor (GLP-1R) agonist compounds
WO2008081418A1 (en) * 2007-01-05 2008-07-10 Covx Technologies Ireland Limited Glucagon-like protein-1 receptor (glp-1r) agonist compounds
KR101224335B1 (en) * 2007-01-05 2013-01-25 씨오브이엑스 테크놀로지스 아일랜드 리미티드 Glucagon-like protein-1 receptor (glp-1r) agonist compounds
TWI413528B (en) * 2007-01-05 2013-11-01 Hanmi Science Co Ltd An insulinotropic complex using an immunoglobulin fragment
EP1972349A1 (en) * 2007-03-21 2008-09-24 Biocompatibles UK Limited GLP-1 fusion peptides conjugated to polymer(s), their production and use
WO2008113601A1 (en) * 2007-03-21 2008-09-25 Biocompatibles Uk Ltd. Glp-1 fusion peptides conjugated to polymer(s), their production and use
WO2008131242A1 (en) 2007-04-18 2008-10-30 Zymogenetics, Inc. Single chain fc, methods of making and methods of treatment
US8986684B2 (en) 2007-07-25 2015-03-24 Alexion Pharmaceuticals, Inc. Methods and compositions for treating autoimmune disease
US8557769B2 (en) 2007-08-03 2013-10-15 Eli Lilly And Company Co-administration of FGF-21 and GLP-1 to treat diabetes and lower blood glucose
US8895694B2 (en) 2007-09-05 2014-11-25 Novo Nordisk A/S Glucagon-Like Peptide-1 derivatives and their pharmaceutical use
US9409966B2 (en) 2007-09-05 2016-08-09 Novo Nordisk A/S Glucagon-like peptide-1 derivatives and their pharmaceutical use
US9657079B2 (en) 2007-09-05 2017-05-23 Novo Nordisk A/S Truncated GLP-1 derivatives and their therapeutical use
US9067977B2 (en) 2007-09-05 2015-06-30 Novo Nordisk A/S Peptides derivatized with A-B-C-D- and their therapeutical use
US10182984B2 (en) 2007-09-21 2019-01-22 The Regents Of The University Of California Targeted interferons demonstrate potent apoptotic and anti-tumor activities
US9534033B2 (en) 2007-09-21 2017-01-03 The Regents Of The University Of California Targeted interferons demonstrating potent apoptotic and anti-tumor activities
TWI403331B (en) * 2007-11-29 2013-08-01 Hanmi Science Co Ltd A pharmaceutical composition for treating obesity-related disease comprising insulinotropic peptide conjugate
WO2009121804A1 (en) 2008-03-31 2009-10-08 Glaxo Group Limited Drug fusions and conjugates
US20110020345A1 (en) * 2008-03-31 2011-01-27 Christopher Herring Drug fusions and conjugates
WO2010011096A2 (en) 2008-07-23 2010-01-28 Hanmi Pharmaceutical Co., Ltd. A polypeptide complex comprising non-peptidyl polymer having three functional ends
US8865868B2 (en) 2008-08-06 2014-10-21 Novo Nordisk Healthcare Ag Conjugated proteins with prolonged in vivo efficacy
US10466252B2 (en) 2008-12-05 2019-11-05 Glaxo Group Limited Methods for selecting protease resistant polypeptides
US10302655B2 (en) 2008-12-05 2019-05-28 Glaxo Group Limited Methods for selecting protease resistant polypeptides
US8513192B2 (en) 2009-01-22 2013-08-20 Novo Nordisk A/S Stable growth hormone compounds resistant to proteolytic degradation
US8779103B2 (en) 2009-03-27 2014-07-15 Glaxo Group Limited Drug fusions and conjugates
WO2010108937A2 (en) 2009-03-27 2010-09-30 Glaxo Group Limited Drug fusions and conjugates
US8841249B2 (en) 2009-08-06 2014-09-23 Novo Nordisk A/S Growth hormones with prolonged in-vivo efficacy
WO2011039096A1 (en) 2009-09-30 2011-04-07 Glaxo Group Limited Drug fusions and conjugates with extended half life
US8541373B2 (en) 2009-12-08 2013-09-24 Teva Pharmaceutical Industries Ltd. BChE albumin fusions for the treatment of cocaine abuse
US9695226B2 (en) 2010-01-22 2017-07-04 Novo Nordisk Healthcare Ag Growth hormones with prolonged in-vivo efficacy
US9211342B2 (en) 2010-01-22 2015-12-15 Novo Nordisk Healthcare Ag Stable growth hormone compounds resistant to proteolytic degradation
US8779109B2 (en) 2010-01-22 2014-07-15 Novo Nordisk Health Care Ag Growth hormones with prolonged in-vivo efficacy
WO2011122921A2 (en) 2010-04-02 2011-10-06 Hanmi Holdings Co., Ltd. An insulin conjugate using an immunoglobulin fragment
US10744187B2 (en) 2010-04-02 2020-08-18 Hanmi Science Co., Ltd. Insulin conjugate using an immunoglobulin fragment
US9981017B2 (en) 2010-04-02 2018-05-29 Hanmi Science Co., Ltd. Insulin conjugate using an immunoglobulin fragment
US9492507B2 (en) 2010-04-02 2016-11-15 Hanmi Science Co., Ltd. Insulin conjugate using an immunoglobulin fragment
US9168288B2 (en) 2010-04-09 2015-10-27 Mount Sinai Hospital Methods for treating disorders of the gastrointestinal tract using a GLP-1 agonist
WO2011123943A1 (en) 2010-04-09 2011-10-13 Mount Sinai Hospital Methods for treating disorders of the gastrointestinal tract using a glp-1 agonist
WO2011136361A1 (en) 2010-04-30 2011-11-03 株式会社 三和化学研究所 Peptide for improving in vivo stability of physiologically active substance or the like and physiologically active substance with improved in vivo stability
US8889619B2 (en) 2010-06-11 2014-11-18 Beijing Dongfang Biotech Co., Ltd. Fusion protein of Exendin-4 and its analog, preparation method and use thereof
WO2011153965A1 (en) 2010-06-11 2011-12-15 北京精益泰翔技术发展有限公司 Fusion protein of exendin-4 and its analog, preparation method and use thereof
EP3248611A1 (en) 2010-07-21 2017-11-29 Hanmi Science Co., Ltd. Novel long-acting glucagon conjugate and pharmaceutical composition comprising the same for the prevention and treatment of obesity
US11382957B2 (en) 2010-12-16 2022-07-12 Novo Nordisk A/S Solid compositions comprising a GLP-1 agonist and a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid
US10960052B2 (en) 2010-12-16 2021-03-30 Novo Nordisk A/S Solid compositions comprising a GLP-1 agonist and a salt of N-(8-(2-hydroxybenzoyl) amino) caprylic acid
WO2012118042A1 (en) 2011-02-28 2012-09-07 独立行政法人国立循環器病研究センター Medicinal agent for inhibiting metastasis of malignant tumor
EP3189835A1 (en) 2011-02-28 2017-07-12 National Cerebral and Cardiovascular Center Medical agent for suppressing malignant tumor metastasis
WO2012136792A2 (en) 2011-04-07 2012-10-11 Glaxo Group Limited Cck compositions
WO2012136790A1 (en) 2011-04-07 2012-10-11 Glaxo Group Limited Compositions comprising fusion proteins or conjugates with an improved half -life
US11117947B2 (en) 2011-04-12 2021-09-14 Novo Nordisk A/S Double-acylated GLP-1 derivatives
US11034746B2 (en) 2011-04-12 2021-06-15 Novo Nordisk A/S Double-acylated GLP-1 derivatives
WO2013027680A1 (en) 2011-08-19 2013-02-28 独立行政法人国立循環器病研究センター Drug for preventing exacerbation of malignant tumor, comprising combination of natriuretic peptide receptor gc-a agonist and gc-b agonist
US11434271B2 (en) 2011-11-04 2022-09-06 Hanmi Science Co., Ltd. Method for preparing physiologically active polypeptide complex
WO2013066106A1 (en) 2011-11-04 2013-05-10 Hanmi Science Co., Ltd. Method for preparing physiologically active polypeptide complex
WO2013083826A2 (en) 2011-12-09 2013-06-13 Novo Nordisk A/S Glp-1 agonists
US9504757B2 (en) 2011-12-30 2016-11-29 Hanmi Science Co., Ltd. Site-specific GLP-2 conjugate using an immunoglobulin fragment
WO2013100704A1 (en) 2011-12-30 2013-07-04 Hanmi Science Co., Ltd. A site-specific glp-2 conjugate using an immunoglobulin fragment
US11168109B2 (en) 2012-03-08 2021-11-09 Hanmi Science Co., Ltd. Process for preparation of physiologically active polypeptide complex
US11123296B2 (en) 2012-03-22 2021-09-21 Novo Nordisk A/S Compositions comprising a delivery agent and preparation thereof
US10933120B2 (en) 2012-03-22 2021-03-02 Novo Nordisk A/S Compositions of GLP-1 peptides and preparation thereof
US11759502B2 (en) 2012-03-22 2023-09-19 Novo Nordisk A/S Compositions of GLP-1 peptides and preparation thereof
US11759503B2 (en) 2012-03-22 2023-09-19 Novo Nordisk A/S Compositions of GLP-1 peptides and preparation thereof
US11759501B2 (en) 2012-03-22 2023-09-19 Novo Nordisk A/S Compositions of GLP-1 peptides and preparation thereof
WO2013171570A1 (en) 2012-05-16 2013-11-21 Glaxo Group Limited Polypeptide loaded poca nanoparticles for oral administration
US9884124B2 (en) 2012-05-17 2018-02-06 Extend Biosciences, Inc. Carriers for improved drug delivery
US11033499B2 (en) 2012-06-20 2021-06-15 Novo Nordisk A/S Tablet formulation comprising a GLP-1 peptide and a delivery agent
US10987424B2 (en) 2012-07-25 2021-04-27 Hanmi Pharm. Co., Ltd. Liquid formulation of long-acting insulin conjugate
US10975158B2 (en) 2012-12-07 2021-04-13 The Regents Of The University Of California CD138-targeted interferon demonstrates potent apoptotic and anti-tumor activities
US9803021B2 (en) 2012-12-07 2017-10-31 The Regents Of The University Of California CD138-targeted interferon demonstrates potent apoptotic and anti-tumor activities
US9409662B2 (en) 2013-01-15 2016-08-09 Teva Pharmaceutical Industries, Ltd. Formulations of albu-BChE, preparation and uses thereof
US11045523B2 (en) 2013-04-05 2021-06-29 Novo Nordisk Healthcare Ag Formulation of growth hormone albumin-binder conjugate
US10822427B2 (en) 2013-05-29 2020-11-03 The Regents Of The University Of California Anti-CSPG4 fusions with interferon for the treatment of malignancy
US10093745B2 (en) 2013-05-29 2018-10-09 The Regents Of The University Of California Anti-CSPG4 fusions with interferon for the treatment of malignancy
US20160280762A1 (en) * 2013-08-01 2016-09-29 Jiangsu T-Mab Biopharma Co., Ltd Glp-1 analog fusion protein and preparation method and use thereof
US10253103B2 (en) 2013-08-13 2019-04-09 Gmaz Biopharm LLC Antibody specifically binding to GLP-1R and fusion protein thereof with GLP-1
US10059773B2 (en) 2013-08-13 2018-08-28 Gmax Biopharm Llc. Antibody specifically binding to GLP-1 R and fusion protein thereof with GLP-1
WO2015022420A1 (en) * 2013-08-16 2015-02-19 Medimmune Limited Gip and glp-1 receptor dual-agonists for the treatment of diabetes
EP3889185A3 (en) * 2014-03-31 2022-01-26 Hanmi Pharm. Co., Ltd. Composition for improving the solubility of a protein or peptide by using immunoglobulin fc fragment linkage
WO2015165480A1 (en) 2014-04-30 2015-11-05 Institute For Research In Biomedicine Human cytomegalovirus vaccine compositions and method of producing the same
US10968266B2 (en) 2014-09-05 2021-04-06 University Of Copenhagen GIP peptide analogues
US9988430B2 (en) 2014-10-10 2018-06-05 Novo Nordisk A/S Stable GLP-1 based GLP-1/glucagon receptor co-agonists
WO2016055610A1 (en) * 2014-10-10 2016-04-14 Novo Nordisk A/S Stable glp-1 based glp-1/glucagon receptor co-agonists
US10406202B2 (en) 2014-10-22 2019-09-10 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US9616109B2 (en) 2014-10-22 2017-04-11 Extend Biosciences, Inc. Insulin vitamin D conjugates
US10420819B2 (en) 2014-10-22 2019-09-24 Extend Biosciences, Inc. Insulin vitamin D conjugates
US10702574B2 (en) 2014-10-22 2020-07-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US11116816B2 (en) 2014-10-22 2021-09-14 Extend Biosciences, Inc. Therapeutic vitamin d conjugates
US9789197B2 (en) 2014-10-22 2017-10-17 Extend Biosciences, Inc. RNAi vitamin D conjugates
WO2016073704A1 (en) * 2014-11-06 2016-05-12 Children's Research Institute, Children's National Medical Center Immunotherapeutics for cancer and autoimmune diseases
US10485870B2 (en) 2015-02-11 2019-11-26 Gmax Biopharm Llc. Stable pharmaceutical solution formulation of GLP-1R antibody fusion protein
US10851144B2 (en) 2015-04-10 2020-12-01 Amgen Inc. Interleukin-2 muteins for the expansion of T-regulatory cells
US11421033B2 (en) 2015-09-04 2022-08-23 The Scripps Research Institute Insulin immunoglobulin fusion proteins
CN107033234B (en) * 2017-01-03 2018-06-26 北京凯因科技股份有限公司 Acylated glp-1 derivatives
CN107033234A (en) * 2017-01-03 2017-08-11 北京凯因科技股份有限公司 The acylated derivatives of GLP 1
WO2018220123A1 (en) 2017-05-31 2018-12-06 University Of Copenhagen Long-acting gip peptide analogues
US11572399B2 (en) 2017-05-31 2023-02-07 University Of Copenhagen Long-acting GIP peptide analogues
US11752198B2 (en) 2017-08-24 2023-09-12 Novo Nordisk A/S GLP-1 compositions and uses thereof
US11761963B2 (en) 2017-09-27 2023-09-19 Alexion Pharmaceuticals, Inc. Biomarker signature for predicting tumor response to anti-CD200 therapy
US11833248B2 (en) 2018-02-02 2023-12-05 Novo Nordisk A/S Solid compositions comprising a GLP-1 agonist and a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid
WO2019201328A1 (en) 2018-04-19 2019-10-24 杭州先为达生物科技有限公司 Acylated glp-1 derivative
US11713345B2 (en) * 2018-10-22 2023-08-01 Janssen Sciences Ireland Unlimited Company Glucagon like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion proteins and uses thereof
US20220089669A1 (en) * 2018-10-22 2022-03-24 Janssen Pharmaceutica Nv Glucagon like peptide 1 (glp1)-growth differentiation factor 15 (gdf15) fusion proteins and uses thereof
WO2020115049A1 (en) 2018-12-03 2020-06-11 Antag Therapeutics Aps Modified gip peptide analogues
WO2020115048A1 (en) 2018-12-03 2020-06-11 Antag Therapeutics Aps Modified gip peptide analogues
WO2020125744A1 (en) 2018-12-21 2020-06-25 江苏恒瑞医药股份有限公司 Bispecific protein
US11318191B2 (en) 2020-02-18 2022-05-03 Novo Nordisk A/S GLP-1 compositions and uses thereof

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