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WO2013148871A1 - Polypeptides génétiquement modifiés - Google Patents

Polypeptides génétiquement modifiés Download PDF

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Publication number
WO2013148871A1
WO2013148871A1 PCT/US2013/034152 US2013034152W WO2013148871A1 WO 2013148871 A1 WO2013148871 A1 WO 2013148871A1 US 2013034152 W US2013034152 W US 2013034152W WO 2013148871 A1 WO2013148871 A1 WO 2013148871A1
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WIPO (PCT)
Prior art keywords
seq
sequence
leptin
amino acids
engineered polypeptide
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PCT/US2013/034152
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English (en)
Inventor
Jonathan David Roth
Soumitra S. Ghosh
Original Assignee
Amylin Pharmaceuticals, Llc
Astrazeneca Pharmaceuticals Lp
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Publication of WO2013148871A1 publication Critical patent/WO2013148871A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • 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
    • 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
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • leptin agonism is able to markedly and synergistically improve the therapeutic benefits already evident with administration of a GLP-1 agonist in non-alcohol steatohepatitis (NASH).
  • NASH non-alcohol steatohepatitis
  • the combination of a ineffective dose of leptin with a moderately effective dose of [ 14 L]-Exendin-4 (SEQ ID NO: l) leads to marked, synergistic improvements in food intake, body weight or adiposity, liver weight, lipid or plasma total cholesterol, or triglycerides.
  • diseases amendable to such treatment include lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes (including type I and type II), NASH, nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X, and Huntington's Disease, or combinations thereof.
  • an engineered polypeptide including a first peptide hormone domain (HD1) which includes an exendin domain sequence, and a second peptide hormone domain (HD2) which includes a humanized chimeric seal leptin sequence.
  • HD1 is covalently bonded to HD2 through a bond, or through a linker LI, as described herein.
  • a method for treating a disease in a subject including administering an engineered polypeptide as disclosed herein to a subject in need thereof in an amount effective to treat the disease.
  • the disease can be diabetes, overweight, obesity, Alzheimer's disease, short bowel syndrome, fatty liver disease, dyslipidemia, coronary artery disease, stroke, hyperlipidemia, NASH or Parkinson's disease.
  • composition including an engineered polypeptide disclosed herein and a pharmaceutically acceptable excipient.
  • Obsity and “overweight” refer to mammals having a weight greater than normally expected, and may be determined by, e.g., physical appearance, body mass index (BMI) as known in the art, waist-to-hip circumference ratios, skinfold thickness, waist circumference, and the like.
  • BMI body mass index
  • the Centers for Disease Control and Prevention (CDC) define overweight as an adult human having a BMI of 25 to 29.9; and define obese as an adult human having a BMI of 30 or higher. Additional metrics for the determination of obesity exist. For example, the CDC states that a person with a waist-to-hip ratio greater than 1.0 is overweight.
  • Lean body mass refers to the fat-free mass of the body, i.e., total body weight minus body fat weight is lean body mass. Lean body mass can be measured by methods such as hydrostatic weighing, computerized chambers, dual-energy X-ray absorptiometry, skin calipers, magnetic resonance imaging (MRI) and bioelectric impedance analysis (BIA) as known in the art.
  • MRI magnetic resonance imaging
  • BIOA bioelectric impedance analysis
  • mammal refers to warm-blooded animals that generally have fur or hair, that give live birth to their progeny, and that feed their progeny with milk. Mammals include humans; companion animals (e.g., dogs, cats); farm animals (e.g., cows, horses, sheep, pigs, goats); wild animals; and the like.
  • the mammal is a female. In one embodiment, the mammal is a female human. In one embodiment, the mammal is a male. In one embodiment, the mammal is a male human. In one embodiment, the mammal is a cat or dog. In one embodiment, the mammal is a diabetic mammal, e.g., a human having type 2 diabetes. In one embodiment, the mammal is an obese diabetic mammal, e.g., an obese mammal having type 2 diabetes.
  • subject in the context of methods described herein refers to a mammal.
  • fragment in the context of polypeptides refers herein in the customary chemical sense to a portion of a polypeptide.
  • a fragment can result from N-terminal deletion or C-terminal deletion of one or more residues of a parent polypeptide, and/or a fragment can result from internal deletion of one or more residues of a parent polypeptide.
  • “Fragment” in the context of an antibody refers to a portion of an antibody which can be linked to a biologically active molecule to modulate solubility, distribution within a subject, and the like.
  • Exendin-4(l-30) describes a biologically active fragment of Exendin-4 where the exendin C- terminal "tail" of amino acids 31-39 is deleted.
  • polypeptides refers, in the customary sense, to a polypeptide which serves as a reference structure prior to modification, e.g., insertion, deletion, addition and/or substitution.
  • conjugate in the context of engineered polypeptides described herein refers to covalent linkage between component polypeptides, e.g., HD1, HD2, linkers and the like.
  • fusion in the context of engineered polypeptides described herein refers to covalent linkage between component polypeptides, e.g., HD1, HD2 and the like, via either or both terminal amino or carboxy functional group of the peptide backbone.
  • Engineered polypeptides can be synthetically or recombinantly made.
  • fusions are made using recombinant biotechnology, however, can also be made by chemical synthesis and conjugation methods.
  • "Analog” as used herein in the context of polypeptides refers to a compound that has insertions, deletions and/or substitutions of amino acids relative to a parent compound.
  • "Analog sequence” as used herein in the context of polypeptides refers to an amino acid sequence that has insertions, deletions and/or substitutions of amino acids relative to a parent amino acid sequence (e.g., wild-type sequence, native sequence). An analog may have superior stability, solubility, efficacy, half-life, and the like.
  • an analog is a compound having at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even higher, sequence identity to the parent compound.
  • the analog has from 1 to 5 amino acid modifications selected independently from an insertion, deletion, addition and substitution.
  • the exendin analog can have from 1 to 5 amino acid modifications selected independently from any one or combination of an insertion, deletion, addition and substitution, and preferably retains at least 50%>, for example 50%>, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even higher, sequence identity to the parent compound, and even more preferably at least 80%>, 85%, 90%>, 95%, 98%, or even higher, sequence identity to the parent compound, and preferably the parent compound is Exendin-4, Exendin-4(l-38), Exendin-4(l-37). Exendin-4(l-36), Exendin-4(l-35), Exendin-4(l-34).
  • at least amino acids corresponding to positions 1, 4, 6, 7 and 9 of Exendin-4 are those as in native Exendin-4, and further the one to five modifications are conservative amino acid substitutions at positions other than positions 1, 4, 6, 7 and 9 of Exendin-4.
  • an exendin analog retains the amino acid at least as found in position 3, 4, 6, 5, 7, 8, 9, 10, 11, 13, 15, 18, 19, 22, 23, 25, 26, and/or 30 of Exendin-4, and further preferably has no more than 1 to 5 of the remaining positions substituted with another amino acid, most preferably a chemically conservative amino acid.
  • any substitution or modification at positions 1 and/or 2 will retain resistance to DPP-IV cleavage while retaining or improving insulinotropic activity as is known in the art for Exendin-4 analogs, such as desamino-histidyl-Exendin-4.
  • the term “conservative” in the context of amino acid substitutions refers to substitution which maintains properties of charge type (e.g., anionic, cationic, neutral, polar and the like), hydrophobicity or hydrophilicity, bulk (e.g., van der Waals contacts and the like), and/or functionality (e.g., hydroxy, amine, sulfhydryl and the like).
  • charge type e.g., anionic, cationic, neutral, polar and the like
  • hydrophobicity or hydrophilicity e.g., van der Waals contacts and the like
  • functionality e.g., hydroxy, amine, sulfhydryl and the like
  • derivative in the context of a compound disclosed herein refers to a compound afforded by chemical modification, e.g., by the bonding of one or more derivatizing moieties as described herein.
  • identity refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 50% identity, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a sequence comparison algorithms as known in the art, for example BLAST or BLAST 2.0.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized
  • BLAST and BLAST 2.0 are used, as known in the art, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the web site of the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., Id.).
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al., Id.).
  • These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • Cumulative scores are calculated using, e.g., for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always>0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • IC 50 refers in the customary sense to the half maximal inhibitory concentration of a compound inhibiting a biological or biochemical function. Accordingly, in the context of receptor binding studies, IC 50 refers to the concentration of a test compound which competes half of a known ligand from a specified receptor.
  • EC 50 refers in the customary sense to the effective concentration of a compound which induces a response halfway between a baseline response and maximum response, as known in the art.
  • peptide and "polypeptide” in the context of components of the engineered polypeptides described herein are synonymous.
  • a indicates a D-amino acid (e.g., D-Ala).
  • square brackets indicate separate fragments and Crosshatch (“#") indicates linking positions.
  • an engineered polypeptide including a first peptide hormone domain (HD1) which includes an exendin domain sequence, and a second peptide hormone domain (HD2) which includes a humanized chimeric seal leptin sequence.
  • HD1 is covalently bonded to HD2 through a bond, or through a linker LI, as described herein.
  • engineered polypeptide refers, in the customary sense, to polypeptides and derivatives thereof which are not naturally occurring polypeptides, and/or which have been synthesized by chemical or biological processes, or combinations thereof.
  • exendin domain refers to a polypeptide having the sequence of an exendin, an exendin analog, an exendin active fragment, or an exendin analog active fragment, and derivatives thereof, as disclosed herein.
  • exendin domain expressly refers to an exendin, an exendin analog, an exendin active fragment, or an exendin analog active fragment.
  • sequence in the context of a polypeptide refers to the amino acid sequence of the polypeptide.
  • exendin domain sequence refers to the amino acid sequence of an exendin domain
  • humanized chimeric seal leptin sequence refers to the amino acid sequence of a humanized chimeric seal leptin.
  • active fragment refers, in the customary sense, to a fragment of a parent polypeptide, which fragment demonstrates biological activity, e.g., binding, agonism or antagonism, in a biological assay.
  • biologically active compound and the like refer, in the customary sense, to compounds, e.g., polypeptides, non-polypeptides, and the like, which can provide biological activity.
  • humanized chimeric seal leptin refers to polypeptides having significant sequence identity, e.g., 50% to 95% sequence identity, to a seal leptin, a seal leptin analog, an active fragment of a seal leptin, an active fragment of a seal leptin analog, and derivatives thereof, wherein at least one region of contiguous amino acids of a parent seal leptin has been replaced by a corresponding region from human leptin.
  • humanized chimeric seal leptin expressly refers to a seal leptin, a seal leptin analog, a seal leptin active fragment, or a seal leptin analog active fragment, wherein at least one region of contiguous amino acids of a parent seal leptin has been replaced by a corresponding region from human leptin.
  • corresponding region and the like in the context of the comparison of polypeptides refer, in the customary sense, to an alignment of polypeptides, as known in the art. It is understood that the absolute numbering of corresponding amino acids can differ between compared polypeptides.
  • linker refers to a divalent chemical moiety which can covalently bond to both HDl and HD2.
  • peptide linker refers to a divalent peptide which bonds together two chemical entities, e.g., HDl and HD2.
  • the engineered polypeptide has greater binding at a human leptin receptor relative to seal leptin binding.
  • the engineered polypeptide has greater solubility relative to human leptin solubility.
  • linker LI is a bond. In one embodiment, linker LI is a linker as described herein which covalently links HDl and HD2.
  • the engineered polypeptide includes HDl as an N-terminal moiety and HD2 as a C-terminal moiety.
  • N-terminal moiety refers, in the customary sense, to the relative positioning of a region, e.g., HDl, of a polypeptide, e.g., an engineered polypeptide disclosed herein, toward the N-terminal of the polypeptide.
  • C-terminal moiety refers, in the customary sense, to the relative positioning of a region of a polypeptide toward the C-terminal of the polypeptide.
  • the engineered polypeptide has the structure HD1-HD2.
  • the engineered polypeptide has the structure HD 1 -L 1 -HD2.
  • the engineered polypeptide is to be read in the N- terminus to C-terminus orientation.
  • the terms HD1-HD2, HD1-L1-HD2, and the like mean, in the absence of an express indication of the N-terminus and/or the C-terminus, that the HD1 resides at the N-terminus of the engineered polypeptide, and the HD2 resides at the C-terminus.
  • the engineered polypeptide is to be read according to the express indication of the termini.
  • the terms HDlc-term-HD2, HDl-Ll-HD2N-term and the like mean that HD2 resides at the N-terminus of the engineered polypeptide, and HD1 resides at the C-terminus.
  • the HD1 sequence includes an exendin domain sequence, as disclosed herein. In one embodiment, the HD1 sequence consists of an exendin domain sequence, as disclosed herein.
  • Exendins are peptides that are found in the salivary secretions of the Gila monster and the Mexican Bearded Lizard, which are reptiles endogenous to Arizona and Northern Mexico.
  • Exendin-3 is present in the salivary secretions of Heloderma horridum (Mexican Beaded Lizard)
  • Exendin-4 is present in the salivary secretions of Heloderma suspectum (Gila monster). See Eng et al, 1990, J. Biol. Chem., 265:20259-62; Eng et al, 1992, J. Biol. Chem., 267:7402-7405.
  • HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH 2 (SEQ ID NO: 3).
  • Exendin-4 peptide analog has been reported that is a full-length C-terminally amidated Exendin-4 peptide analog with a single nucleotide difference at position 14 compared to native Exendin-4. See e.g., Hargrove et al, 2007, Regulatory Peptides, 141: 113-119.
  • the sequence of [ 14 Leu]Exendin-4 is HGEGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS- NH 2 (SEQ ID NO: l).
  • Exendin-4 peptide analog is a chimera of the first 32 amino acids of Exendin-4 having amino acid substitutions at positions 14 and 28 followed by a 5 amino acid sequence from the C-terminus of a non- mammalian (frog) GLP1 : [ 14 Leu, 28 Gln]Exendin-4(l-32)-fGLP- 1(33-37) with sequence: HGEGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS (SEQ ID NO:4).
  • Exendin- 4 C-terminally truncated, biologically active forms of Exendin- 4, such as Exendin-4(l-28) (SEQ ID NO:5), Exendin-4(l-29) (SEQ ID NO:6), Exendin-4(l-30) (SEQ ID NO:7), Exendin-4(1-31) (SEQ ID NO:8), Exendin-4(l-32) (SEQ ID NO:9) and their amidated forms.
  • Exendin analogs are suitable as exendin domains of the engineered polypeptides disclosed herein.
  • square brackets i.e., "[]" in a peptidic compound name indicate substitution of the residue or chemical feature within the square brackets.
  • [ 14 Leu]Exendin-4, [ 14 Leu]Ex-4, and the like refer to Exendin-4 having leucine at position 14.
  • the numeric position of an amino acid can be indicated by prepended or postpended numbers in a variety of ways routinely employed in the art.
  • the terms 14 Leu, Leul4, 14Leu, Leu 14 and the like are synonymous in referring to a leucine at position 14.
  • a C-terminal amide, or other C-terminal capping moiety can be present in compounds described herein.
  • each polypeptide disclosed herein having a C-terminal is understood to represent both C-terminal acid and C-terminal amide forms of the polypeptide, unless such C-terminal capping would prevent formation of an engineered polypeptide disclosed herein. Accordingly, HD1 would not be C-terminally capped if HD2 or a linker to HD2 is attached at the C-terminal of HD1.
  • the exendins have some sequence similarity to several members of the glucagon-like peptide (GLP-1) family, the highest homology (53%) being to GLP-1(7-36)NH 2 . See e.g., Goke et al, 1993, J. Biol. Chem., 268: 19650-55.
  • the sequence of GLP-1 (7-37)NH 2 (also sometimes referred to as "GLP-1") is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 10).
  • This peptide has an insulinotropic effect stimulating insulin secretion from pancreatic beta-cells. It is understood, however, that exendins are not GLP-1 homo logs. For example,
  • Exendin-4 can act at GLP-1 receptors in vitro on certain insulin-secreting cells, however, it has also been reported that Exendin-4 may act at receptors not acted upon by GLP-1. Moreover, Exendin-4 shares some but not all biological properties in vivo with GLP-1, and it has a significantly longer duration of action than GLP-1. Based on their insulinotropic activities, the use of Exendin-3 and Exendin-4 for the treatment of diabetes mellitus and the prevention of hyperglycemia has been proposed (Eng, U.S. Pat. No.
  • Exendin-4 has been approved in the United States and in Europe for use as a therapeutic for treating type 2 diabetes.
  • exendins are not the species homo log of mammalian GLP-1 as was reported by Chen and Drucker who cloned the exendin gene from the Gila monster. See e.g., J. Biol. Chem. 1997, 272:4108-15.
  • Novel exendin agonist compound sequences useful in the engineered polypeptides described herein are described in WO 99/07404 (i.e., PCT/US98/16387 filed Aug. 6, 1998), in WO 99/25727 (i.e., PCT/US98/24210, filed Nov. 13, 1998), in WO 99/25728 (i.e.,
  • exendins include exendin fragments: Exendin-4(l-28) (SEQ ID NO:5), Exendin-4(l-29) (SEQ ID NO:6),
  • Exendin-4(l-30) SEQ ID NO:7), Exendin-4(1-31) (SEQ ID NO:8)and Exendin-4(l-32) (SEQ ID NO:9).
  • Analogs thereof include substitution at the 14 Met position (i.e., 14 Met) with a non- oxidizing amino acid, e.g., leucine. Examples include [ 14 Leu]Exendin-4(l-28) (SEQ ID NO: l 1), [ 14 Leu]Exendin-4(l-29) (SEQ ID NO: 12), [ 14 Leu]Exendin-4(l-30) (SEQ ID NO: 13),
  • Exendin analog agonists for use as exendin domains in the engineered polypeptides described herein include those described in US Patent No. 7,223,725 (incorporated herein by reference and for all purposes), such as compounds of the formula: Xaai Xaa 2 Xaa 3 Gly Xaas Xaa 6 Xaa 7 Xaa 8 Xaa 9 Xaaio Xaan Xaai 2 Xaai 3 Xaai 4 Xaais Xaai 6 Xaa ⁇ Ala Xaaig Xaa 20 Xaa 2 i Xaa 22 Xaa 23 Xaa 24 Xaa 2 5 Xaa 26 Xaa 27 Xaa 2 g-Zi; wherein Xaai is His, Arg or Tyr; Xaa 2 is Ser, Gly, Ala or Thr; Xaa 3 is Ala, Asp or Glu
  • Zi is absent, Gly, Gly-Gly, Gly-Gly-Xaa 3 i, Gly-Gly-Xaa 3 i-Ser, Gly-Gly-Xaa 3 i-Ser-Ser (SEQ ID NO:391), Gly-Gly-Xaa 3 Ser-Ser-Gly (SEQ ID NO:392), Gly-Gly-Xaa 3 Ser-Ser-Gly-Ala (SEQ ID NO:393), Gly-Gly-Xaa 3 i-Ser-Ser-Gly-Ala-Xaa 36 (SEQ ID NO:394),
  • each exendin analog agonist can be a C-terminal acid or C-terminal amine.
  • any and each of the exendin analogs described above also specifically contemplated are those wherein a replacement for the histidine corresponding to Xaai is made with any of D-histidine, desamino-histidine, 2- amino-histidine, beta-hydroxy-histidine, homohistidine.
  • exendin analogs described herein wherein a replacement for the glycine at Xaa2 is made with any of D-Ala, Val, Leu, Lys, Aib, (1 -amino cyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, l-aminocyclopentyl)carboxylic acid,
  • exemplary compounds include those of the above formula wherein: Xaai is His or Arg; Xaa 2 is Gly or Ala; Xaa 3 is Asp or Glu; Xaas is Ala or Thr; Xaa 6 is Ala or Phe; Xaa 7 is Thr or Ser; Xaa 8 is Ala, Ser or Thr; Xaa 9 is Asp or Glu; Xaaio is Ala, or Leu; Xaan is Ala or Ser; Xaai 2 is Ala or Lys; Xaai 3 is Ala or Gin; Xaai 4 is Ala or Leu; Xaais is Ala or Glu; Xaaa
  • each exendin analog agonist can be a C-terminal acid or C-terminal amine.
  • exendin analogs described above also specifically contemplated are those wherein a replacement for the histidine corresponding to position Xaal is made with any of D-histidine, desamino-histidine, 2-amino-histidine, beta-hydroxy-histidine,
  • exemplary compounds include those set forth in WO 99/25727 identified therein as compounds 2-23.
  • Xaai 4 is Leu, He, or Val, more preferably Leu, and/or Xaa 25 is Trp, Phe or Tyr, more preferably Trp or Phe. It is believed that these compounds will be less susceptive to oxidative degradation, both in vitro and in vivo, as well as during synthesis of the compound.
  • exendin analogs suitable as exendin domains for use in the present engineered polypeptides include those described in United States Patent 6528486 published March 4, 2003 (incorporated herein by reference and for all purposes).
  • exendin analogs include those consisting of an exendin or exendin analog having at least 90% homology to Exendin-4 having optionally between one and five deletions at positions 34-39, and a C-terminal extension of a peptide sequence of 4-20 amino acid units covalently bound to said exendin wherein each amino acid unit in said peptide extension sequence is selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, and Met.
  • the extension is a peptide sequence of 4-20 amino acid residues, e.g., in the range of 4-15, more preferably in the range of 4-10 in particular in the range of 4-7 amino acid residues, e.g., of 4, 5, 6, 7, 8 or 10 amino acid residues, where 6 amino acid residues are preferred.
  • the extension peptide contains at least one Lys residue, and is even more preferably from 3 to 7 lysines and even most preferably 6 lysines.
  • Exemplary exendin analogs useful as exendin domains include:
  • HGEGTFTSDLSKQMEEEAVRLFIEWLWLKNGGPSSGAS H-[des 36 Pro, 37 ' 38 Pro]Exendin- 4(l-39)-NH 2 ) (SEQ ID NO: 18);
  • repetition of an amino acid can be indicated by a subscripted number setting forth the number of repetitions; i.e., Lys 6 , (Lys) 6 and the like refer to hexalysyl (SEQ ID NO:24).
  • a subscripted number can also indicate the position of a residue within a sequence; e.g., "AAi AA 2 AA 3 " refers to amino acids 1-3 of a polypeptide sequence.
  • any and each of the exendin analogs described above specifically contemplated are those wherein a replacement for the histidine corresponding to position 1 is made with any of D-histidine, desamino-histidine, 2-amino-histidine, beta- hydroxy-histidine, homohistidine.
  • N-alpha-acetyl-histidine alpha-fluoromethyl-histidine, alpha- methyl-histidine, 3-pyridylalanine, 2-pyridylalanine, 4-pyridylalanine, 4-imidazoacetyl, des- amino-histidyl (or imidazopropionyl), beta-hydroxy-imidazopropionyl, N-dimethyl-histidyl or beta-carboxy-imidazopropionyl.
  • exendin analogs described herein wherein a replacement for the glycine at position 2 is made with any of D-Ala, Val, Leu, Lys, Aib, (l-aminocyclopropyl)carboxylic acid, (l-aminocyclobutyl)carboxylic acid, l-aminocyclopentyl)carboxylic acid, (1 -amino cyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid.
  • exendin analogs suitable as exendin domains for use in the engineered polypeptide constructs are those described in published PCT application WO2004035623 (incorporated herein by reference and for all purposes), particularly those comprised of naturally-occurring amino acids, which describes exendin analogs having at least
  • Additional such analogs further include a 1 -7 amino acid C-terminal extension that includes at least one Lys amino acid and more preferably at least five Lys amino acid units such as six or seven Lys amino acid units.
  • exendin analogs suitable as exendin domains for use in the engineered polypeptide constructs are those described in published PCT application
  • WO/2010/120476 (incorporated herein by reference and for all purposes), which describes exendin analogs having modified amino acid residues in the N-terminal portion of an exendin or exendin analog to create a high beta-turn characteristic in that region.
  • analogs are designed to mimic amino acid residues Hisi Gly 2 Glu 3 by creating a conformationally constrained region, include exendin analogs containing a thiazolidine-proline peptide mimetic at Hisi Gly 2 Glu 3 , which can be used as a modification in Exendin-4 or other analogs described herein.
  • exendins in any and each of the exendins, exendin analogs and formulas described herein, specifically contemplated are those wherein a replacement for the histidine corresponding to position 1 is made with any of L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, beta-hydroxy-histidine, homohistidine.
  • preferred exendin analogs for use in engineered polypeptide conjugates as described herein wherein the Hisi position is modified are
  • exendins or exendin analogs described herein wherein a replacement for the glycine at position 2 is made with any of D-Ala, Val, Leu, Lys, Aib, (l-aminocyclopropyl)carboxylic acid, (1- aminocyclobutyl)carboxylic acid, l-aminocyclopentyl)carboxylic acid, (1 -amino
  • cyclohexyl)carboxylic acid (l-aminocycloheptyl)carboxylic acid, or (1-aminocyclooctyl) carboxylic acid.
  • an exendin domain can have at least 65%, for example 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%o or even higher, sequence identity relative to a parent exendin sequence.
  • the parent exendin is Exendin-4
  • the exendin analog may have at least 65%, for example 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even higher, sequence identity relative to Exendin-4.
  • HD1 includes an exendin domain sequence having at least 65% identity with an Exendin-4 sequence (SEQ ID NO:3).
  • HD1 consists of an exendin domain sequence having at least 65% identity with an Exendin-4 sequence (SEQ ID NO:3).
  • the exendin domain sequence is an Exendin-4 sequence (SEQ ID NO:3).
  • the exendin domain sequence is a [ 14 Leu] Exendin-4 sequence (SEQ ID NO: l).
  • the exendin domain sequence has at least 90% identity with an Exendin-4(l-32) sequence (SEQ ID NO: 15).
  • the exendin domain sequence is a [ 14 Leu]Exendin-4(l-32) sequence (SEQ ID NO: 15).
  • the exendin domain sequence is the sequence of Exendin-4(l-28) (SEQ ID NO:5), Exendin-4(l-29) (SEQ ID NO:6), Exendin-4(l-30) (SEQ ID NO:7), Exendin- 4(1-31) (SEQ ID NO:8) or Exendin-4(l-32) (SEQ ID NO:9).
  • the exendin domain sequence includes a sequence following: Exendin-4 (SEQ ID NO:3), [ 14 Leu]Exendin-4 (SEQ ID NO: l), [ 14 Leu, 28 Gln]Exendin-4(l-32)- fGLP-l(33-37) (SEQ ID NO:4), [ 28 Gln]Exendin-4(l-32) (SEQ ID NO:25),
  • the exendin domain sequence has at least 70% identity with an Exendin-4 sequence (SEQ ID NO:3) or to a sequence selected from the group consisting of any of sequences Exendin-4 (SEQ ID NO:3), [ 14 Leu] Exendin-4 (SEQ ID NO: l), [ 14 Leu,
  • the exendin domain sequence includes a sequence following: HGEGTFTSDLSKQMEEEAVRLFIEWLK GGPSSGAPPPS (SEQ ID NO:3),
  • HGEGTFTSDLSKQLEEEAVRLFIEWLK GGPSSGAPPPS (SEQ ID NO: l)
  • HGEGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS (SEQ ID NO:4)
  • HGEGTFTSDLSKQMEEEAVRLFIEWLKQGGPSKEIIS (SEQ ID NO:32),
  • HGEGTFTSDLSKQLEEEAVRLFIEWLK GGPSSGAPPS (SEQ ID NO:33),
  • HGEGTFTSDLSKQMEEEAVRLFIEWLK GGPSSGAPPS (SEQ ID NO:34),
  • HGEGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIISKKKKK (SEQ ID NO:35)
  • HGEGTFTSDLSKQMEEEAVRLFIEWLK GGPSSGAPPPSKKKKKKK (SEQ ID NO:36), HGEGTFTSDLSKQMEEEAVRLFIEWLKQGGPSKEIISKKKKK (SEQ ID NO:37), HGEGTFTSDLSKQLEEEAVRLFIEWLK GGPSSGAPPSKKKKK (SEQ ID NO:38), HGEGTFTSDLSKQMEEEAVRLFIEWLK GGPSSGAPPSKKKKK (SEQ ID NO:39).
  • the exendin domain sequence consists of a sequence selected from (SEQ ID NO:3), (SEQ ID NO: l), (SEQ ID NO:4), and SEQ ID NOS:32-39.
  • the exendin domain sequence has at least 70% identity with a sequence selected from (SEQ ID NO:3), (SEQ ID NO: l), (SEQ ID NO:4), and SEQ ID NOS:32- 39.
  • the exendin domain sequence includes from 1 to 5 amino acid modifications relative to an Exendin-4 sequence (SEQ ID NO: 3), which 1 to 5 amino acid modifications are each independently selected from an insertion, deletion, addition or substitution. [0061] In one embodiment, the exendin domain sequence includes from 1 to 5 amino acid modifications relative to an Exendin-4(l-32) sequence (SEQ ID NO:9), which 1 to 5 amino acid modifications are each independently selected from an insertion, deletion, addition or substitution.
  • the exendin domain sequence includes from 1 to 5 amino acid modifications relative to an Exendin-4(l-28) sequence (SEQ ID NO:5), which 1 to 5 amino acid modifications are each independently selected from an insertion, deletion, addition or substitution.
  • Leptins refer to leptins, leptin active fragments, leptin analogs, and leptin derivatives; and a leptin, a leptin active fragment, a leptin analog, and a leptin derivative; respectfully.
  • Exemplary leptins include those which elicit one or more biological responses known in the art to be elicited when leptins are administered to subjects. See, e.g., published U.S. Patent application Nos. US 2007/0020284 and US 2008/0207512, U.S. Patent Nos. 6,309,853, and US 7,183,254, and PCT Published
  • Exemplary leptins include the compounds described in U.S. Patent Nos. US 5,594,101, US 5,851,995, US 5,691,309, US 5,580,954, US 5,554,727, US 5,552,523, US 5,559,208, US 5,756,461, US 6,309,853, published U.S. Patent application No. US 2007/0020284, and PCT Published Application Nos. WO 96/23517, WO 96/005309, WO 98/28427, WO 2004/039832, WO 98/55139, WO 98/12224, and WO 97/02004, each of which is incorporated herein in its entirety and for all purposes.
  • Leptin activity includes leptin binding activity and leptin functional activity.
  • leptin analog compounds can have an IC 50 of about 200 nM or less, about 100 nM or less, or about 50 nM or less, or about 5 nM or less, or about 1 nM or less, in a leptin binding assay, such as that described herein.
  • Leptin analog compounds can have an EC 50 of about 20 nM or less, about 10 nM or less, about 5 nM or less, about 1 nM or less, or about 0.1 nM or less, in a leptin functional assay, such as that described herein.
  • a leptin functional assay such as that described herein.
  • chimeric polypeptides are based on a wild type seal leptin polypeptide wherein at least one contiguous region of 1-30 amino acids of a wild type seal leptin sequence has been replaced with a contiguous region of 1-30 amino acids of a mature human leptin sequence.
  • a wild type seal leptin sequence includes the sequence of wild type seal leptin (SEQ ID NO:40) and the sequence of wild type seal leptin with an N-terminal methionine (SEQ ID NO:42). Accordingly, it is understood that SEQ ID NO:40 and SEQ ID NO:42 are seal leptins, SEQ ID NO:43 and SEQ ID NO:44 are human leptins, and the like.
  • a mature human leptin sequence useful for chimerizing wild type seal leptin as provided herein, includes the following sequences: mature human leptins (SEQ ID NO:45), mature human leptins with N-terminal methionine (SEQ ID NO:46), mature human leptin form 1 (SEQ ID NO:43), mature human leptin form 2 (SEQ ID NO:47), mature human leptin form 3 (SEQ ID NO:48), mature human leptin form 4 (SEQ ID NO:49), mature human leptin form 1 with N-terminal methionine (Metreleptin, or A100, SEQ ID NO:44), mature human leptin form 2 with N-terminal methionine (SEQ ID NO:50), mature human leptin form 3 with N-terminal methionine (SEQ ID NO:51), mature human leptin form 4 with N-terminal methionine (SEQ ID NO:52), A200 (SEQ ID NO:45
  • a series of chimeric polypeptides are described wherein at least one contiguous region of 1-30 amino acids of a wild type seal leptin sequence (SEQ ID NO:40 or SEQ ID NO:42) has been replaced with a contiguous region of 1-30 amino acids of A100 (SEQ ID NO:44).
  • the engineered polypeptide includes a humanized chimeric seal leptin sequence having the sequence of SEQ ID NO:40, wherein 5% to 55% of SEQ ID NO:40 is substituted with a corresponding human leptin sequence.
  • the engineered polypeptide includes a humanized chimeric seal leptin sequence having the sequence of SEQ ID NO:41, wherein 5% to 55% of SEQ ID NO:41 is substituted with a corresponding human leptin sequence.
  • a contiguous region e.g., 1-30, 2-30, 3- 30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, of amino acids can include any naturally or non- naturally occurring amino acid. Any combination of amino acids can be employed without restriction. That is, two or more amino acids in a contiguous region can be replaced with a naturally occurring amino acid, a non-naturally occurring amino acid, a conservative
  • substitution refers, as customary in the art, to an amino acid substitution which retains charge type and/or size, as known in the art.
  • non-conservative substitution refers, as customary in the art, to an amino acid substitution which changes charge type and/or size, as known in the art.
  • chimeric polypeptides described herein have demonstrated biological activity, in addition to enhanced physical properties.
  • a humanized chimeric seal leptin can have leptin activity in vitro and in vivo.
  • a humanized chimeric seal leptins can also demonstrate enhanced stability and solubility compared to the mature human leptin polypeptides which are used to derive the sequence.
  • leptins include the following: [0073] Unprocessed Full-length Human Leptin (i.e., includes 21-residue N-terminal signal sequence):
  • Leptin A100 Variants Variants of Leptin A100 with the following residue substitutions follow:
  • Le tin A200 is an Fc antibody fragment condensation product with leptin, as known in the art. See e.g., Lo et al., 2005, Protein Eng. Design & Selection, 18: 1-10.
  • the amino acid sequence of A200 is as follows:
  • Leptin A300 Leptin A300 is metreleptin with substitutions Wl 01 Q and Wl 39Q (N-terminal ⁇ et counted as residue 1): MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAV YQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYS TEVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO:54).
  • Leptin A400 is metreleptin with the serine residue at position 78 replaced with a cysteine residue, as set forth following:
  • Leptin A500 Research by a number of investigators including the inventors has focused on the effects on aggregation of residue substitution in leptin. See e.g., Ricci et al, 2006.
  • the HD2 sequence of the engineered polypeptide includes an analog of the humanized chimeric seal leptin sequence or an active fragment of an analog of the humanized chimeric seal leptin sequence.
  • Seal leptin A sequence of seal leptin follows:
  • SEQ ID NO:40 is leptin from
  • Exemplary polypeptides incorporating sequence regions from seal leptin and human leptins, and analogs thereof, include the following.
  • Helix 1 region of a mature human leptin polypeptide spans a contiguous region of 20 amino acids.
  • Helix 1 and Helix 3 are antiparallel helices that form part of Binding Site II of leptin to its receptor. This site interacts with the cytokine receptor homology domain (CRH) of the leptin receptor and is thought to be a major receptor binding site, but not involved in receptor activation.
  • CSH cytokine receptor homology domain
  • references herein to helices and other secondary structural features refer to regions within an X-ray crystallographic model of human leptin and the corresponding regions within seal leptins, as known in the art.
  • Helix 1 of seal leptin corresponds to Helix 1 of human leptin
  • Helix 2 of seal leptin corresponds to Helix 2 of human leptin
  • Helix 3 of seal leptin corresponds to Helix 3 of human leptin
  • Helix 4 of seal leptin corresponds to Helix 4 of human leptin
  • AB Loop of seal leptin corresponds to AB Loop of human leptin
  • Loop 3-4 of seal leptin corresponds to Loop 3-4 of human leptin.
  • human Helix 1 encompasses residues 5-25; human Helix 2 encompasses residues 52-67; human Helix 3 encompasses residues 73-94; human Helix 4 encompasses residues 122-143; the human AB Loop encompasses residues 25-51; and the human Loop 3-4 encompasses residues 95-121.
  • the present disclosure relates to chimeric polypeptides that are based on wild type seal leptin with an incorporated Helix 1 sequence from mature human leptin.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 3-22 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 5-24 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence set forth in SEQ ID NO:72 following.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 3-22 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 5-24 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO:73 following.
  • Helix 2 region of a mature human leptin polypeptide spans a region of 16 contiguous amino acids. This helix is buried in the 4-helix bundle as described in the crystallographic literature. See e.g., Zhang et al, 1997, Nature 387:206.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 50-65 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 52-67 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO: 74 following.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 50-65 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 52-67 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO:75.
  • Helix 3 region of a mature human leptin polypeptide spans a contiguous region of 22 amino acids.
  • Helix 3 and Helix 1 are antiparallel helices that form part of Binding Site II of leptin to its receptor. This site interacts with the cytokine receptor homology domain (CRH) of the leptin receptor and is thought to be a major receptor binding site, but not involved in receptor activation.
  • CSH cytokine receptor homology domain
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO:76 following.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO:77 following.
  • Helix 4 region of a mature human leptin polypeptide spans a contiguous region of 22 amino acids. Helix 4 is thought to form parts of Binding Site I and Binding Site III of leptin, both of which are important for receptor activation. See, for example, Peelman et al., 2004, Id.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 120-141 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 122-143 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO:78 following.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 120-141 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 122-143 of A 100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO:79 following.
  • the AB Loop region of a mature human leptin polypeptide spans a contiguous region of 27 amino acids.
  • the AB Loop is thought to form part of Binding Site III as well as a small portion of Binding Site I of leptin. See, for example, Peelman et al., 2004, J. Biol. Chem. 279: 41038. This region also contains the absolutely conserved motif GLDFIP.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO: 80 following.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO:81 following.
  • Loop 3-4 region of a mature human leptin polypeptide spans a contiguous region of 27 amino acids. Loop 3-4 is thought to contain a part of Binding Site III of leptin to its receptor. See, for example, Peelman et al, 2004, Id.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO: 82 following.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:44).
  • a chimeric polypeptide includes the sequence described in SEQ ID NO: 83 following.
  • a chimeric combination polypeptide is included in the humanized chimeric seal leptin.
  • the term "chimeric combination polypeptide" refers to a chimeric peptide wherein two or more contiguous regions of 1-30 amino acids of a wild type seal leptin sequence (for example, SEQ ID NO:40 or SEQ ID NO:42) have been replaced at each region with a corresponding contiguous region of 1-30 amino acids of a mature human leptin sequence.
  • Chimeric combination polypeptides can be engineered to demonstrate enhanced physical properties compared to the mature human leptin polypeptides which are used to derive the sequences, while retaining the biological activity of human leptin.
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 3-22 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 5-24 of A100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID NO:44).
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO: 84 following.
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 3-22 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 5-24 of A 100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 73- 94 of A100 (SEQ ID NO:44
  • the present disclosure relates to chimeric combination polypeptides that are based on wild type seal leptin with an incorporated helix 3 sequence and an incorporated AB Loop sequence from mature human leptin.
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 23- 49 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID NO:44).
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO: 86 following.
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A 100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 25- 51 of A100 (SEQ ID NO:44).
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO: 87 following.
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 93- 119 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:4).
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO:88 following.
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A 100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:44).
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO: 89 following.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 120- 141 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 122-143 of A100 (SEQ ID NO:44).
  • SEQ ID NO:40 wild type seal leptin polypeptide
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO: 90 following. [0158] Seal leptin with amino acids 23-49 replaced with amino acids 25-51 (AB loop) of metreleptin, and with amino acids 120-141 replaced with amino acids 122-143 (helix 4) of metreleptin, respectively:
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A 100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 120-141 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 122-143 of A100 (SEQ ID NO:44).
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO:91 following.
  • the present disclosure relates to chimeric combination polypeptides that are based on wild type seal leptin with an incorporated AB Loop sequence and an incorporated Loop 3-4 sequence from mature human leptin.
  • a chimeric polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 93- 119 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:44).
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO: 92 following.
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:44).
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO:93 following.
  • the present disclosure relates to chimeric combination polypeptides that are based on wild type seal leptin with an incorporated AB Loop sequence, an incorporated Loop 3-4 sequence, and an incorporated helix 3 sequence from mature human leptin.
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide (SEQ ID NO:40), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A100 (SEQ ID NO:44), the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 71-92 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID NO:44).
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO:94 following.
  • a chimeric combination polypeptide includes the amino acid sequence of a wild type seal leptin polypeptide with an N-terminal methionine (SEQ ID NO:42), wherein the contiguous region spanning the amino acids at positions 23-49 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 25-51 of A 100 (SEQ ID NO:44), the contiguous region spanning the amino acids at positions 93-119 of SEQ ID NO:42 has been replaced with a contiguous region spanning the amino acids at positions 95-121 of A100 (SEQ ID NO:44), and the contiguous region spanning the amino acids at positions 71- 92 of SEQ ID NO:40 has been replaced with a contiguous region spanning the amino acids at positions 73-94 of A100 (SEQ ID NO:44).
  • SEQ ID NO:42 N-terminal methionine
  • a chimeric combination polypeptide includes the sequence described in SEQ ID NO: 95 following.
  • the chimeric polypeptides provided by the invention contain a Cys to Ser amino acid substitution at position 30 of the wild type seal polypeptide sequence.
  • chimeric polypeptides or chimeric combination polypeptides are provided:
  • each of the polypeptides disclosed herein are also contemplated to include a methionine at the N-terminus in frame with the naturally -occurring first amino acid thereof, e.g., Met-Exendin-4, which is Exendin-4 with an added N-terminal methionine. It is further understood that where a C-terminal Gly appears in a engineered polypeptide sequence set forth herein, the residue may be lost during subsequent amidation.
  • Some embodiments are intermediates in synthesis, for example, such as those having a "His tag" which is used for affinity purification as is known in the art, and that can optionally be subsequently removed to yield a mature engineered polypeptide suitable for therapeutic use.
  • HD2 has at least 50% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:40, SEQ ID NO:76, SEQ ID NO: 100, SEQ ID NO:42, SEQ ID NO:77, SEQ ID NO: 101, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:40, SEQ ID
  • HD2 has at least 90% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:40, SEQ ID NO:76, SEQ ID NO: 100, SEQ ID NO:42, SEQ ID NO:77, SEQ ID NO: 101, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:40, SEQ ID NO:76, SEQ ID
  • HD2 of the engineered polypeptide has the amino acid sequence of SEQ ID NO:77.
  • HD2 has an amino acid sequence selected from the group consisting of: SEQ ID NO:40, SEQ ID NO:76, SEQ ID NO: 100, SEQ ID NO:42, SEQ ID NO:77, SEQ ID NO: 101, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 102, SEQ ID NO:40,
  • an engineered polypeptide wherein the HD2 sequence includes a seal leptin sequence (SEQ ID NO:40); wherein at least one contiguous region of from 8 to 30 amino acids of the seal leptin sequence has been replaced with at least one contiguous region of from 8 to 30 amino acids of a human leptin sequence (SEQ ID NO:44); wherein the seal leptin sequence comprises a seal leptin Helix 1 sequence, a seal leptin Helix 2 sequence, a seal leptin Helix 3 sequence, a seal leptin Helix 4 sequence, a seal leptin AB Loop sequence, and a seal leptin Loop 3-4 sequence; and wherein the human leptin sequence comprises a human leptin Helix 1 sequence, a human leptin Helix 2 sequence, a human leptin Helix 3 sequence, a human leptin Helix 4 sequence, a human leptin AB Loop sequence, and
  • At least one contiguous region of from 8 to 30 amino acids of the seal leptin sequence corresponds to the at least one contiguous region of from 8 to 30 amino acids of the human leptin sequence.
  • the engineered polypeptide further includes from 1 to 5 amino acid modifications not encompassed within the at least one contiguous region of 8 to 30 amino acids of the seal leptin sequence, the 1 to 5 amino acid modifications each independently selected from an insertion, deletion, addition or substitution.
  • At least one of the 1 to 5 amino acid modification not encompassed within the at least one contiguous region of from 8 to 30 amino acids of the seal leptin sequence is substitution of cysteine at a position corresponding to position 30 of the seal leptin sequence.
  • the seal leptin Helix 1 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Helix 1 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Helix 1 sequence is replaced by the human leptin Helix 1 sequence.
  • the seal leptin Helix 2 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Helix 2 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Helix 2 sequence is replaced by the human leptin Helix 2 sequence.
  • the seal leptin Helix 3 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Helix 3 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Helix 3 sequence is replaced by the human leptin Helix 3 sequence.
  • the seal leptin Helix 4 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Helix 4 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Helix 4 sequence is replaced by the human leptin Helix 4 sequence.
  • the seal leptin AB Loop sequence includes the at least one contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin AB Loop sequence includes the at least one contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • the seal leptin AB Loop sequence is replaced by the human leptin AB Loop sequence.
  • the seal leptin Loop 3-4 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Loop 3-4 sequence includes the at least one contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Loop 3-4 sequence is replaced by the human leptin Loop 3-4 sequence.
  • the engineered polypeptide includes two contiguous regions of from 8 to 30 amino acids of the seal leptin sequence which have replaced with two contiguous regions of from 8 to 30 amino acids of a human leptin sequence (SEQ ID NO:44).
  • a first contiguous region of from 8 to 30 amino acids of the seal leptin sequence has been replaced with a first contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • a second contiguous region of from 8 to 30 amino acids of the seal leptin sequence has been replaced with a second contiguous region of from 8 to 30 amino acids of the human leptin sequence.
  • the seal leptin Helix 1 sequence includes the first contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Helix 1 sequence includes the first contiguous region of 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Helix 3 sequence includes the second contiguous region of from 8 to 30 amino acids of said seal leptin sequence
  • the human leptin Helix 3 sequence includes the second contiguous region of from 8 to 30 amino acids of said human leptin sequence.
  • the seal leptin Helix 3 sequence includes the first contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Helix 3 sequence includes the first contiguous region of 8 to 30 amino acids of the human leptin sequence
  • the seal leptin AB Loop sequence includes the second contiguous region of from 8 to 30 amino acids of said seal leptin sequence
  • the human leptin AB Loop sequence includes the second contiguous region of from 8 to 30 amino acids of said human leptin sequence.
  • the seal leptin Helix 3 sequence includes the first contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Helix 3 sequence includes the first contiguous region of 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Loop 3-4 sequence includes the second contiguous region of from 8 to 30 amino acids of said seal leptin sequence
  • the human leptin Loop 3-4 sequence includes the second contiguous region of from 8 to 30 amino acids of said human leptin sequence.
  • the seal leptin Helix 4 sequence includes the first contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Helix 4 sequence includes the first contiguous region of 8 to 30 amino acids of the human leptin sequence
  • the seal leptin AB Loop sequence includes the second contiguous region of from 8 to 30 amino acids of said seal leptin sequence
  • the human leptin AB Loop sequence includes the second contiguous region of from 8 to 30 amino acids of said human leptin sequence.
  • the seal leptin AB Loop sequence includes the first contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin AB Loop sequence includes the first contiguous region of 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Loop 3-4 sequence includes the second contiguous region of from 8 to 30 amino acids of said seal leptin sequence
  • the human leptin Loop 3-4 sequence includes the second contiguous region of from 8 to 30 amino acids of said human leptin sequence.
  • a first contiguous region of from 8 to 30 amino acids of the seal leptin sequence has been replaced with a first contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • a second contiguous region of from 8 to 30 amino acids of the seal leptin sequence has been replaced with a second contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • a third contiguous region of from 8 to 30 amino acids of the seal leptin sequence has been replaced with a third contiguous region of from 8 to 30 amino acids of the human leptin sequence.
  • the seal leptin AB Loop sequence includes the first contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin AB Loop sequence includes the first contiguous region of 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Loop 3-4 sequence includes the second contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Loop 3-4 sequence includes the second contiguous region of from 8 to 30 amino acids of the human leptin sequence
  • the seal leptin Helix 3 sequence includes the third contiguous region of from 8 to 30 amino acids of the seal leptin sequence
  • the human leptin Helix 3 sequence includes the third contiguous region of 8 to 30 amino acids of the human leptin sequence.
  • linkers In some embodiments, engineered polypeptides are provided having a linker LI, as described herein, covalently linking a polypeptide hormone domains HDl and HD2. In some embodiments, a first linker (LI) covalently links HDl within the engineered polypeptide. In some embodiments, LI is a bond. In some embodiments, the polypeptide hormone domain (e.g., HDl as described herein) can be covalently linked to the HD2 peptide via a peptide linker. Any linker is optional; i.e., any linker may simply be a bond. In one embodiment the linker includes from 1 to 30 amino acids linked by peptide bonds.
  • the amino acids can be selected from the 20 naturally occurring (i.e., physiological) amino acids.
  • non-natural amino acids can be incorporated either by chemical synthesis, post-translational chemical modification or by in vivo incorporation by recombinant expression in a host cell. Some of these amino acids may be glycosylated.
  • the 1 to 30 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine, and further from aspartate and glutamate.
  • the linker is made up of a majority of amino acids that are sterically unhindered, such as glycine, alanine and/or serine.
  • “Sterically unhindered” refers, in the customary sense, to a amino acid having a small side chain, e.g., 0-2 non-hydrogen atoms, such that steric hinderance is minimized relative to amino acids having larger side chains, e.g., Leu, Trp, Tyr, Phe, and the like.
  • Polyglycines are particularly useful, e.g. (Gly) 3 , (Gly) 4 , (Gly) 5 , as are polyalanines, poly(Gly-Ala) and poly(Gly-Ser).
  • Charged polyglycines can be useful, and include e.g., poly (Glytrap -Glu) (SEQ ID NO: l 10), poly(Glytraining-Lys), (SEQ ID NO: l 1 1), poly(Glywoven - Asp) (SEQ ID NO: 112), and poly(Gly n -Arg) (SEQ ID NO: 113) motifs (where n can be 1 to 6).
  • linkers include (Gly) 3 Lys(Gly) 4 (SEQ ID NO: l 14);
  • GlyProAsnGlyGly (SEQ ID NO: 117). Combinations of Gly and Ala are particularly useful as are combination of Gly and Ser.
  • the peptide linker is selected from the group of a glycine rich peptide, e.g., Gly-Gly-Gly; the sequences [Gly-Ser] n (SEQ ID NO: 118), [Gly- Gly- Ser] n (SEQ ID NO: 119), [Gly-Gly-Gly- Ser] n (SEQ ID NO: 120) and [Gly- Gly-Gly-Gly-Gly-Ser] n (SEQ ID NO: 121), where n is 1, 2, 3, 4, 5 or 6, for example [Gly-Gly-Gly- Gly Ser] 3 .
  • Glycine rich peptide refers to a polypeptide which includes a plurality of glycine residues, preferably a majority of glycine residues, more preferably a pre
  • charged linkers may be used.
  • Such charges linkers may be contain a significant number of acidic residues (e.g., Asp, Glu, and the like), or may contain a significant number of basic residues (e.g., Lys, Arg, and the like), such that the linker has a pi lower than 7 or greater than 7, respectively.
  • acidic residues e.g., Asp, Glu, and the like
  • basic residues e.g., Lys, Arg, and the like
  • Such linkers may impart advantageous properties to the engineered polypeptides disclosed herein, such as modifying the peptides pi (isoelectric point) which can in turn improve solubility and/or stability characteristics of such polypeptides at a particular pH, such as at physiological pH (e.g., between pH 7.2 and pH 7.6, inclusive), or in a pharmaceutical composition including such polypeptides.
  • solubility for a peptide can be improved by formulation in a composition having a pH that is at least or more than plus or minus one pH unit from the pi of the peptide.
  • an “acidic linker” is a linker that has a pi of less than 7; between 6 and 7, inclusive; between 5 and 6, inclusive; between 4 and 5, inclusive; between 3 and 4, inclusive; between 2 and 3, inclusive; or between 1 and 2, inclusive.
  • a “basic linker” is a linker that has a pi of greater than 7; between 7 and 8, inclusive; between 8 and 9, inclusive; between 9 and 10, inclusive; between 10 and 11, inclusive; between 11 and 12 inclusive, or between 12 and 13, inclusive.
  • an acidic linker contains a sequence selected from the group of [Gly-Glu] n (SEQ ID NO: 122); [Gly-Gly-Glu] n (SEQ ID NO: 123); [Gly-Gly-Gly-Glu] n (SEQ ID NO: 124); [Gly-Gly-Gly-Gly-Glu] n (SEQ ID NO: 125), [Gly-Asp] n (SEQ ID NO: 126); [Gly-Gly-Asp] n (SEQ ID NO: 127); [Gly-Gly-Gly-Asp] n (SEQ ID NO: 128); [Gly-Gly-Gly-Gly-Gly- Asp] posting (SEQ ID NO: 129), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Gly-Gly- Glu] 6 .
  • a basic linker will contain a sequence that is selected from the group of [Gly-Lys] n (SEQ ID NO: 130); [Gly-Gly-Lys] n (SEQ ID NO: 131); [Gly-Gly-Gly-Lys] n (SEQ ID NO: 132); [Gly-Gly-Gly-Gly-Lys] n (SEQ ID NO: 133), [Gly- Arg] n (SEQ ID NO: 134); [Gly-Gly- Arg] n (SEQ ID NO: 135); [Gly-Gly-Gly-Arg] n (SEQ ID NO: 136); [Gly-Gly-Gly-Gly- Arg] n (SEQ ID NO: 137) where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Gly-Gly- Lys] 6 .
  • linkers may be prepared which possess certain structural motifs or characteristics, such as an alpha helix.
  • a linker may contain a sequence that is selected from the group of [Glu- Ala-Ala- Ala-Lys] n (SEQ ID NO: 138), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Glu-Ala-Ala-Ala-Lys]3, [Glu-Ala-Ala-Ala-Lys]4, or [Glu- Ala- Ala- Ala-Lys] 5.
  • a biocompatible linker other than a peptide linker may be used to covalently attach terminii of HD1 and HD2, a side chain of HD1 with a terminus of HD2, a side chain of HD2 with a terminus of HD1, or a side chain of HD1 with a side chain of HD2.
  • the linker can be a biocompatible polymer, preferably water soluble, and more preferably about 50kDa to about 5000kDa, or about 50KDa to 500kDa, or about lOOkDa to 500kDa.
  • biocompatible, water soluble polymer linker is a PEG linker, such as -(CH 2 -CH 2 -0) n - where n is such that the PEG linker can have a molecular weight of 100 to 5000 kDa, preferably 100 to 500 kDa.
  • a linker may be -NH-CH 2 -CH 2 -(0-CH 2 -CH 2 ) n -0-CH 2 -CO-, where n is such that the PEG linker molecular weight is lOOkDa to 5000kDa, preferably lOkDa to 500kDa.
  • biocompatible polymers can be used, such as including but not limited to polysaccharides, polypropylene glycol, and co-polymers of propylene and ethylene glycols.
  • a linker will include a reactive group at each end that can be the same or different reactive group.
  • Such linkers with reactive groups are known and available.
  • the reactive group is reactive with either an N-terminal amino or C-terminal carboxy group of a peptide.
  • a reactive group can be an a butylaldehyde, a propionaldehyde, an aldehyde, a succinimide or a maleimide moiety, as is known in the art.
  • linkers suitable for use in accordance with the invention may possess one or more of the characteristics and motifs described above and herein.
  • a linker may include an acidic linker as well as a structural motif, such as an alpha helix.
  • a linker may include a basic linker and a structural motif, such as an alpha helix.
  • a linker may include an acidic linker, a basic linker, and a structural motif, such as an alpha helix.
  • engineered polypeptides in accordance with the invention may possess more than one linker, and each such linker may possess one or more of the characteristics described herein.
  • linkers described herein are exemplary, and linkers within the scope of this invention may be much longer and may include other residues.
  • the engineered polypeptide includes a linker LI which is a peptide linker including from 1 to 30 amino acids.
  • linker LI includes amino acids selected from the 20 naturally occurring amino acids. In one embodiment, linker LI consists of amino acids selected from the 20 naturally occurring amino acids.
  • linker LI includes a non-natural amino acid incorporated by chemical synthesis, post-translational chemical modification or by in vivo incorporation by recombinant expression in a host cell.
  • amino acids of linker LI are selected from glycine, alanine, proline, asparagine, glutamine, and lysine.
  • linker LI includes a majority of amino acids that are sterically unhindered.
  • sterically unhindered refers, in the customary sense, to relative conformational freedom of an amino acid due to reduced side chain bulk or side chain to back bone constraint.
  • linker LI includes polyglycine, polyalanine, poly(Gly-Ala) or poly(Gly-Ser). [0221] In one embodiment, linker LI includes the sequence (Gly) 3 , (Gly) 4 (SEQ ID NO: 139), or (Gly) 5 (SEQ ID NO: 140). [0222] In one embodiment, linker LI includes the sequence GGG, GGS, GGGG (SEQ ID NO: 139), or GGGS (SEQ ID NO: 141).
  • linker LI includes the sequence (Gly) 3 Lys(Gly) 4 (SEQ ID NO:
  • linker LI includes combinations of Gly and Ala.
  • linker LI includes combinations of Gly and Ser.
  • linker LI is a glycine rich peptide.
  • linker LI includes an N-terminal dipeptide, which N-terminal dipeptide includes amino acids residues T, A, S or G. In one embodiment, linker LI includes an N-terminal TG dipeptide.
  • linker LI includes a C-terminal dipeptide, which C-terminal dipeptide includes amino acids residues T, A, S or G.
  • linker LI includes a C-terminal AS dipeptide.
  • linker LI includes an N-terminal TG dipeptide and a C-terminal AS dipeptide.
  • linker LI includes a sequence selected from the group consisting of TG-(GGG)i (SEQ ID NO: 142), TG-(GGGG)i (SEQ ID NO: 143), TG-(GGGGG)i (SEQ ID NO: 144), TG-(GGG) 2 (SEQ ID NO: 145), TG-(GGGGGGG) i (SEQ ID NO: 146), TG-(GGGG) 2 (SEQ ID NO: 147), TG-(GGG) 3 (SEQ ID NO: 148), (GGG) i -AS (SEQ ID NO: 149), (GGGG)i- AS (SEQ ID NO: 150), (GGGGG)i-AS (SEQ ID NO: 151), (GGG) 2 -AS (SEQ ID NO: 152),
  • linker LI includes a sequence selected from the group consisting of TG-(GGS)i (SEQ ID NO: 163), TG-(GGGS)i (SEQ ID NO: 164), TG-(GGGGS)i (SEQ ID NO: 165), TG-(GGS) 2 (SEQ ID NO: 166), TG-(GGGGGGS)i (SEQ ID NO: 167), TG-(GGGS) 2 (SEQ ID NO: 168), TG-(GGS) 3 (SEQ ID NO: 169), (GGS)i-AS (SEQ ID NO: 170), (GGGS)i-AS (SEQ ID NO: 171), (GGGGS) AS (SEQ ID NO: 172), (GGS) 2 -AS (SEQ ID NO: 173),
  • linker LI consists of a sequence selected from the group consisting of TG-(GGG)i (SEQ ID NO: 142), TG-(GGGG)i (SEQ ID NO: 143), TG-(GGGGG)i (SEQ ID NO: 144), TG-(GGG) 2 (SEQ ID NO: 145), TG-(GGGGGGG) i (SEQ ID NO: 146), TG-(GGGG) 2 (SEQ ID NO: 147), TG-(GGG) 3 (SEQ ID NO: 148), (GGG)i-AS (SEQ ID NO: 149), (GGGG)i- AS (SEQ ID NO: 150), (GGGGG) AS (SEQ ID NO: 151), (GGG) 2 -AS (SEQ ID NO: 152), (GGGGGGG)i-AS (SEQ ID NO: 153), (GGGG) 2 -AS (SEQ ID NO: 154), (GGG) 3 -AS (SEQ ID NO: 142
  • linker LI consists of a sequence selected from the group consisting of TG-(GGS)i (SEQ ID NO: 163), TG-(GGGS)i (SEQ ID NO: 164), TG-(GGGGS)i (SEQ ID NO: 165), TG-(GGS) 2 (SEQ ID NO: 166), TG-(GGGGGGS)i (SEQ ID NO: 167), TG-(GGGS) 2 (SEQ ID NO: 168), TG-(GGS) 3 (SEQ ID NO: 169), (GGS)i-AS (SEQ ID NO: 170), (GGGS)i-AS (SEQ ID NO: 171), (GGGGS)i-AS (SEQ ID NO: 172), (GGS) 2 -AS (SEQ ID NO: 173),
  • a engineered polypeptide described herein is superior to a corresponding compound having a different moiety that can extend plasma half-life (e.g., PEG or of Fc or albumin) conjugated with a hormone domain(s).
  • the term "superior” refers to a variety of functional properties which could be weighed in the evaluation of a treatment for a disease or disorder.
  • the engineered polypeptide described herein could require less biologically active (hormone domain) component, for example IX, 2X, 3X, 4X, 5X, or even less, than the corresponding compound having a different moiety conjugated with the hormone domain(s).
  • the engineered polypeptide described herein could have higher potency, for example, 1.5X, 2X, 3X, 4X, 5X, 10X, 20X, 50X, or even higher potency.
  • Engineered polypeptide compounds contemplated herein include the compounds as set forth in Table 1 following.
  • Compounds with sequence of SEQ ID NOS: 184-375 include an exendin domain which is Exendin-4(l-28), [ 14 L]Exendin-4(l-28), Exendin-4(l-32), or
  • a linker is present, having the sequence TGGGGAS, TGGGSAS, TGGGGGAS, TGGGGSAS, TG-(GGGG) 4 -AS, and TG-(GGGS) 4 -AS.
  • the humanized chimeric seal leptin component of the engineered polypeptides has the sequence of SEQ ID NO: 72, SEQ ID NO:94, SEQ ID NO:74, SEQ ID NO:98, SEQ ID NO:76, SEQ ID NO: 100, SEQ ID NO:78, or SEQ ID NO: 102.
  • HGEGTFTSDLSKQLEEEAVRLFIEWLKNTGGGGASPIQRVQDDTKTLIKTIITRINDISPP 235 QGVSSRPRVAGLDFIPRVQSVRTLSKMDQTLAVYQQILTSLQSRSVVQIANDLANLRA LLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRN PGC
  • HGEGTFTSDLSKQLEEEAVRLFIEWLKNTGGGSASPIQRVQDDTKTLIKTIITRINDISPP 243 QGVSSRPRVAGLDFIPRVQSVRTLSKMDQTLAVYQQILTSLQSRSVVQIANDLANLRA LLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRN PGC
  • HGEGTFTSDLSKQLEEEAVRLFIEWLKNTGGGSASPIQRVQDDTKTLIKTIITRINDISPP 246 QGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRSVVQIANDLANLRAL LRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLQGSLQDMLWQLDLNP GC
  • HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSTGGGGSASPIQKVQDDTKTLIKTIVTR 304 INDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRSVVQIANDL ANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLR QLDRNPGC
  • HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSTGGGGSGGGSGGGSGGGSASPIQRVQ 326 DDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSL QSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVAL SRLQGSLQDMLWQLDLNPGC
  • HGEGTFTSDLSKQLEEEAVRLFIEWLKNGGPSTGGGGSGGGSGGGSGGGSASPIQRVQ 375 DDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQ SRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALS RLQGSLQDMLWQLDLNPGC
  • engineered polypeptides disclosed herein include those that have been derivatized. Such derivatized engineered polypeptides include conjugation to one or more polymer moieties, such as polyethylene glycol (PEG) or fatty acid chains of various lengths (e.g., stearyl, palmitoyl, octanoyl, etc.), or by the addition of polyamino acids, such as poly-his, poly-arg, poly-lys, and poly-ala. Modifications can also include small molecules moieties, such as short alkyls and constrained alkyls (e.g., branched, cyclic, fused, adamantyl) and aromatic groups.
  • PEG polyethylene glycol
  • fatty acid chains of various lengths
  • polyamino acids such as poly-his, poly-arg, poly-lys, and poly-ala.
  • Modifications can also include small molecules moieties, such as short alkyls and constrained alkyls (e.
  • the polymer moieties will typically have a molecular weight from about 500 to about 60,000 Daltons.
  • the polymer may be linear or branched.
  • Such derivatizations can take place at the N- or C-terminus or at a side chain of an amino acid residue, e.g. lysine epsilon amino group, aspartic acid, glutamic acid, cysteine sulfhydryl group, within the Polypeptide Conjugate.
  • derivatization can occur at multiple sites throughout the conjugate polypeptide.
  • substitution of one or more amino acids with, or addition of, a lysine, aspartic acid, glutamic acid or cysteine can be done. See for example U.S. Patents 5824784 and 5824778, which are incorporated by reference herein.
  • the engineered polypeptides can be conjugated to one, two, or three polymer moieties. In one embodiment, the engineered polypeptides are linked to one
  • the polyethylene glycol can have a molecular weight from about 200 daltons to about 80,000 daltons; from about 5,000 daltons to about 60,000 daltons; from about 10,000 daltons to about 50,000 daltons; or from about 15,000 daltons to about 40,000 daltons.
  • the polyethylene glycol may be linear or branched.
  • the pegylated engineered polypeptides includes a lysine side chain to which is covalently attached via the lysine epsilon amino group a
  • the total molecular weight of the PEG moiety is at least about 10,000 Daltons, at least about 20,000 Daltons, at least about 40,000 Daltons or at least about 60,000 Daltons.
  • engineered polypeptides are linked to one or two polyethylene glycols, where the polyethylene glycol is further linked to a lipophilic moiety.
  • the polyethylene glycol in this case may have a molecular weight from about 200 to about 7,000 daltons or from about 500 to about 5,000 daltons.
  • the lipophilic moiety may be an alkyl group (e.g., Ci_ 2 o alkyl group; C 1-10 alkyl group; Ci_ 6 alkyl group; Ci_ 4 alkyl group), a fatty acid (e.g., C 4 _ 28 fatty acid chain; C 8 _ 24 fatty acid chain; Ci 0 _ 2 o fatty acid chain), cholesteryl, adamantyl, and the like.
  • the alkyl group may be linear or branched, preferably linear.
  • the fatty acid is an acetylated fatty acid or an esterified fatty acid.
  • the - (polyethylene glycol)-(lipophilic moiety) may be linked to the compound at a C-terminal amino acid residue, an N-terminal amino acid residue, an internal amino acid residue (e.g., an internal Lys amino acid residue), or a combination thereof (e.g., the compound is linked at the N-terminal and C-terminal amino acid residues).
  • the engineered polypeptides are linked to a polyamino acid.
  • Exemplary polyamino acids include poly-lysine, poly-aspartic acid, poly-serine, poly-glutamic acid, and the like.
  • the polyamino acid may be in the D or L form, preferably the L form.
  • the polyamino acids may comprise from 1 to 12 amino acid residues; from 2 to 10 amino acid residues; or from 2 to 6 amino acid residues.
  • the engineered polypeptides are linked to a fatty acid.
  • the fatty acid may be a C 4 -C 2 8 fatty acid chain, a Cs-C 24 fatty acid chain, or a Cio-C 2 o fatty acid chain.
  • the fatty acid is an acetylated fatty acid.
  • the fatty acid is an esterified fatty acid.
  • the engineered polypeptides are linked to albumin.
  • the albumin may be a recombinant albumin, serum albumin, or recombinant serum albumin.
  • the compounds are linked to an albumin-fatty acid (i.e., an albumin linked to a fatty acid).
  • the engineered polypeptides are linked to an immunoglobulin or an immunoglobulin Fc region.
  • the immunoglobulin may be IgG, IgE, IgA, IgD, or IgM.
  • the compounds are linked to an IgG Fc region or an IgM Fc region.
  • the immunoglobulin Fc region is (i) the heavy chain constant region 2(C H 2) of an immunoglobulin; (ii) the heavy chain constant region 3(C H 3) of an immunoglobulin; or (iii) both the heavy chain constant regions 2(C H 2) and 3(C H 3) of an immunoglobulin.
  • the immunoglobulin Fc region may further comprise the hinge region at the heavy chain constant region.
  • Other embodiments for the immunoglobulin Fc region that can be linked to exendin analog peptides are described in WO 2008/082274, the disclosure of which is incorporated by reference herein.
  • any linking group known in the art can be used.
  • the linking group may comprise any chemical group(s) suitable for linking the peptide to the polymer.
  • engineered polypeptides can be directly attached to the polymer without any linking group.
  • Exemplary linking groups include amino acids, maleimido groups, dicarboxylic acid groups, suecinimide groups, or a combination of two or more thereof.
  • an engineered polypeptide including one or more duration enhancing moieties linked thereto, optionally through a linker.
  • Linkage of the duration enhancing moiety to the peptide can be through a linker as described herein.
  • linkage of the duration enhancing moiety to the peptide can be via a direct covalent bond.
  • the duration enhancing moiety can be a water soluble polymer, or a long chain aliphatic group, as described herein.
  • a plurality of duration enhancing moieties are attached to the peptide, in which case each linker to each duration enhancing moiety is independently selected from the linkers described herein.
  • the terms "duration enhancing moiety" and the like refer, in the customary sense, to chemical species which lengthen the duration of biological activity of the attached engineered polypeptide.
  • a duration enhancing moiety is covalently bonded to an amino acid side chain of the peptide, or to a backbone atom or moiety thereof.
  • backbone moieties include a free amine at the N-terminal, and a free carboxyl or carboxylate at the C- terminal.
  • an amino acid side chain or a backbone atom or moiety is covalently bonded to a polyethylene glycol, a long chain aliphatic group, or a derivative thereof.
  • the duration enhancing moiety is a water-soluble polymer.
  • a "water soluble polymer” means a polymer which is sufficiently soluble in water under physiologic conditions of e.g., temperature, ionic concentration and the like, as known in the art, to be useful for the methods described herein.
  • a water soluble polymer can increase the solubility of a peptide or other biomolecule to which such water soluble polymer is attached. Indeed, such attachment has been proposed as a means for improving the circulating life, water solubility and/or antigenicity of administered proteins, in vivo. See, e.g., U.S. Pat. No.
  • the duration enhancing moiety includes a polyethylene glycol.
  • Polyethylene glycol (“PEG”) has been used in efforts to obtain therapeutically usable peptides. See, e.g., Zalipsky, S., 1995, Bioconjugate Chemistry 6: 150-165; Mehvar, R., 2000, J. Pharm. Pharmaceut. Sci. 3: 125-136.
  • PEG backbone As appreciated by one of skill in the art, the PEG backbone
  • PEG polyethylene glycol polymer
  • Methods for attaching polymer moieties, such as PEG and related polymers, to reactive groups found on a peptides and proteins are well known in the art.
  • Typical attachment sites in proteins include primary amino groups, such as those on lysine residues or at the N-terminus, thiol groups, such as those on cysteine side-chains, and carboxyl groups, such as those on glutamate or aspartate residues or at the C-terminus. Common sites of attachment are to the sugar residues of glycoproteins, cysteines or to the N-terminus and lysines of the target peptide.
  • pegylated and the like refer to covalent attachment of polyethylene glycol to a peptide or other biomolecule, optionally through a linker as described herein and/or as known in the art.
  • a PEG moiety in an engineered polypeptide described herein has a nominal molecular weight within a specified range.
  • the size of a PEG moiety is indicated by reference to the nominal molecular weight, typically provided in kilodaltons (kDa).
  • the molecular weight is calculated in a variety of ways known in the art, including number, weight, viscosity and "Z" average molecular weight. It is understood that polymers, such as PEG and the like, exist as a distribution of molecule weights about a nominal average value.
  • the term "mPEG40KD" refers to a methoxy polyethylene glycol polymer having a nominal molecular weight of 40 kilodaltons. Reference to PEGs of other molecular weights follows this convention.
  • the PEG moiety has a nominal molecular weight in the range 10-100 kDa, 20-80 kDa, 20-60 kDa, or 20-40 kDa.
  • the PEG moiety has a nominal molecular weight of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or even 100 kDa.
  • the PEG moiety has a molecular weight of 20, 25, 30, 40, 60 or 80 kDa.
  • PEG molecules useful for derivatization of engineered polypeptides are typically classified into linear, branched and comb (i.e., PolyPEG®) classes of PEGs, as known in the art.
  • polyPEG® linear, branched and comb
  • two arm branched Y-shaped
  • comb refers to branched PEG moieties, as known in the art.
  • comb in the context of PEGs, also known as “comb” or “comb-type” PEGs, refers to a variety of multi-arm PEGs attached to a backbone, typically poly(methacrylate), as known in the art.
  • Covalent attachment of PEG can be conveniently achieved by a variety of methods available to one skilled in the synthetic chemical arts.
  • PEG reagents are typically reacted under mild conditions to afford the pegylated compound.
  • additional steps including but not limited to reduction are employed.
  • N-hydroxylsuccinimide (NHS) functionalized mPEG can be mixed with peptide having a free amine in a suitable solvent (e.g., dry DMF) under nitrogen in the presence of DIPEA (e.g., 3 equivalents per TFA counterion) for a suitable time (e.g., 24 hrs).
  • a suitable solvent e.g., dry DMF
  • DIPEA e.g., 3 equivalents per TFA counterion
  • the conjugate can be precipitated by the addition of a precipitation reagent (e.g., cold diethyl ether).
  • a precipitation reagent e.g., cold diethyl ether
  • the precipitate can be isolated by centrifugation and dissolved in water followed by lyophilization. Purification can be afforded by a variety of chromatographic procedures (e.g., MacroCap SP cation exchange column using gradient 0.5 M NaCl). Purity can be checked by SDS-PAGE.
  • Mass spectrometry e.g., MALDI
  • MALDI mass spectrometry
  • PEG-SS succinimidyl succinate
  • PEG-SS reacts with amine groups under mild conditions to form the amide, as shown in Scheme 1.
  • NHS functionalization provides amino reactive PEG derivatives that can react with primary amine groups at pH 7 ⁇ 9 to form stable amide bonds. Reaction can be finished in 1 hour or even less time. Exemplary reactions follow in Schemes 1 and 2.
  • PEG-SG succinimidyl glutarate
  • PEG-SG reacts with amine groups to form the corresponding amide, as shown in Scheme 2.
  • PEG-NPC p-nitrophenyl carbonate
  • PEG-isocyanate As shown in Scheme 4, PEG-isocyanate can react with amine to form the resultant relatively stable urethane linkage.
  • PEG-aldehyde A variety of PEG-aldehyde reactions with amine can afford the imine, which can be further reduced to afford the pegylated amine.
  • the reaction pH may be important for target selectivity.
  • N -terminal amine pegylation may be at around pH 5.
  • reaction of mPEG-propionaldehyde with peptide amine, followed by reduction affords the compound depicted in Scheme 5 following.
  • Thiol pegylation PEG-maleimide. Pegylation is conveniently achieved at free thiol groups by a variety of methods known in the art. For example, as shown in Scheme 9 following, PEG-maleimide pegylates thiols of the target compound in which the double bond of the maleimic ring breaks to connect with the thiol. The rate of reaction is pH dependent and best conditions are found around pH 8.
  • PEG-vinylsulfone is useful for the pegylation of free thiol.
  • PEG-orthopyridyl-disulfide OPSS
  • OPSS PEG-orthopyridyl-disulfide
  • PEG-iodoacetamide pegylates thiols to form stable thioether bonds in mild basic media. This type of conjugation presents an interesting aspect in that by strong acid analysis the pegylated cysteine residue of the protein can give rise to carboxymethylcysteine which can be evaluated by a standard amino acid analysis (for example, amino acid sequencing), thus offering a method to verify the occurrence of the reaction.
  • a typical reaction scheme is depicted in Scheme 12 following.
  • the duration enhancing moiety includes a long chain aliphatic group, and the resulting compound is a long chain peptide conjugate.
  • the term "long chain engineered polypeptide conjugate" as used herein refers to an engineered
  • a further strategy for modulating the duration of activity and potency of peptide and protein therapeutic agents involves derivatizing with long chain aliphatic (e.g., fatty acid) chains of various lengths, for example but not limited to C6-C24, C8-C20, Cio-Cig, C 12 -C 16 , and the like.
  • long chain aliphatic e.g., fatty acid
  • a "fatty acid” as used herein means a long chain aliphatic moiety terminated with a carboxyl functionality. It is understood that long chain aliphatic groups can be fully hydrogenated or partially dehydrogenated.
  • C x refers to a carbon chain containing "x" carbon atoms.
  • the carboxyl functionality of a fatty acid is available for bonding with the peptide.
  • acylation of amino groups is a common means employed for chemically modifying proteins, and general methods of acylation are known in the art and include the use of activated esters, acid halides, or acid anhydrides. See, e.g.,
  • Fatty acid chain(s) may be linked to an amino, carboxyl, or thiol group, and may be linked by N or C terminus, or at the side chains of lysine, aspartic acid, glutamic acid, or cysteine, as known in the art and/or as described herein.
  • the fatty acid moieties may be linked with diamine and dicarboxylic groups, as known in the art.
  • long chain (e.g. , C 6 -C24) aliphatic groups preferably fatty acid chains
  • the long chain aliphatic group is C 16 , C 18 , C20, C22 or even C24.
  • the long chain aliphatic group is fully hydrogenated.
  • the long chain aliphatic group contains one or more double bonds.
  • oligonucleotides can be provided for multistep PCR, as known in the art. These oligonucleotides can be used in multiple PCR reactions under conditions well known in the art to build the cDNA encoding the protein of interest. For one example is IX AmpliTaq® Buffer, 1.3 mM MgC , 200uM dNTPs, 4 U AmpliTaq® Gold, 0.2 uM of each primer (AmpliTaq Gold, ABI), with cycling parameters: (94C:30s, 58C: 1 min, 72C: lmin), 35 cycles.
  • Restriction sites can be added to the ends of the PCR products for use in vector ligation as known in the art.
  • Specific sites can include Ndel and Xhol, such that the cDNA can then be in the proper reading frame in a pET45b expression vector (Novagen). By using these sites, any N-terminal His Tag that are in this vector can be removed as the translation start site would then be downstream of the tag.
  • verification can be conduct by sequencing using e.g., T7 promoter primer, T7 terminator primer and standard ABI BigDye® Term v3.1 protocols as known in the art.
  • Sequence information can be obtained from e.g., an ABI 3730 DNA Analyzer and can be analyzed using Vector NTI® v.10 software (Invitrogen). Expression constructs can be designed in a modular manner such that linker sequences can be easily cut out and changed, as known in the art.
  • Protease recognition sites known in the art or described herein, can be incorporated into constructs useful for the design, construction, manipulation and production of recombinant engineering polypeptides described herein.
  • Codon optimized nucleotide sequences for all proteins can be generated by overlap PCR and subcloned into a modified pET32 vector (EK cleavage site replaced with TEV cleavage site) at Kpnl and Xhol restriction sites. Sequence verified vector DNA can then be transformed to BL21 cells (Novagen), and induced at 30C O/N in MAGIC MEDICTM autoinducing media (Invitrogen).
  • the engineered polypeptide compounds described herein may be prepared using biological, chemical, and/or recombinant DNA techniques that are known in the art. Exemplary methods are described herein and in US Patent No. 6,872,700; WO 2007/139941; WO 2007/140284; WO 2008/082274; WO 2009/011544; and US Publication No. 2007/0238669, the disclosures of which are incorporated herein by reference in their entireties and for all purposes. Other methods for preparing the compounds are set forth herein.
  • the engineered polypeptides compounds described herein may be prepared using standard solid-phase peptide synthesis techniques, such as an automated or semiautomated peptide synthesizer.
  • HD1 and HD2 can be made separately and then conjugated together or can be made as a single polypeptide.
  • HD1 and HD2 may alternatively be produced by non-biological peptide synthesis using amino acids and/or amino acid derivatives having reactive side-chains protected, the non-biological peptide synthesis including step-wise coupling of the amino acids and/or the amino acid derivatives to form a polypeptide according to the first aspect having reactive side-chains protected, removing the protecting groups from the reactive side-chains of the polypeptide, and folding of the polypeptide in aqueous solution.
  • normal amino acids e.g.
  • glycine, alanine, phenylalanine, isoleucine, leucine and valine) and pre -protected amino acid derivatives are used to sequentially build a polypeptide sequence, in solution or on a solid support in an organic solvent.
  • the protecting groups are removed and the polypeptide is allowed to fold in an aqueous solution.
  • the engineered polypeptides according to the present disclosure reversibly fold.
  • the engineered polypeptide may be produced by a method including producing HD1 and HD2 according to any method, e.g. as described herein, such as by non-biological peptide synthesis, and conjugating the produced HD1 and HD2 components which then fold completely reversibly.
  • This can be assessed by a variety of methods, e.g., circular dichroism spectra analysis. For example, one spectrum can be taken at 20°C and a second spectrum after heating to 90 °C followed by return to 20 °C.
  • an alpha-N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at room temperature (RT) in an inert solvent (e.g., dimethylformamide, N-methylpyrrolidinone, methylene chloride, and the like) in the presence of coupling agents (e.g., dicyclohexylcarbodiimide, 1- hydroxybenzo- triazole, and the like) in the presence of a base (e.g., diisopropylethylamine, and the like).
  • an inert solvent e.g., dimethylformamide, N-methylpyrrolidinone, methylene chloride, and the like
  • coupling agents e.g., dicyclohexylcarbodiimide, 1- hydroxybenzo- triazole, and the like
  • a base e.g., diisopropylethylamine, and the like
  • the alpha-N-carbamoyl protecting group is removed from the resulting peptide -resin using a reagent (e.g., trifluoroacetic acid, piperidine, and the like) and the coupling reaction repeated with the next desired N-protected amino acid to be added to the peptide chain.
  • a reagent e.g., trifluoroacetic acid, piperidine, and the like
  • Suitable N-protecting groups are well known in the art, such as t-butyloxycarbonyl (tBoc)
  • fluorenylmethoxycarbonyl Fmoc
  • the solvents, amino acid derivatives and 4- methylbenzhydryl-amine resin used in the peptide synthesizer may be purchased from Applied Biosystems Inc. (Foster City, CA).
  • Solid phase peptide synthesis can be used for the engineered polypeptides, since in general solid phase synthesis is a straightforward approach with excellent scalability to commercial scale, and is generally compatible with relatively long engineered polypeptides.
  • Solid phase peptide synthesis may be carried out with an automatic peptide synthesizer (Model 43 OA, Applied Biosystems Inc., Foster City, CA) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry (See APPLIED BIOSYSTEMS USER'S MANUAL FOR THE ABI 430A PEPTIDE SYNTHESIZER, Version 1.3B Jul. 1, 1988, section 6, pp.
  • Boc-peptide -resins may be cleaved with HF (-5°C to 0°C, 1 hour).
  • the peptide may be extracted from the resin with alternating water and acetic acid, and the filtrates lyophilized.
  • the Fmoc-peptide resins may be cleaved according to standard methods (e.g., Introduction to Cleavage Techniques, Applied Biosystems, Inc., 1990, pp. 6-12).
  • Peptides may also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).
  • Non-peptide compounds may be prepared by art-known methods.
  • phosphate-containing amino acids and peptides containing such amino acids may be prepared using methods known in the art, such as described in Bartlett et al, 1986, Biorg. Chem., 14:356-377.
  • Compounds can be conjugated using art methods or as described herein [0277]
  • the engineered polypeptides may alternatively be produced by recombinant techniques well known in the art. See, e.g., Sambrook et al, 1989 ⁇ Id.). These engineered polypeptides produced by recombinant technologies may be expressed from a polynucleotide.
  • polynucleotides including DNA and RNA, that encode such engineered polypeptides may be obtained from the wild-type cDNA, e.g. Exendin-4, taking into consideration the degeneracy of codon usage, and may further engineered as desired to incorporate the indicated substitutions.
  • These polynucleotide sequences may incorporate codons facilitating transcription and translation of mRNA in microbial hosts. Such manufacturing sequences may readily be constructed according to the methods well known in the art. See, e.g., WO 83/04053, incorporated herein by reference in its entirety and for all purposes.
  • the polynucleotides above may also optionally encode an N-terminal methionyl residue.
  • Non- peptide compounds useful in the present invention may be prepared by art-known methods.
  • phosphate-containing amino acids and peptides containing such amino acids may be prepared using methods known in the art. See, e.g., Bartlett and Landen, 1986, Bioorg. Chem. 14: 356-77.
  • a variety of expression vector/host systems may be utilized to contain and express a engineered polypeptide coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus
  • Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), WI 38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC 12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the protein are described herein and/or are known in the art.
  • polynucleotide sequences are useful in generating new and useful viral and plasmid DNA vectors, new and useful transformed and transfected procaryotic and eucaryotic host cells (including bacterial, yeast, and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells capable of expression of the present engineered polypeptides.
  • the polynucleotide sequences encoding engineered polypeptides herein may be useful for gene therapy in instances where underproduction of engineered polypeptides would be alleviated, or the need for increased levels of such would be met.
  • the present invention also provides for processes for recombinant DNA production of the present engineered polypeptides.
  • a process for producing the engineered polypeptides from a host cell containing nucleic acids encoding the engineered polypeptide including: (a) culturing the host cell containing polynucleotides encoding the engineered polypeptide under conditions facilitating the expression of the DNA molecule; and (b) obtaining the engineered polypeptides.
  • Host cells may be prokaryotic or eukaryotic and include bacteria, mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkey cells, baby hamster kidney cells, cancer cells or other cells), yeast cells, and insect cells.
  • mammalian host systems for the expression of the recombinant protein also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post- translational processing which cleaves a "prepro" form of the protein, may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like, have specific cellular machinery and characteristic mechanisms for such post-translational activities, and may be chosen to ensure the correct modification and processing of the introduced foreign protein.
  • a yeast system may be employed to generate the engineered polypeptides of the present invention.
  • the coding region of the engineered polypeptides DNA is amplified by PCR.
  • a DNA encoding the yeast pre-pro-alpha leader sequence is amplified from yeast genomic DNA in a PCR reaction using one primer containing nucleotides 1-20 of the alpha mating factor gene and another primer complementary to nucleotides 255-235 of this gene (Kurjan and
  • pre-pro-alpha leader coding sequence and engineered polypeptide coding sequence fragments are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter, such that the promoter directs expression of a fusion protein consisting of the pre-pro-alpha factor fused to the mature engineered polypeptide.
  • ADH2 yeast alcohol dehydrogenase
  • the vector further includes an ADH2 transcription terminator downstream of the cloning site, the yeast "2 -micron" replication origin, the yeast leu-2d gene, the yeast REPl and REP2 genes, the E. coli beta- lactamase gene, and an E. coli origin of replication.
  • the beta-lactamase and leu-2d genes provide for selection in bacteria and yeast, respectively.
  • the leu-2d gene also facilitates increased copy number of the plasmid in yeast to induce higher levels of expression.
  • the REP1 and REP2 genes encode proteins involved in regulation of the plasmid copy number.
  • the DNA construct described in the preceding paragraph is transformed into yeast cells using a known method, e.g., lithium acetate treatment (Steams et al., 1990,. Meth. Enz. 185: 280- 297).
  • the ADH2 promoter is induced upon exhaustion of glucose in the growth media (Price et al, 1987, Gene 55:287).
  • the pre-pro-alpha sequence effects secretion of the fusion protein from the cells.
  • the yeast KEX2 protein cleaves the pre-pro sequence from the mature engineered polypeptides (Bitter et al, 1984, Proc. Natl. Acad. Sci. USA 81:5330-5334).
  • Engineered polypeptides of the invention may also be recombinantly expressed in yeast, e.g. Pichia, using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, CA), following the manufacturer's instructions. This system also relies on the pre-pro-alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol.
  • AOX1 alcohol oxidase
  • the secreted engineered polypeptide is purified from the yeast growth medium by, e.g., the methods used to purify said engineered polypeptide from bacterial and mammalian cell supernatants.
  • the DNA encoding a engineered polypeptide may be cloned into a baculovirus expression vector, e.g., pVL1393 (PharMingen, San Diego, CA). This engineered- polypeptide-encoding vector is then used according to the manufacturer's directions
  • the DNA sequence encoding the predicted mature engineered polypeptide may be cloned into a plasmid containing a desired promoter and, optionally, a leader sequence (see, e.g., Better et al, 1988, Science 240: 1041-1043). The sequence of this construct may be confirmed by automated sequencing.
  • the plasmid is then transformed into E. coli, strain MCI 061, using standard procedures employing CaCl 2 incubation and heat shock treatment of the bacteria (Sambrook et al., Id.). The transformed bacteria are grown in LB medium supplemented with carbenicillin, and production of the expressed protein is induced by growth in a suitable medium.
  • the leader sequence will affect secretion of the mature engineered polypeptide and be cleaved during secretion.
  • the secreted recombinant engineered polypeptide is purified from the bacterial culture media by the method described herein.
  • the engineered polypeptides may be expressed in an insect system.
  • Insect systems for protein expression are well known to those of skill in the art.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the engineered polypeptide coding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of a engineered polypeptide will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S.
  • the DNA sequence encoding the engineered polypeptides may be amplified by PCR and cloned into an appropriate vector, for example, pGEX-3X (Pharmacia, Piscataway, New Jersey).
  • the pGEX vector is designed to produce a fusion protein including glutathione-S-transferase (GST), encoded by the vector, and a protein encoded by a DNA fragment inserted into the vector's cloning site.
  • the primers for the PCR may be generated to include, for example, an appropriate cleavage site.
  • the recombinant fusion protein may then be cleaved from the GST portion of the fusion protein.
  • the pGEX-3X/ engineered polypeptide construct is transformed into E.
  • coli XL-1 Blue cells (Stratagene, La Jolla, CA), and individual transformants are isolated and grown at 37 degrees C in LB medium (supplemented with carbenicillin) to an optical density at wavelength 600 nm of 0.4, followed by further incubation for 4 hours in the presence of 0.5 mM Isopropyl beta-D-Thiogalactopyranoside (Sigma Chemical Co., St. Louis, Missouri). Plasmid DNA from individual transformants is purified and partially sequenced using an automated sequencer to confirm the presence of the desired engineered polypeptide-encoding gene insert in the proper orientation.
  • the fusion protein when expected to be produced as an insoluble inclusion body in the bacteria, may be purified as described above or as follows. Cells are harvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/mL lysozyme (Sigma Chemical Co.) for 15 min. at RT. The lysate is cleared by sonication, and cell debris is pelleted by centrifugation for 10 min. at 12,000xg. The fusion protein-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min.
  • the pellet is resuspended in standard phosphate buffered saline solution (PBS) free of Mg ++ and Ca ++ .
  • PBS phosphate buffered saline solution
  • the fusion protein is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in 0.4 M KC1 to visualize the protein, which is excised and
  • GST/engineered polypeptide fusion protein is produced in bacteria as a soluble protein, it may be purified using the GST Purification Module (Pharmacia Biotech).
  • the fusion protein may be subjected to digestion to cleave the GST from the mature engineered polypeptide.
  • the digestion reaction (20-40 ⁇ g fusion protein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 mL PBS) is incubated 16-48 hrs. at RT and loaded on a denaturing SDS-PAGE gel to fractionate the reaction products. The gel is soaked in 0.4 M KC1 to visualize the protein bands.
  • the identity of the protein band corresponding to the expected molecular weight of the engineered polypeptide may be confirmed by partial amino acid sequence analysis using an automated sequencer (Applied Biosystems Model 473 A, Foster City, CA).
  • mammalian 293 cells may be co-transfected with plasmids containing the engineered polypeptides cDNA in the pCMV vector (5 ' CMV promoter, 3 ' HGH poly A sequence) and pSV2neo (containing the neo resistance gene) by the calcium phosphate method.
  • the vectors should be linearized with Seal prior to transfection.
  • an alternative construct using a similar pCMV vector with the neo gene incorporated can be used.
  • Stable cell lines are selected from single cell clones by limiting dilution in growth media containing 0.5 mg/mL G418 (neomycin-like antibiotic) for 10-14 days. Cell lines are screened for engineered polypeptides expression by ELISA or Western blot, and high-expressing cell lines are expanded for large scale growth.
  • G418 neomycin-like antibiotic
  • the transformed cells are used for long-term, high-yield protein production and as such stable expression is desirable.
  • the cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the selectable marker is designed to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
  • Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell.
  • a number of selection systems may be used to recover the cells that have been transformed for recombinant protein production.
  • selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
  • anti-metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the
  • aminoglycoside, G418 also, that confers resistance to chlorsulfuron; and hygro, that confers resistance to hygromycin.
  • Additional selectable genes that may be useful include trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Markers that give a visual indication for identification of transformants include anthocyanins, beta-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.
  • the engineered polypeptides of the present invention may be produced using a combination of both automated peptide synthesis and recombinant techniques.
  • either or both the HD1 and HD2, and optionally a linker can be made synthetically or recombinantly and then ligated together using methods known in the art, such as "native chemical ligation" and known variations thereof in which an amide bond is formed joining the parent compounds. See, e.g., United States Patent No. 6326468, which is incorporated herein by reference and for all purposes.
  • an engineered polypeptide of the present invention may contain a combination of modifications including deletion, substitution, insertion and derivatization by PEGylation (or other moiety, e.g.
  • an engineered polypeptide may be produced in stages.
  • an intermediate engineered polypeptide containing the modifications of deletion, substitution, insertion, and any combination thereof may be produced by recombinant techniques as described.
  • the intermediate engineered polypeptide is PEGylated (or subjected to other chemical derivatization, e.g., acylation, C-terminal amidation) through chemical modification with an appropriate PEGylating reagent (e.g., from NeKtar Transforming Therapeutics, San Carlos, CA) to yield the desired engineered polypeptide derivative.
  • an appropriate PEGylating reagent e.g., from NeKtar Transforming Therapeutics, San Carlos, CA
  • C-terminal amidation can be achieved by use of a glycine amino acid-C-terminally extended precursor, synthesized for example in yeast (e.g. Pichia) as alpha-factor fusion protein that will be secreted into culture medium. After purification, the C-terminal glycine of the engineered polypeptide precursor can be converted to amide by enzymatic amidation, e.g.
  • PAM peptidylglycine alpha-amidating monooxygenase
  • Peptides may be purified by any number of methods known in the art, including as described herein In one method peptides are purified by RP-HPLC (preparative and analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10 ⁇ , 2.2X25 cm; Vydac, Hesperia, CA) may be used to isolate peptides, and purity may be determined using a C4, C8 or C18 analytical column (5 ⁇ , 0.46X25 cm; Vydac).
  • Amino acid analyses may be performed on the Waters Pico Tag system and processed using the Maxima program.
  • Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115°C, 20-24 h). Hydrolysates may be derivatized and analyzed by standard methods (Cohen et al, THE PICO TAG METHOD: A MANUAL OF ADVANCED
  • Engineered polypeptide expression assay Methods are available for assaying the level of protein expression by a host cell. Procedures useful for assaying the level of protein expression by a host cell are exemplified in the following typical protocol. About 25 ul BL21 E. coli cells are transformed with 2ul plasmid DNA (expression vector for the engineered polynucleotide). Cells can be plated and incubated overnight at 37 degrees C or at room temperature (RT) over a 48-hr period.
  • RT room temperature
  • a single colony can be selected and used to grow starter culture in 4 ml LB media with appropriate antibiotic for ⁇ 6 hrs.
  • Glycerol stocks can be prepared by adding lOOul 80% sterile glycerol to 900ul stock, which can then be mixed gently and stored at -80C.
  • a 250 ul sample can be removed for TCP uninduced sample.
  • An aliquot, for example, 2 ml of Magic media containing appropriate antibiotic can be inoculated with 5 ul starter culture, which can then be incubated overnight (up to 24 hrs) at 37C, 300 rpm.
  • Magic Media is autoinducing.
  • 60 ml Magic Media containing appropriate antibiotic can be inoculated with 60 ul starter culture in a 250ml or 125 ml Thompson flask, which can then be incubated overnight (up to 24 hrs) at 30C, 300rpm. After incubation, 250 ul culture can be removed from each tube and the cells pelleted. The cell can be resuspended in 1 ml 50 mM Tris pH 8, 150mM NaCl, to which can be added 0.1 volumes (lOOul) POP culture reagent and 1 ul r-lysozyme (1 :750 dilution in r-lysozyme buffer). The mixture can be mixed well and incubated at least 10 min at RT.
  • the preparation can then be centrifuge 10 min at 14000 x G.
  • the supernatant (soluble fraction) can be removed and retained, and samples can be prepared for gel analysis (15 ul + 5 ul LDS).
  • the remaining inclusion body pellet can be resuspended in 1ml 1% SDS with sonication.
  • the sample can be prepared for gel analysis (15ul + 5 ul LDS).
  • 1.0 volumes POP culture reagent and 1 ul r-lysozyme (1 :750 dilution in r-lysozyme buffer) can be added.
  • the mixture can be mixed well and incubated at least 10 min at RT. These samples may not need to be centrifuged.
  • the sample can then be prepared for gel analysis (15ul + 5 ul LDS).
  • NU-PAGE gels (4-12%) non-reduced in 1XMES buffer can be run and stained with SIMPLYBLUETM microwave protocol. Destaining can be conducted overnight, as known in the art. A gel image can be retained, and analyzed to determine protein expression levels.
  • Engineered polypeptides can be expressed and isolated as follows. A protein sequence of the desired engineered polypeptide can be designed and back translated using commercial software to a DNA sequence for cloning into an E. coli expression vector. Nucleic acid sequences can be either obtained as oligonucleotides and ligated using standard PCR
  • telomere sequences expressing the protein of interest can be placed in plasmid pET45 with a T7 promoter for inducible expression. After constructs are verified by sequencing, the vector DNA can be purified and transformed into an expression host, typically BL21(DE3). A single colony can be selected to grow a starter culture in 4 ml LB media for ⁇ 6 hrs. Glycerol stocks can be prepared by adding lOOul 80% glycerol to 900ul stock and stored at -80C. Optionally, 500 ul of un-induced sample can be retained for gel analysis. A 60 ml culture (e.g. MAGICMEDIATM E.
  • coli Expression Medium can be inoculated using 60ul starter culture in a 125ml Thompson flask and incubated at 30 degrees C overnight. Removed 250ul sample for analysis. The cells can be collected as a pellet by centrifuging, and frozen for later processing. Preparation of cell extract and first pass purification with Nickel resin can be performed as follows. E. coli cell pellets can be completely resuspended in a volume of lysis buffer (50 mM TrisHCl, 150 mM NaCl, pH 8.0) equal to the starting culture volume.
  • lysis buffer 50 mM TrisHCl, 150 mM NaCl, pH 8.0
  • Cells can then be subjected to a microfluidizer (Microfluidics, MA) at 100 psi for three times.
  • Cell extracts can be centrifuged for 30 minutes at 16,000 x g to remove debris.
  • EGTA 150mM stock
  • EGTA 150mM stock
  • the lysate can then be applied to a Ni-NTA SUPERFLOWTM column that has been washed and pre-equilibrated.
  • Protein bound to the column can then be washed with lysis buffer plus EGTA (50 mM TrisHCl, 150 mM NaCl, pH8.0, 3 mM EGTA) before the bound protein is eluted with 50 mL of elution buffer (25 mM TrisHCl, 50 mM NaCl, 250 mM Imidazol, pH8.0).
  • Cleavage of His-Tag and subsequent purification can be as follows.
  • the eluted protein can be concentrated with Amicon® Ultral5 centrifugal filter unit (Millipore, USA) and then diluted with 25 mM TrisHCl, pH8.0, 50 mM NaCl to prepare for protease digestion which removes the HisTag from the N-terminus of the desired protein.
  • Added can be 0.1% of ⁇ -mercaptoethanol and 1% of Turbo TEV protease (2 mg/mL, 10,000 units/mg; Excellgen, USA) to the protein solution, which can be mixed and incubated at room temperature for 4 hours and then at 4°C over night.
  • An Ni-NTA Ni-NTA
  • SUPERFLOWTM column (Qiagen, USA) can be pre-equilibrated with 50 mM TrisHCl, 100 mM NaCl, 45 mM imidazole, pH8.0.
  • the TEV digest reaction can be diluted 2-fold with 50 mM TrisHCl, 150 mM NaCl, pH8.0.
  • the diluted digest reaction can be carefully applied to the top of Ni-NTA column and flow-through can be collected.
  • To the column can be added 10 mL of 50 mM trisHCl, 100 mM NaCl, 45 mM imidazole, pH8.0 to elute any unbound protein.
  • the eluted proteins from the column can be collected and combined, and then further purified using size exclusion chromatography (2x with Superdex® 75 HiLoad 26/60 column; GE Healthcare Biosciences, USA). Any remaining bacterial endotoxin can be removed using EndoTrap® Red (Lonza, Switzerland) according to manufacturer's instructions.
  • Inclusion Body preparation For engineered polypeptides that are found in the inclusion body fraction, the following procedure can be beneficial. The cell pellet can be resuspended in a minimum of 100 ml Lysis buffer for each 50 ml culture.
  • a 10ml pipette can be used to resuspend, then the tube can be washed out with an additional 70ml.
  • the resuspended cell solution can be multiply run, e.g., 4 passes, through a microfluidizer@ 100 PSI (min) taking care to keep chamber in ice water through the entire process.
  • the fluidized slurry can be centrifuged at 14000 x g, 20 min (e.g., JLA 10.5,
  • the inclusion body pellet can be resuspended on ice in chilled lysis buffer with stir bar and stir plate for 1 hour at 4C after disruption with pipette tip.
  • the pellet can be resuspended a second time in distilled H 2 0 with stir bar and stir plate for 1 hour at 4C after disruption with pipette tip, followed by centrifugation at 14000 x g, 15 min.
  • the supernatant can be removed and discarded.
  • the resultant can be stored at -80C.
  • Inclusion body pellets can be solubilized in appropriate volume of solubilization buffer (8M urea or 8M guanidine, 50 mM Tris, 10 mM DTT, pH 7.75) for 1 hour at RT. The solubilized pellets can be centrifuged for 20 min at 27 OOOg.
  • solubilization buffer 8M urea or 8M guanidine, 50 mM Tris, 10 mM DTT, pH 7.75
  • Filtered (e.g., 0.4 um) supernatant can be transferred drop by drop into appropriate volume of refolding buffer (50 mM Tris-HCl, 1 M urea, 0.8 M arginine, 4 mM cysteine, 1 mM cystamine; pH 8) at RT. The result can then be placed at 4°C overnight or longer with gentle mixing. Samples can be concentrated and run on a gel filtration column (SUPERDEXTM 75 26/60) at 1-2 ml/min in 4C environment using a GE Healthsciences AKTA FPLCTM. Appropriate protein containing fractions can be identified via SDS-PAGE, pooled and run through a second gel filtration column.
  • a protein sample can then be concentrated in Amicon filter to appropriate concentration and assayed for endotoxin levels using, e.g., ENDOSAFE® PTSTM Reader (Charles River), as known in the art.
  • ENDOSAFE® PTSTM Reader (Charles River)
  • Quality control assays can include analytical HPLC-SEC, non reducing SDS PAGE and RP HPLC - MS to obtain approximate mass.
  • Proteins can be obtained in lxPBS (137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodium phosphate, 1.4 mM monopotassium phosphate, pH7.2), distributed into aliquots and flash frozen for storage at -70 to -80 °C.
  • Obesity and overweight are common and serious public health problems in the United States and throughout the world. Upper body obesity is the strongest risk factor known for type 2 diabetes mellitus and is a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovarian syndrome, cancers of the breast, prostate, and colon, and increased incidence of complications of general anesthesia. See, e.g., Kopelman, 2000, Nature 404:635-43.
  • Obesity reduces life-span and carries a serious risk of the co-morbidities listed above, as well disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholelithiasis, orthopedic injury, and thromboembolic disease. See e.g., Rissanen et al, 1990, Br. Med. J, 301:835-7. Obesity is also a risk factor for the group of conditions called insulin resistance syndrome, or "Syndrome X" and metabolic syndrome. The worldwide medical cost of obesity and associated disorders is enormous.
  • the pathogenesis of obesity is believed to be multi-factoral. A problem is that, in obese subjects, nutrient availability and energy expenditure do not come into balance until there is excess adipose tissue.
  • the central nervous system (CNS) controls energy balance and
  • hypothalamus coordinates a variety of behavioral, autonomic and endocrine activities appropriate to the metabolic status of the animal.
  • the mechanisms or systems that control these activities are broadly distributed across the forebrain (e.g., hypothalamus), hindbrain (e.g., brainstem), and spinal cord.
  • metabolic (i.e., fuel availability) and cognitive (i.e., learned preferences) information from these systems is integrated and the decision to engage in appetitive (food seeking) and consummatory (ingestion) behaviors is either turned on (meal procurement and initiation) or turned off (meal termination).
  • the hypothalamus is thought to be principally responsible for integrating these signals and then issuing commands to the brainstem.
  • Brainstem nuclei that control the elements of the consummatory motor control system e.g., muscles responsible for chewing and swallowing.
  • these CNS nuclei have literally been referred to as constituting the "final common pathway" for ingestive behavior.
  • Neuroanatomical and pharmacological evidence support that signals of energy and nutritional homeostasis integrate in forebrain nuclei and that the consummatory motor control system resides in brainstem nuclei, probably in regions surrounding the trigeminal motor nucleus. There are extensive reciprocal connection between the hypothalamus and brainstem.
  • CNS-directed anti-obesity therapeutics e.g., small molecules and peptides
  • Obesity remains a poorly treatable, chronic, essentially intractable metabolic disorder. Accordingly, a need exists for new therapies useful in weight reduction and/or weight maintenance in a subject. Such therapies would lead to a profound beneficial effect on the subject's health.
  • Diabetes and cardiovascular disease Diabetes mellitus is recognized as a complex, chronic disease in which 60% to 70% of all case fatalities among diabetic patients are a result of cardiovascular complications. Diabetes is not only considered a coronary heart disease risk equivalent but is also identified as an independent predictor of adverse events, including recurrent myocardial infarction, congestive heart failure, and death following a cardiovascular incident.
  • diabetic patients are two to three times more likely to experience an acute myocardial infarction than non-diabetic patients, and diabetic patients live eight to thirteen years less than non-diabetic patients.
  • Coronary Syndrome Summit A State of the Art Approach, Kansas City, MO, September 21, 2002.
  • a logistic regression model of glucose control over the entire hospitalization was most predictive of mortality.
  • In a smaller cohort of consecutive diabetic/ ACS patients there was a graded increase in mortality at one year with increasing glucose levels upon hospital admission.
  • the ACC/AHA guidelines suggest initiation of aggressive insulin therapy to achieve lower blood glucose during hospitalization.
  • Dyslipidemia is a disruption in the normal lipid component in the blood. It is believed that prolonged elevation of insulin levels can lead to dyslipidemia. Hyperlipidemia is the presence of raised or abnormal levels of lipids and/or lipoproteins in the blood.
  • Fatty liver disease e.g., nonalcoholic fatty liver disease (NAFLD) refers to a wide spectrum of liver disease ranging from simple fatty liver (steatosis), to nonalcoholic fatty liver disease
  • NASH steatohepatitis
  • cirrhosis irreversible, advanced scarring of the liver. All of the stages of NAFLD have in common the accumulation of fat (fatty infiltration) in the liver cells (hepatocytes).
  • exendins can be well suited as a treatment modality for this condition.
  • engineered polypeptides described herein which include an exendin or biologically active (hormone domain) peptide component, or fragment or analog thereof, can be useful in the treatment of fatty liver disorders.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • GLP-1 has been reported to play an important role in regulating neuronal plasticity and cell survival.
  • GLP-1 has been reported to induce neurite outgrowth and to protect against excitotoxic cell death and oxidative injury in cultured neuronal cells.
  • GLP-1 and Exendin-4 were reported to reduce endogenous levels of amyloid-beta peptide (A-beta protein) in mouse brain and to reduce levels of beta-amyloid precursor protein (beta- APP) in neurons. See, e.g., Perry et al, 2004, Curr. Drug Targets 5(6):565-571. Treatment with the engineered compounds disclosed herein can provide benefit to the therapeutic targets associated with Alzheimer's disease. [0314] Parkinson's disease.
  • Parkinson's disease is the synonym of "primary parkinsonism", i.e. isolated parkinsonism due to a neurodegenerative process without any secondary systemic cause. Parkinsonism is characterized by symptoms of tremor, stiffness, and slowing of movement caused by loss of dopamine. Without wishing to be bound by any theory, it is believed that Exendin-4 may act as a survival factor for dopaminergic neurons by
  • Exendin-4 may be a valuable therapeutic agent for neurodegenerative diseases such as PD.
  • Metabolic syndrome X is characterized by insulin resistance, dyslipidemia, hypertension, and visceral distribution of adipose tissue, and plays a pivotal role in the pathophysiology of type 2 diabetes. It has also been found to be strongly correlated with NASH, fibrosis, and cirrhosis of the liver. Accordingly, engineered polypeptides described herein can be useful in the treatment of metabolic syndrome X.
  • Glucocorticoids are well known to affect carbohydrate metabolism. In response to exogenous glucocorticoid administration, increased hepatic glucose production and reduced insulin secretion and insulin-stimulated glucose uptake in peripheral tissues is observed. Furthermore, glucocorticoid treatment alters the proinsulin(Pl)
  • hyperglycemia induced by glucocorticoids in subjects without diabetes include a minimal elevation of fasting blood glucose, exaggerated postprandial hyperglycemia, insensitivity to exogenous insulin, and non-responsiveness to metformin or sulfonylurea therapy.
  • engineered polypeptides described herein which include an exendin biologically active (hormone domain) peptide component, or fragment or analog thereof, can be useful in the treatment of steroid induced diabetes.
  • HIV Human Immunodeficiency Virus
  • HAV-l protease inhibitors Pro
  • PT use with the development of hyperglycemia began to appear. While approximately 1% to 6% of HIV-infected subjects who are treated with Pis will develop diabetes mellitus, a considerably larger proportion will develop insulin resistance and impaired glucose tolerance. Accordingly, engineered polypeptides described herein which include an exendin biologically active (hormone domain) peptide component, or fragment or analog thereof, can be useful in the treatment of HIV treatment-induced diabetes.
  • LAD A Latent Autoimmune Diabetes in Adults
  • Progressive autoimmune diabetes also known as latent autoimmune diabetes in adults (LAD A)
  • LAD A latent autoimmune diabetes in adults
  • LADA patients have circulating antibodies to either islet cell cytoplasmic antigen or, more frequently, glutamic acid
  • engineered polypeptides described herein which include an exendin biologically active (hormone domain) peptide component, or fragment or analog thereof, can be useful in the treatment of LADA.
  • hypoglycemia Unawareness HU. Defective glucose counterregulation can occur even after only a single recent episode of hypoglycemia. Subjects who experience repeated episodes of hypoglycemia often lose their capacity to recognize the symptoms typically associated with hypoglycemia or impending insulin shock, a condition called "hypoglycemia unawareness". Because the-patient doesn't appreciate his or her own status, blood glucose levels can then fall so low that serious neurological problems ensue, including coma and seizure.
  • engineered polypeptides described herein which include an exendin biologically active (hormone domain) peptide component, or fragment or analog thereof, can be useful in the treatment of HU.
  • GLP 1 receptor has been localized in the lung. Exendins can elicit a biological response via GLP-1 receptor.
  • sarcoidosis is a systemic granulomatous disease that frequently involves the lung. Although classically thought of as a restrictive lung disease, airway obstruction has become a recognized feature of the disease in the past years. Sarcoidosis can affect the airway at any level and when the involvement includes small airways, it can resemble more common obstructive airway diseases, such as asthma and chronic bronchitis.
  • engineered polypeptides described herein which include an exendin biologically active (hormone domain) peptide component, or fragment or analog thereof, can be useful in the treatment of restrictive lung disease because such hormone domain peptide can improve elasticity of lung or delay rigidity.
  • hormone domain biologically active
  • SBS Short Bowel Syndrome
  • Exendin-4 has been reported as effective for the treatment of short bowel syndrome. See Kunkel et al. Neurogastroenterol. Motil. (2011).
  • SBS is a serious clinical disorder characterized by diarrhea and nutritional deprivation.
  • GLP-1 Glucagon- like peptide- 1
  • GLP-1 levels may be deficient.
  • Exenatide improved the nutritional state and intestinal symptoms of patients with SBS. Accordingly, SBS patients are amenable to treatment with the engineered polypeptides described herein.
  • Improvement in bowel frequency and form and obtaining bowel movements that are no longer meal-related can be achieved.
  • An additional benefit is that total parenteral nutrition can be stopped.
  • These compounds herein will provide substantial improvement in the bowel habits, nutritional status and quality of life of SBS patients, and further may reduce the need for parenteral nutrition and small bowel transplant.
  • NASH Non-alcoholic steatohepatitis
  • NASH Non-alcohol Fatty Liver Disease
  • NASH is now considered to be one of the most common liver diseases in western countries. Fatty infiltration is a typical response of the liver to a wide array of noxious stimuli, including hypoxia, toxins, systemic inflammation, malignancies, and various metabolic derangements. Although NASH itself is generally considered to be a benign condition, it may lead to liver fibrosis, cirrhosis, and ultimately failure. NASH is a subcategory of NAFLD characterized histologically by macrovesicular steatosis, ballooning degeneration, hepatocyte necrosis, fibrosis, occasional Mallory bodies, and infiltration of inflammatory cells (American Gastroenterological Association, Technical review on nonalcoholic fatty liver disease.
  • Gastroenterology 123: 1705-1725 (2002)) Although NAFLD and NASH are often asymptomatic, elevated concentrations of serum alanine aminotransferase (ALT), a biochemical marker of liver injury, are indicative of NAFLD, but cannot distinguish between NAFLD and NASH (American Gastroenterological Association, Medical position statement: Nonalcoholic fatty liver disease. Gastroenterology 123: 1702-1704 (2002)). Serum concentrations of aspartate aminotransferase (AST) may be higher than ALT, especially in the presence of hepatic cirrhosis, and serum alkaline phosphatase (ALP) concentrations may also be elevated (American Gastroenterological Association, Medical position statement: Nonalcoholic fatty liver disease. Gastroenterology 123: 1702-1704 (2002)). However, measures of hepatic functional capacity do not become abnormal until cirrhosis has developed and liver failure is imminent (American
  • Gastroenterological Association Medical position statement: Nonalcoholic fatty liver disease. Gastroenterology 123: 1702-1704 (2002)). In obese T2DM, progressive hepatomegaly due to NAFLD occurs frequently and may be accompanied by right upper quadrant discomfort.
  • NAFLD can cause progressive fibrosis leading to cirrhosis and its complications, including portal hypertension and liver failure (American Gastroenterological Association, Medical position statement: Nonalcoholic fatty liver disease. Gastroenterology 123: 1702-1704 (2002)).
  • NASH is associated with decreased insulin-mediated suppression of lipolysis and the resulting elevation in serum free fatty acid concentrations that contribute to impaired pancreatic .beta.-cell function and increased cardiovascular morbidity and mortality (Yki-Jarvinen et al., Curr. Molec. Med. 5:287-295 (2005); American Gastroenterological Association, Medical position statement: Nonalcoholic fatty liver disease. Gastroenterology 123: 1702-1704 (2002); Raz et al, Diabetes/Metab. Res. Rev. 21 :3-14 (2005)).
  • a method for treating a disease in a subject is in need of treatment for the disease.
  • the disease is diabetes, overweight, obesity, Alzheimer's disease, short bowel syndrome, fatty liver disease, dyslipidemia, coronary artery disease, stroke, hyperlipidemia, NASH or Parkinson's disease.
  • the subject is need of treatment is obese.
  • the subject has diabetes.
  • Diabetes can include type I, type II, gestational or pre-diabetes as well as HIV or steroid induced diabetes.
  • the method of treatment includes administration to the subject of a engineered polypeptide as described herein in an amount effective to treatment the disease.
  • Particularly useful for these diseases are compounds described herein having glucose lowering activity (e.g. HD1 fragments or analogs linked to an HD2), having reduction of body weight or reduction of food intake activity, lowering of HbAlc, delaying of gastric emptying, lowering of plasma glucagon, and/or intestinal motility benefit.
  • the disease or disorder is diabetes, overweight, obesity, short bowel syndrome, NASH or Parkinson's disease.
  • the disease is type I diabetes, type II diabetes or prediabetes.
  • the disease is type II diabetes.
  • the disease is dyslipidemia or hyperlipidemia.
  • the disease or disorder can be diabetes, overweight, obesity, dyslipidemia, Alzheimer's disease, fatty liver disease, SBS, hyperlipidemia, Parkinson's disease or cardiovascular disease or other diseases described herein.
  • the engineered polypeptide may include an exendin or fragment or analog thereof. Accordingly, the engineered polypeptide can have one of the following structures: HD1-HD2 or HD1-L1-HD2.
  • the exendin is Exendin-4.
  • the exendin fragment is a fragment of Exendin-4.
  • the exendin analog has at least 70%, for example 70%, 75%>, 80%>, 85%>, 90%), 95%o or even higher, identity with Exendin-4.
  • glucose lowering activity e.g. Exendin-4 or its fragments or analogs linked to a humanized chimeric seal leptin
  • having reduction of body weight or reduction of food intake activity e.g. HbAlc
  • delaying of gastric emptying lowering of plasma glucagon, or intestinal motility benefit.
  • exendin domain is Exendin-4.
  • exendin domain is a fragment of Exendin-4.
  • the exendin domain is an analog having at least 70%, e.g., 70%>, 75%, 80%>, 85%, 90%, 95% or even higher, identity with Exendin-4.
  • amylin Receptor Binding Assays The amylin receptor binding assay is a ligand binding assay measuring the potency of test compounds, e.g., polypeptides disclosed herein, in displacing 125 I-amylin (rat) from human amylin receptor 3 (AMY3) ectopically expressed in a cell line, e.g., a Codex ACTONETM cell line.
  • This cell line can be generated using ACTONETM HEK293-CNG-hCalcR cell line (CB-80200-258) stably expressing human RAMP3 (NCBI protein database CAA04474) to produce the human AMY3 receptor.
  • Crude membranes from AMY3 cell cultures can be prepared by homogenization in ice cold 20 mM HEPES containing protease inhibitors (Roche Cat#l 1873580001). The crude membranes can be incubated with 20 pM 125 I- amylin (Perkin Elmer Cat#NEX4480) (2000 Ci/mmol) and increasing concentration of test peptide. Incubation can be carried out in 20 mM HEPES with 5 mM MgCl 2 and 1 mM CaCl 2 for 60 minutes at ambient temperature in 96-well polystyrene plates (Costar Cat#3797).
  • Incubations can be terminated by rapid filtration through UniFilter® 96 plates GF/B (Perkin Elmer, Cat#6005199), pre-soaked for at least 30 minutes in 0.5% polyethylenimine.
  • the Unifilter® plates can be washed several times using ice cold PBS using a MicroMate 96 Cell Harvester (Perkin Elmer). Unifilter plates can then be dried, scintillant added, MICROSCINTTM 20(Perkin Elmer Cat#6013621) and CPM determined by reading on a Perkin Elmer/Wallac TriLux multiwell scintillation counter capable of reading radiolabeled iodine.
  • the potency (IC 50 ) of test peptide is determined by the analysis of a concentration- response curve using non-linear regression analysis fitted to a 4-parameter curve. Binding affinities can be calculated using GraphPad Prism® software (GraphPad Software, Inc., San Diego, CA).
  • Calcitonin Receptor Binding Assays The calcitonin receptor binding assay is a ligand binding assay measuring the potency of test compounds in displacing 125 I-calcitonin (human) from HEK293 cells stably expressing the rat C la calcitonin receptor. The methodology is the same as for AMY3, wherein 125 I-calcitonin (Perkin-Elmer NEX4220) can be used at a concentration of 50 pM.
  • CGRP Receptor Binding Assays The calcitonin gene -related peptide (CGRP) receptor binding assay is a ligand assay measuring the potency of test compounds in displacing 125 I-CGRP(human) from SK MC cells, as known in the art, endogenously expressing the human CGRP receptor.
  • the methodology is the same as for AMY3, wherein 125 I-CGRP (Perkin-Elmer NEX3540) can be used at a concentration of 50 pM.
  • Adrenomedulin Receptor Binding Assays The adrenomedulin receptor binding assay is a ligand binding assay measuring the potency of test compounds in displacing
  • I-adrenomedulin (Perkin-Elmer NEX4270) can be used at a concentration of 25 pM.
  • the GLP-1 receptor binding assay is a ligand binding assay measuring the potency of test compounds in displacing 125 I-GLP-1 (human) from 6-23 (clone 6) cells, as known in the art, endogenously expressing the rat GLP-1 receptor.
  • the methodology is the same as for AMY3, wherein 125 I-GLP-1 (Perkin-Elmer NEX3080) can be used at a concentration of 60 pM.
  • the leptin receptor binding assay is a ligand binding assay measuring the potency of test compounds in displacing 125 I-leptin (murine) from 32D-OBECA cells, as known in the art, stably expressing the human Leptin receptor.
  • the methodology is the same as for AMY3, wherein 125 I-Leptin(Perkin-Elmer NEX3400) can be used at a concentration of 100 pM, and wherein the incubation time can be increased from 60 minutes to 180 minutes.
  • amylin Functional Assays The amylin functional assay can be used to measure increases in cyclic- AMP (cAMP) in the Codex ACTONETMHEK293-CNG-hCalcR cell line (CB-80200-258) stably transfected with human RAMP3 (NCBI protein database CAA04474) to produce the human AMY3 receptor. Accumulation of cAMP can be measured following 30 minute incubation with test compounds. Efficacy of peptides is determined relative to cell treatment with lOuM forskolin (a constitutive activator of adenylate cyclase). Potency (EC 50 ) of compounds can be determined by the analysis of a concentration-response curve using non-linear regression analysis fitted to a 4-parameter model, using e.g., GraphPad Prism® software.
  • cAMP cyclic- AMP
  • Peptides can be diluted in assay buffer(HBSS, 01%BSA) and incubated with cells in the presence of 250 uM IBMX(Calbiochem 410957).
  • the cAMP can be measured using the cAMP Dynamic 2 assay (Cisbio) as per the manufacturer's instructions.
  • cAMP can be detected by a decrease in time -resolved fluorescence energy transfer (TR-FRET) using an GeniousPro plate reader (Tecan).
  • Calcitonin Functional Assays The calcitonin functional assay can be used to measure increases in cyclic-AMP (cAMP) in HEK293 cells stably expressing the rat C la calcitonin receptor. Accumulation of cAMP can be measured following 30 minute incubation with test compounds. Efficacy of peptides is determined relative to cell treatment with lOuM forskolin (a constitutive activator of adenylate cyclase), and potency (EC 50 ) of peptides is determined by the analysis of a concentration-response curve using non-linear regression analysis fitted to a 4-parameter model.
  • cAMP cyclic-AMP
  • Peptides can be diluted in assay buffer(HBSS, 01%BSA) and incubated with cells in the presence of 250 uM IBMX(Calbiochem 410957).
  • the cAMP can be measured using the cAMP Dynamic 2 assay(Cisbio) as per the manufacturer's instructions.
  • cAMP can be detected by a decrease in time -resolved fluorescence energy transfer (TR-FRET) using an GeniousPro plate reader (Tecan).
  • TR-FRET time -resolved fluorescence energy transfer
  • Tecan GeniousPro plate reader
  • Accumulation of cAMP can be measured following 30 minute incubation with test compounds. Efficacy of peptides can be determined relative to cell treatment with lOuM forskolin (a constitutive activator of adenylate cyclase), and potency (EC 50 ) of peptides can be determined by the analysis of a concentration-response curve using non-linear regression analysis fitted to a 4-parameter model.
  • lOuM forskolin a constitutive activator of adenylate cyclase
  • potency (EC 50 ) of peptides can be determined by the analysis of a concentration-response curve using non-linear regression analysis fitted to a 4-parameter model.
  • Peptides can be diluted in assay buffer(HBSS, 01%BSA) and incubated with cells in the presence of 250 uM IBMX(Calbiochem 410957).
  • the cAMP can be measured using the cAMP Dynamic 2 assay(Cisbio) as per the manufacturer's instructions.
  • cAMP can be detected by a decrease in time-resolved fluorescence energy transfer (TR-FRET) using an GeniousPro plate reader (Tecan).
  • TR-FRET time-resolved fluorescence energy transfer
  • Tecan GeniousPro plate reader
  • the measurement of pSTAT5 in the cell lysates can be determined using the Perkin Elmer AlphaScreen® SureFire® pSTAT5 assay kit in a 384-well format (PROXIPLATETM 384 Plus). Efficacy of peptides can be determined relative to the maximal signal in cell lysates from cells treated with Human leptin (lOOnM). The EC50 of the peptides can be determined by the analysis of a concentration- response curve using non-linear regression analysis fitted to a 4-parameter model.
  • In vivo assays for activity, duration of action and pharmacokinetics can be done using known methods. For example, duration can be performed using an oral glucose tolerance test (OGTT) in which the drug is administered to the subject at a desired time point before the glucose is administered orally (to measure drug duration of action; OGTT DOA) and glucose blood levels are measured (e.g. readily done in mice). Activity and duration can also be measured using an intravenous glucose tolerance test (IVGTT) in which the drug is administered to the subject at a desired time point before the glucose is administered IV (IVGTT DOA) and blood glucose levels are measured (e.g. can readily be done in rats).
  • IVGTT intravenous glucose tolerance test
  • IVGTT DOA intravenous glucose tolerance test
  • DOA duration of activity
  • test means are compared to the control mean using Dunnett's test (Prism® v. 4.01, GraphPad Software Inc., San Diego, CA). Blood glucose can measured with a OneTouch® Ultra® (LifeScan, Inc., a Johnson & Johnson Company, Milpitas, CA). * p ⁇ 0.05 vs. vehicle control; ANOVA, Dunnett's test. Other parameters can also be measured.
  • the engineered polypeptides may be tested for their duration and extent of appetite suppression and for their duration and extent of effect on body weight loss in various known methods.
  • the polypeptides may be tested for appetite suppression in the mouse food intake assay and for their effect on body weight gain in diet-induced obesity (DIO) mice.
  • DIO diet-induced obesity
  • mice Female NIH/Swiss mice (8-24 weeks old) are group housed with a 12: 12 hour ligh dark cycle with lights on at 0600. Water and a standard pelleted mouse chow diet are available ad libitum, except as noted. Animals are fasted starting at approximately 1500 hrs, 1 day prior to experiment. The morning of the experiment, animals are divided into
  • Body Weight, fat redistribution, and lean body mass Assays can also be performed as follows. Diet-induced obesity (DIO) in the in the Sprague-Dawley rat is a valuable model for the study of obesity and regulation of energy homeostasis. These rats were developed from a line of (Crl:CD®(SD)BR) rats that are prone to become obese on a diet relatively high in fat and energy. See, for example, Levin, 1994, Am. J. Physiol. 267:R527-R535, Levin et al, 1997, Am. J. Physiol. 273:R725-R730.
  • DIO male rats are obtained from Charles River Laboratories, Inc. (Wilmington, MA). The rats are housed individually in shoebox cages at 22 °C in a 12/12-hour light dark cycle. Rats are maintained ad- libitum on a moderately high fat diet (32% kcal from fat; Research Diets D1226B). The animals typically achieve a mean body weight of about 500 g. Levin DIO rats are habituated to caging environment for 7 days. During the 3 nights of habituation, animals receive a single
  • IP intraperitoneal
  • rats are administered a single IP injection of compound or vehicle (e.g. 10% DMSO) at the onset of the dark cycle.
  • Food intake is measured by an automated food intake measuring system (BioDAQ, Research Diets) at 5 sec intervals throughout the course of the study. Body weight is recorded nightly.
  • Body composition can be measured prior to and after drug treatment using NMR (Echo Medical Systems, Houston, TX).
  • NMR Echo Medical Systems, Houston, TX
  • rats are briefly placed ( ⁇ 1 min) in a well-ventilated plexiglass tube that was then inserted into a specialized rodent
  • a pharmaceutical composition including an engineered polypeptide described herein in combination with a pharmaceutically acceptable excipient (e.g., carrier).
  • a pharmaceutically acceptable carrier refers to pharmaceutical excipients, for example, pharmaceutically, physiologically, acceptable organic or inorganic carrier substances suitable for enteral or parenteral application that do not deleteriously react with the active agent.
  • Suitable pharmaceutically acceptable carriers include water, salt solutions (e.g., Ringer's solution and the like), alcohols, oils, gelatins, and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrrolidine.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • a pharmaceutical composition which includes a engineered polypeptide as described herein in combination with a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is a long lasting pharmaceutical composition.
  • the term "long lasting" in the context of administration of a pharmaceutical composition refers to duration of action. Accordingly, a long lasting
  • compositions may be administered at intervals of, for example, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or even longer.
  • administration is twice a day (i.e., "twice daily").
  • the pharmaceutical composition is a parenteral pharmaceutical composition. In one embodiment, the pharmaceutical composition is a sustained release or long lasting pharmaceutical composition. In one embodiment, the pharmaceutical composition is formulated as a twice daily pharmaceutical composition. In one embodiment, the pharmaceutical composition is formulated as a once daily pharmaceutical composition. In one embodiment, the pharmaceutical composition is formulated as a once weekly pharmaceutical composition.
  • the pharmaceutical composition is useful for treating a disease in a subject.
  • the disease is diabetes, overweight, obesity, Alzheimer's disease, fatty liver disease, short bowel syndrome, dyslipidemia, coronary artery disease, stroke, hyperlipidemia, NASH or Parkinson's disease.
  • the disease is diabetes, overweight, obesity, short bowel syndrome, or Parkinson's disease.
  • the disease is type I diabetes, type II diabetes or prediabetes.
  • the pharmaceutical composition includes an engineered
  • polypeptide as set forth in Table 1 (SEQ ID NOS: 184-375). In one embodiment, the amino acids listed in Table 1 (SEQ ID NOS: 184-375).
  • composition is an engineered polypeptide as set forth in Table 1 (SEQ ID NO: 1
  • the pharmaceutical composition includes an engineered
  • the engineered polypeptides described herein can be administered alone or can be coadministered to a subject.
  • Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound).
  • a combination therapy including leptin (e.g., metreleptin) and an amylin (e.g., pramlintide). See e.g., U.S. Published Appl. No. 2008/0207512.
  • an engineered polypeptide described herein useful for treatment of e.g., obesity and overweight can be administered alone to achieve such treatment or co-administered with either a leptin or leptin agonist, e.g. metreleptin, and/or an amylin or amylin agonist, e.g. pramlintide.
  • a leptin or leptin agonist e.g. metreleptin
  • an amylin or amylin agonist e.g. pramlintide
  • the formulations and methods described herein further provide that the engineered polypeptide is co-administered with one or more anti-diabetic agents, such as anti-hyperglycemia agents, e.g. insulin (including regular, short acting, long-acting, and basal insulins), amylins, pramlintide, metformin and thiazolidinediones (including rosiglitazone and pioglitazone).
  • anti-diabetic agents such as anti-hyperglycemia agents, e.g. insulin (including regular, short acting, long-acting, and basal insulins), amylins, pramlintide, metformin and thiazolidinediones (including rosiglitazone and pioglitazone).
  • the formulations and methods described herein further provide that the engineered polypeptide is co-administered with one or more cholesterol and/or triglyceride lowering agents.
  • exemplary agents include HMG CoA reductase inhibitors (e.g., atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin); bile ace sequestrants (e.g., colesevelam, cholestyramine, colestipol); fibrates (e.g., fenofibrate, clofibrate,
  • gemfibrozil ezetimibe, nicotinic acid, probucol, a lovastatin/niacin combination; an
  • compositions for use as a medicament, i.e. for use in therapy, since the exendin compound is a therapeutically active compound, and surprisingly retains activity when fused to a humanized chimeric seal leptin or analog or fragment thereof.
  • Compositions including an engineered polypeptide, either liquid or dry form, and optionally at least one pharmaceutically acceptable carrier and/or excipient are also specifically contemplated herein.
  • Co-administration can be achieved by separately administering the exendin, exendin agonist, or exendin analog agonist engineered polypeptide with the second agent, or by administering a single pharmaceutical formulation including the exendin, exendin agonist, or exendin analog agonist engineered polypeptide and the second agent.
  • Appropriate dosage regimens for the second agents are generally known in the art.
  • the preparations can also be co-administered, when desired, with other active substances (e.g. to reduce metabolic degradation) as known in the art or other therapeutically active agents.
  • An exendin engineered polypeptide described herein can be administered with other active anti-diabetes or anti-obesity agents, such as leptin or leptin agonists and amylin or amylin agonist compounds, e.g. the amylins, including davalintide and their analogs.
  • Amylins is a peptide hormone synthesized by pancreatic ⁇ -cells that is co- secreted with insulin in response to nutrient intake.
  • the sequence of amylin is highly preserved across mammalian species, with structural similarities to calcitonin gene-related peptide (CGRP), the calcitonins, the intermedins, and adrenomedulin, as known in the art.
  • CGRP calcitonin gene-related peptide
  • the glucoregulatory actions of amylin complement those of insulin by regulating the rate of glucose appearance in the circulation via suppression of nutrient-stimulated glucagon secretion and slowing gastric emptying.
  • pramlintide a synthetic and equipotent analogue of human amylin, reduces postprandial glucose excursions by suppressing
  • Davalintide also known as "AC-2307” is a potent amylin agonist useful in the treatment of a variety of disease indications. See WO 2006/083254 and WO 2007/114838, each of which is incorporated by reference herein in its entirety and for all purposes.
  • Davalintide is a chimeric peptide, having an N-terminal loop region of amylin or calcitonin and analogs thereof, an alpha-helical region of at least a portion of an alpha-helical region of calcitonin or analogs thereof or an alpha-helical region having a portion of an amylin alpha-helical region and a calcitonin alpha-helical region or analog thereof, and a C-terminal tail region of amylin or calcitonin.
  • the sequences of human calcitonin, salmon calcitonin and davalintide follow:
  • KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO:381.
  • davalintide, and fragment and analogs thereof can require C-terminal amidation to elicit a full biological response. It is understood that amylin compounds such as those described herein which include amylins and/or davalintide, and fragment and analogs thereof, can be amidated at the C-terminal.
  • Amylin agonist compounds include native amylin peptides, amylin analog peptides, and other compounds (e.g., small molecules) that have amylin agonist activity.
  • the "amylin agonist compounds” can be derived from natural sources, can be synthetic, or can be derived from recombinant DNA techniques.
  • Amylin agonist compounds have amylin agonist receptor binding activity and may include amino acids (e.g., natural, unnatural, or a combination thereof), peptide mimetics, chemical moieties, and the like. The skilled artisan will recognize amylin agonist compounds using amylin receptor binding assays or by measuring amylin agonist activity in soleus muscle assays.
  • amylin agonist compounds will have an IC 50 of about 200 nM or less, about 100 nM or less, or about 50 nM or less, in an amylin receptor binding assay, such as that described herein, in US Patent No. 5,686,411, and US Publication No. 2008/0176804, the disclosures of which are incorporated by reference herein in their entireties and for all purposes.
  • amylin agonist compounds will have an EC 50 of about 20 nM or less, about nM 15 or less, about nM 10 or less, or about nM 5 or less in a soleus muscle assay, such as that described herein and in US Patent No. 5,686,411.
  • the amylin agonist compound has at least 90% or 100% sequence identity to
  • the amylin agonist compound is a peptide chimera of amylin (e.g., human amylin, rat amylin, and the like) and calcitonin (e.g., human calcitonin, salmon calcitonin, and the like).
  • amylin e.g., human amylin, rat amylin, and the like
  • calcitonin e.g., human calcitonin, salmon calcitonin, and the like.
  • Suitable and exemplary amylin agonist compounds are also described in US Publication No. 2008/0274952, the disclosure of which is incorporated by reference herein in its entirety and for all purposes.
  • the compounds can be administered simultaneously or sequentially, together or separately formulated. Since the engineered compounds herein are inherently long-acting, they are suitable for once daily, once weekly or longer administration. Accordingly, the other agent may be administered either in one or multiple doses, e.g. once daily, twice daily, three times daily, once weekly, as needed, during the period of dosing for the exendin engineered polypeptide, e.g. once weekly.
  • These pharmaceutical compounds may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang et al. (1988) J. of Parenteral Sci. and Tech., Technical Report No. 10, Supp. 42:2 S.
  • the engineered polypeptides may be formulated into a stable, safe pharmaceutical composition for administration to a patient.
  • Pharmaceutical formulations contemplated for use in the methods of the invention may include approximately 0.01 to 1.0% (w/v), in certain cases 0.05 to 1.0%, of the engineered polypeptide, approximately 0.02 to 0.5% (w/v) of an acetate, phosphate, citrate or glutamate buffer allowing a pH of the final composition of from about 3.0 to about 7.0; approximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcohol tonicifier and, optionally, approximately 0.005 to 1.0% (w/v) of a preservative selected from the group of m-cresol, benzyl alcohol, methyl, ethyl, propyl and butyl parabens and phenol. Such a preservative is generally included if the formulated peptide is to be included in a multiple use product.
  • a pharmaceutical formulation of the present engineered polypeptides may contain a range of concentrations of the compound(s), e.g., between about
  • 0.01% to about 98%o w/w or between about 1 to about 98%> w/w, or preferably between 80%> and 90%) w/w, or preferably between about 0.01% to about 50%> w/w, or more preferably between about 10% to about 25% w/w in these embodiments.
  • a sufficient amount of water for injection may be used to obtain the desired concentration of solution.
  • Additional tonicifying agents such as sodium chloride, as well as other known excipients, may also be present, if desired. In some cases, such excipients are useful in maintenance of the overall tonicity of the compound. An excipient may be included in the presently described formulations at various concentrations.
  • an excipient may be included in the concentration range from about 0.02% to about 20% w/w, preferably between about 0.02% and 0.5% w/w, about 0.02% to about 10% w/v, or about 1% to about 20% w/w.
  • an excipient may be included in solid (including powdered), liquid, semi-solid or gel form.
  • the pharmaceutical formulations may be composed in various forms, e.g., solid, liquid, semisolid or liquid.
  • solid as used herein, is meant to encompass all normal uses of this term including, for example, powders and lyophilized formulations.
  • the presently described formulations may be lyophilized.
  • buffer when used with reference to hydrogen-ion concentration or pH, refer to the ability of a system, particularly an aqueous solution, to resist a change of pH on adding acid or alkali, or on dilution with a solvent.
  • Characteristic of buffered solutions which undergo small changes of pH on addition of acid or base, is the presence either of a weak acid and a salt of the weak acid, or a weak base and a salt of the weak base.
  • An example of the former system is acetic acid and sodium acetate.
  • the change of pH is slight as long as the amount of hydronium or hydroxyl ion added does not exceed the capacity of the buffer system to neutralize it.
  • a variety of liquid vehicles are suitable for use in the formulations of engineered polypeptides, for example, water or an aqueous/organic solvent mixture or suspension.
  • the stability of a engineered polypeptide formulation for use as described herein is enhanced by maintaining the pH of the formulation in a range determined by methods known in the art.
  • the pH of the formulation is maintained in the range of about 3.5 to 5.0, or about 3.5 to 6.5, in some embodiments from about 3.7 to 4.3, or about 3.8 to 4.2.
  • pH may be about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, or even higher.
  • pH may be in the physiological range, pH 6-8, preferably pH 7-7.6.
  • the buffer with the engineered polypeptide is an acetate buffer (preferably at a final formulation concentration of from about 1-5 to about 60 mM), phosphate buffer (preferably at a final formulation concentration of from about 1-5 to about to about
  • the buffer is acetate (preferably at a final formulation concentration of from about 5 to about 30 mM).
  • a stabilizer may be included in the formulations but is not necessarily needed. If included, however, a stabilizer useful in the practice of the present invention is a carbohydrate or a polyhydric alcohol.
  • a suitable stabilizer useful in the practice of the present invention is approximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcohol.
  • the polyhydric alcohols and carbohydrates share the same feature in their backbones, i.e.,— CHOH— CHOH— , which is responsible for stabilizing the proteins.
  • the polyhydric alcohols include such compounds as sorbitol, mannitol, glycerol, and polyethylene glycols (PEGs). These compounds are straight-chain molecules.
  • the carbohydrates such as mannose, ribose, sucrose, fructose, trehalose, maltose, inositol, and lactose, on the other hand, are cyclic molecules that may contain a keto or aldehyde group. These two classes of compounds have been demonstrated to be effective in stabilizing protein against denaturation caused by elevated temperature and by freeze-thaw or freeze-drying processes.
  • Suitable carbohydrates include: galactose, arabinose, lactose or any other carbohydrate which does not have an adverse affect on a diabetic patient, i.e., the carbohydrate is not metabolized to form unacceptably large concentrations of glucose in the blood.
  • Such carbohydrates are well known in the art as suitable for diabetics.
  • Sucrose and fructose are suitable for use with the compound in non-diabetic applications (e.g. treating obesity).
  • the compound is stabilized with a polyhydric alcohol such as sorbitol, mannitol, inositol, glycerol, xylitol, and
  • mannitol is the preferred polyhydric alcohol in some embodiments.
  • Another useful feature of the lyophilized formulations of the present invention is the maintenance of the tonicity of the lyophilized formulations described herein with the same formulation component that serves to maintain their stability.
  • mannitol is the preferred polyhydric alcohol used for this purpose.
  • USP United States Pharmacopeia
  • bacteriostatic or fungistatic concentrations must be added to preparations contained in multiple dose containers. They must be present in adequate concentration at the time of use to prevent the multiplication of microorganisms inadvertently introduced into the preparation while
  • Antimicrobial agents should be evaluated to ensure compatibility with all other components of the formula, and their activity should be evaluated in the total formula to ensure that a particular agent that is effective in one formulation is not ineffective in another. It is not uncommon to find that a particular antimicrobial agent will be effective in one formulation but not effective in another formulation.
  • a preservative is, in the common pharmaceutical sense, a substance that prevents or inhibits microbial growth and may be added to pharmaceutical formulations for this purpose to avoid consequent spoilage of the formulation by microorganisms. While the amount of the preservative is not great, it may nevertheless affect the overall stability of the peptide. [0382] While the preservative for use in the pharmaceutical compositions can range from
  • benzyl alcohol 0.1-1.0%), or m-cresol (0.1-0.6%), or phenol (0.1-0.8%) or combination of methyl (0.05-0.25%) and ethyl or propyl or butyl (0.005%-0.03%) parabens.
  • the parabens are lower alkyl esters of para-hydroxybenzoic acid.
  • Engineered polypeptides may not have a tendency to adsorb onto the glass in a glass container when in a liquid form, therefore, a surfactant may not be required to further stabilize the pharmaceutical formulation.
  • a surfactant should be used in their formulation. These formulations may then be lyophilized. Surfactants frequently cause denaturation of protein, both of hydrophobic disruption and by salt bridge separation. Relatively low concentrations of surfactant may exert a potent denaturing activity, because of the strong interactions between surfactant moieties and the reactive sites on proteins. However, judicious use of this interaction can stabilize proteins against interfacial or surface denaturation.
  • Surfactants which could further stabilize the engineered polypeptide may optionally be present in the range of about 0.001 to 0.3% (w/v) of the total formulation and include polysorbate 80 (i.e., polyoxyethylene(20) sorbitan monooleate), CHAPS® (i.e., 3-[(3-cholamidopropyl) dimethylammonio] l- propanesulfonate), Brij® (e.g., Brij® 35, which is (polyoxyethylene (23) lauryl ether), poloxamer, or another non-ionic surfactant.
  • polysorbate 80 i.e., polyoxyethylene(20) sorbitan monooleate
  • CHAPS® i.e., 3-[(3-cholamidopropyl) dimethylammonio] l- propanesulfonate
  • Brij® e.g., Brij® 35, which is (polyoxyethylene (23) lauryl ether), poloxamer, or another non-ionic
  • sodium chloride or other salt may also be desirable to add sodium chloride or other salt to adjust the tonicity of the pharmaceutical formulation, depending on the tonicifier selected. However, this is optional and depends on the particular formulation selected. Parenteral formulations preferably may be isotonic or substantially isotonic.
  • a preferred vehicle for parenteral products is water.
  • Water of suitable quality for parenteral administration can be prepared either by distillation or by reverse osmosis.
  • Water for injection is the preferred aqueous vehicle for use in the pharmaceutical formulations.
  • additional ingredients may include, e.g., wetting agents, emulsifiers, oils, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatin or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine).
  • proteins e.g., human serum albumin, gelatin or proteins
  • a zwitterion e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine.
  • polymer solutions, or mixtures with polymers provide the opportunity for controlled release of the peptide.
  • Such additional ingredients should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.
  • Containers are also an integral part of the formulation of an injection and may be considered a component, for there is no container that is totally inert, or does not in some way affect the liquid it contains, particularly if the liquid is aqueous. Therefore, the selection of a container for a particular injection must be based on a consideration of the composition of the container, as well as of the solution, and the treatment to which it will be subjected. Adsorption of the peptide to the glass surface of the vial can also be minimized, if necessary, by use of borosilicate glass, for example, Wheaton Type I borosilicate glass #33 (Wheaton Type 1-33) or its equivalent (Wheaton Glass Co.).
  • borosilicate glass for example, Wheaton Type I borosilicate glass #33 (Wheaton Type 1-33) or its equivalent (Wheaton Glass Co.).
  • borosilicate glass vials and cartridges acceptable for manufacture include Kimbel Glass Co., West Co., Bunder Glas GMBH and Form a Vitrum.
  • the biological and chemical properties of the compound may be stabilized by formulation and lyophilization in a Wheaton Type 1-33 borosilicate serum vial to a final concentration of 0.1 mg/ml and 10 mg/ml of the compound in the presence of 5% mannitol, and 0.02% Tween 80.
  • each vial is preferably sealed with a rubber stopper closure held in place by an aluminum band.
  • Stoppers for glass vials such as, West 4416/50, 4416/50 (Teflon faced) and 4406/40, Abbott 5139 or any equivalent stopper can be used as the closure for pharmaceutical for injection.
  • these stoppers are compatible with the peptide as well as the other components of the formulation.
  • the inventors have also discovered that these stoppers pass the stopper integrity test when tested using patient use patterns, e.g., the stopper can withstand at least about 100 injections.
  • the peptide can be lyophilized in to vials, syringes or cartridges for subsequent reconstitution. Liquid formulations of the present invention can be filled into one or two chambered cartridges, or one or two chamber syringes.
  • Typical sterilization processes include filtration, steam (moist heat), dry heat, gases (e.g., ethylene oxide,
  • the engineered polypeptides described herein are administered peripherally to the subjects.
  • the liquid pharmaceutical formulations of the present invention are intended for parenteral administration. Suitable routes of administration include intramuscular, intravenous, subcutaneous, intradermal, intraarticular, intrathecal and the like. In some embodiments, the subcutaneous route of administration is preferred.
  • parenteral controlled release delivery can be achieved by forming polymeric microcapsules, matrices, solutions, implants and devices and administering them parenterally or by surgical means.
  • controlled release formulations are described in U.S. Pat. Nos. 6,368,630, 6,379,704, and 5,766,627, which are incorporated herein by reference. These dosage forms may have a lower bioavailability due to entrapment of some of the peptide in the polymer matrix or device. See e.g., U.S. Pat. Nos. 6,379,704, 6,379,703, and 6,296,842, each of which is incorporated herein by reference in its entirety and for all purposes.
  • the compounds may be provided in dosage unit form containing an amount of the engineered polypeptide that will be effective in one or multiple doses.
  • an effective amount of the engineered polypeptide will vary with many factors including the age and weight of the subject, the subject's physical condition, the condition to be treated, and other factors known in the art.
  • An effective amount of the engineered polypeptides will also vary with the particular combination
  • administration of the engineered polypeptides in combination may allow for a reduced amount of any of the administered engineered polypeptides to be an effective amount.
  • compositions provided herein include compositions wherein the active ingredient is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose.
  • the actual amount effective for a particular application will depend, inter alia, on the condition being treated. For example, when administered in methods to treat diabetes, such compositions will contain an amount of active ingredient effective to achieve the desired result (e.g. decreasing fasting blood glucose in a subject).
  • compositions will contain an amount of active ingredient effective to achieve the desired result (e.g. decrease the body mass).
  • the dosage and frequency (single or multiple doses) of compound administered can vary depending upon a variety of factors, including route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated (e.g., the disease responsive to compounds described herein; fasting blood glucose); presence of other diseases or other health-related problems; kind of concurrent treatment; and complications from any disease or treatment regimen.
  • Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of the invention.
  • Therapeutically effective amounts for use in humans may be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals.
  • the dosage in humans can be adjusted by monitoring one or more physiological parameters, including but not limited to blood sugar and body mass, and adjusting the dosage upwards or downwards, as described above and known in the art.
  • Dosages may be varied depending upon the requirements of the patient and the compound being employed.
  • the dose administered to a patient, in the context of the present invention should be sufficient to affect a beneficial therapeutic response in the patient over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side effects. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached.
  • the dosage range is 0.001% to 10% w/v. In another embodiment, the dosage range is 0.1 % to 5% w/v.
  • typical doses may contain from a lower limit of about 1 ug, 5 ug, 10 ug, 50 ug, 100 ug to 150ug per day to an upper limit of about to 50 ug, to 100 ug, to 150 ug, to 200 ug or even to 5 mg of the pharmaceutical compound.
  • the doses may be delivered in discrete unit doses at the desired interval, e.g. daily or weekly.
  • Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
  • an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is entirely effective to treat the clinical symptoms demonstrated by the particular patient.
  • This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration, and the toxicity profile of the selected agent.
  • the ratio between toxicity and therapeutic effect for a particular compound is its therapeutic index and can be expressed as the ratio between LD 50 (the amount of compound lethal in 50% of the population) and ED 50 (the amount of compound effective in 50% of the population).
  • Compounds that exhibit high therapeutic indices are preferred.
  • Therapeutic index data obtained from cell culture assays and/or animal studies can be used in formulating a range of dosages for use in humans.
  • the dosage of such compounds preferably lies within a range of plasma concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. See, e.g. Fingl et al, In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. l, p.l, 1975.
  • the exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition and the particular method in which the compound is used.
  • Codon optimized nucleotide sequences for proteins were generated by overlap PCR and subcloned into a modified pET32 vector (i.e., EK cleavage site replaced with TEV cleavage site) at Kpnl and Xhol restriction sites, as known in the art. Sequence verified vector DNA was then transformed to BL21 cells (Novagen), and induced @ 30C O/N in Magic Media autoinducing media (Invitrogen).
  • Exemplary construct 1 The DNA sequence following encodes the engineered polypeptide having [ 14 L]Ex-4 (SEQ ID NO: l) linked to a humanized seal leptin (SEQ ID NO: 106) through a TGGGGSASG linker:
  • Exemplary construct 2 The DNA sequence following encodes the engineered polypeptide which includes Ex-4(l-28) (SEQ ID NO:5) linked to a humanized seal leptin (SEQ ID NO: 100) through a TGGGGSAS linker:
  • Exemplary construct 3 The DNA sequence following encodes the engineered polypeptide which includes of [ 14 Leu]Exendin-4 (SEQ ID NO: 1) linked to a humanized seal leptin (SEQ ID NO: 100) through a TGGGGSAS linker:
  • TEV protease site suitable for subsequent processing TEV protease site suitable for subsequent processing:
  • humanized chimeric polypeptides including humanized chimeric seal leptins, described herein have comparable, and some even superior, properties compared with metreleptin (SEQ ID NO:44). These properties include biological properties such as leptin binding activity, leptin functional activity, and food intake in mice, and pharmaceutical properties such as solubility in neutral pH.
  • Leptin binding can be assessed by measuring displacement of 125 I-recombinant-Leptin (murine) from the surface membrane expressing chimeric Leptin (Hu) - EPO (Mu) receptor presented by the 32D OBECA cell line. See e.g., J Biol Chem 1998; 273(29): 18365-18373).
  • Purified cell membranes can be prepared by homogenization from harvested confluent cell cultures of 32D OBECA cells. Membranes can be incubated with 125 I-rec-Murine-Leptin and increasing concentrations of test compound for 3 hours at ambient temperature in 96-well polystyrene plates.
  • Bound and unbound ligand fractions can then be separated by rapid filtration onto 96-well GF/B plates pre-blocked for at least 60' in 0.5% PEI (polyethyleneimine). Glass fiber plates can then be dried, scintillant added, and CPM determined by reading on a multiwell scintillation counter capable of reading radiolabeled iodine.
  • PEI polyethyleneimine
  • phosphorylated STAT5 Signal Transducer and Activator of Transcription 5
  • 32D-Keptin cells ectopically expressing chimeric Hu-Leptin/Mu-EPO receptor
  • the 32D-Keptin cells can be leptin weaned overnight and then treated with test compounds in 96-well plates for 30 minutes at 37°C followed by cell extraction.
  • the pSTAT5 levels in the cell lysates can be determined using the Perkin Elmer AlphaScreen ® SureFire ® pSTAT5 assay kit in a 384-well format (PROXIPLATETM 384 Plus).
  • the efficacy of test compounds can be determined relative to the maximal signal in cell lysates from cells treated with Human leptin.
  • Solubility was measured with the following assay: proteins were concentrated at 4C, spun to remove precipitates, then allowed to equilibrate at room temperature overnight. They were filtered to remove precipitates and then the concentration was determined by measuring absorbance at OD 2 8o and using the theoretical molar extinction coefficient.
  • Seal leptin per se (SEQ ID NO:40) binds in the Obeca cell binding assay with reduced affinity relative to metreleptin. However, some of the tested humanized chimeric seal leptin bind with greater affinity relative to metreleptin. Moreover, most of the tested humanized chimeric seal leptins demonstrate similar functional properties in the Obeca Stat5 functional assay.
  • chimeric polypeptides described herein have comparable, and some even superior, physical stability compared with A100 (SEQ ID NO:44).
  • the chimeric polypeptides have comparable or superior purity and potency, compared with A100 (SEQ ID NO:44). Table 3.
  • GLP- 1 [ 14 Leu]Exendin-4, [ 14 Leu]Exendin-4(l -28) demonstrated sub-nanomolar EC50 in the GLP-1 functional assay.
  • the engineered polypeptide (SEQ ID NO:261) demonstrated sub-micromolar functional activity in this assay.
  • this compound demonstrated functional activity in the leptin assay as a level similar to the activity of metreleptin, or to the humanized chimeric seal leptin with structure of SEQ ID NO: 100.
  • engineered polypeptides can provide sub-micromolar GLP-1 functional activity and sub-nanomolar leptin functional activity.
  • GLP-1 and the exendin analogs demonstrate sub-nanomolar binding at the GLP-1 receptor, and the engineered polypeptide demonstrates an affinity within an order of magnitude of GLP-1. Table 5.
  • humanized chimeric seal leptins have unexpected superiority over seal leptin per se with respect to binding and activity at the human leptin receptor. Moreover, humanized chimeric seal leptins demonstrate significantly greater solubility relative to human leptin, and the binding of the engineered polypeptide to the GLP-1 receptor is reduced by less than a factor of 10 relative to GLP-1. Thus, without wishing to be bound by any theory, it is believed that these unexpected advantages of humanized chimeric seal leptins accrue to the exendin agonist engineered polypeptides contemplated herein. Moreover, as known in the art, the levels of leptin required for leptin therapy are high relative to the levels required for
  • GLP-1 agonists in GLP-1 agonist therapy e.g., exendins as described herein. Accordingly, the apparent decrease in GLP-1 functional activity of the engineered polypeptide as disclosed for SEQ ID NO:261 above relative to GLP-1 and the exendins, can be viewed as a mechanism for balancing multiple activities, i.e., GLP-1 and leptin functional activity, in a single engineered polypeptide for therapeutic use.
  • Embodiment 1 An engineered polypeptide comprising: a first peptide hormone domain (HD1) comprising an exendin domain sequence; and a second peptide hormone domain (HD2) comprising a humanized chimeric seal leptin sequence; wherein said HD1 is covalently bonded to said HD2 through a bond, or through a linker LI .
  • HD1 a first peptide hormone domain
  • HD2 a second peptide hormone domain
  • HD2 comprising a humanized chimeric seal leptin sequence
  • Embodiment 2 The engineered polypeptide according to embodiment 1, wherein said humanized chimeric seal leptin sequence has the sequence of SEQ ID NO:40, wherein 5% to 55% of SEQ ID NO:40 is substituted with a corresponding human leptin sequence.
  • Embodiment 3 The engineered polypeptide according to embodiment 1, wherein said humanized chimeric seal leptin sequence has the sequence of SEQ ID NO:41, wherein 5% to 55% of SEQ ID NO:41 is substituted with a corresponding human leptin sequence.
  • Embodiment 4 The engineered polypeptide according to embodiment 1, said engineered polypeptide having greater binding at a human leptin receptor relative to seal leptin binding.
  • Embodiment 5 The engineered polypeptide according to embodiment 1, said engineered polypeptide having greater solubility relative to human leptin solubility.
  • Embodiment 6 The engineered polypeptide according to any one of embodiments 1 to 5, said linker LI covalently linking said HD1 and said HD2.
  • Embodiment 7 The engineered polypeptide according to any one of embodiments 1 to 6, wherein said engineered polypeptide comprises said HD1 as an N-terminal moiety and said HD2 as a C terminal moiety.
  • Embodiment 8 The engineered polypeptide according to embodiment 7, having the structure HD1 HD2.
  • Embodiment 9 The engineered polypeptide according to embodiment 7, having the structure HD1 LI HD2.
  • Embodiment 10 The engineered polypeptide according to any one of embodiments 1 to 9, wherein said HD1 sequence consists of an exendin domain sequence.
  • Embodiment 11 The engineered polypeptide according to embodiment 10, wherein said exendin domain sequence has at least 65% identity with an Exendin-4 sequence (SEQ ID NO:3).
  • Embodiment 12 The engineered polypeptide according to embodiment 10, wherein said exendin domain sequence is an Exendin-4 sequence (SEQ ID NO:3).

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Abstract

L'invention concerne des composés présentant, entre autres, une bonne durée d'action, une puissance élevée et/ou des régimes de dosage pratiques. Ces composés sont des polypeptides génétiquement modifiés qui incorporent un domaine de l'exendine en combinaison avec une leptine chimère humanisée issue de la graisse de phoque. L'invention concerne également des compositions pharmaceutiques et des méthodes de traitement de maladies telles que le diabète, la surcharge pondérale, l'obésité, la maladie d'Alzheimer, le syndrome de l'intestin court, la stéatose hépatique, la dyslipidémie, la coronaropathie, l'accident vasculaire cérébral, l'hyperlipidémie, la stéatohépatite non alcoolique (NASH) ou la maladie de Parkinson.
PCT/US2013/034152 2012-03-28 2013-03-27 Polypeptides génétiquement modifiés WO2013148871A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9670261B2 (en) 2012-12-21 2017-06-06 Sanofi Functionalized exendin-4 derivatives
US9694053B2 (en) 2013-12-13 2017-07-04 Sanofi Dual GLP-1/glucagon receptor agonists
US9750788B2 (en) 2013-12-13 2017-09-05 Sanofi Non-acylated exendin-4 peptide analogues
US9751926B2 (en) 2013-12-13 2017-09-05 Sanofi Dual GLP-1/GIP receptor agonists
US9758561B2 (en) 2014-04-07 2017-09-12 Sanofi Dual GLP-1/glucagon receptor agonists derived from exendin-4
US9771406B2 (en) 2014-04-07 2017-09-26 Sanofi Peptidic dual GLP-1/glucagon receptor agonists derived from exendin-4
US9775904B2 (en) 2014-04-07 2017-10-03 Sanofi Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists
US9789165B2 (en) 2013-12-13 2017-10-17 Sanofi Exendin-4 peptide analogues as dual GLP-1/GIP receptor agonists
US9932381B2 (en) 2014-06-18 2018-04-03 Sanofi Exendin-4 derivatives as selective glucagon receptor agonists
US9982029B2 (en) 2015-07-10 2018-05-29 Sanofi Exendin-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists
EP3139955A4 (fr) * 2014-04-30 2018-06-27 President and Fellows of Harvard College Protéines de fusion pour le traitement du cancer et procédés associés
US10758592B2 (en) 2012-10-09 2020-09-01 Sanofi Exendin-4 derivatives as dual GLP1/glucagon agonists
US10806797B2 (en) 2015-06-05 2020-10-20 Sanofi Prodrugs comprising an GLP-1/glucagon dual agonist linker hyaluronic acid conjugate
US11123405B2 (en) 2015-12-23 2021-09-21 The Johns Hopkins University Long-acting GLP-1R agonist as a therapy of neurological and neurodegenerative conditions

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060293232A1 (en) * 2004-02-11 2006-12-28 Amylin Pharmaceuticals, Inc. Hybrid polypeptides with selectable properties
US20090214534A1 (en) * 2004-12-02 2009-08-27 Steve Holmes Bispecific Domain Antibodies Targeting Serum Albumin And GLP-1 Or PYY
US20100168011A1 (en) * 2006-12-12 2010-07-01 Amylin Pharmaceuticals, Inc. Pharmaceutical Formulations and Methods for Making the Same
US20100292172A1 (en) * 2006-03-21 2010-11-18 Amylin Pharmaceuticals, Inc. Peptide-Peptidase Inhibitor Conjugates and Methods of Using Same
US20110200623A1 (en) * 2008-07-23 2011-08-18 Hanmi Holdings Co., Ltd. Polypeptide complex comprising non-peptidyl polymer having three functional ends

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060293232A1 (en) * 2004-02-11 2006-12-28 Amylin Pharmaceuticals, Inc. Hybrid polypeptides with selectable properties
US20090214534A1 (en) * 2004-12-02 2009-08-27 Steve Holmes Bispecific Domain Antibodies Targeting Serum Albumin And GLP-1 Or PYY
US20100292172A1 (en) * 2006-03-21 2010-11-18 Amylin Pharmaceuticals, Inc. Peptide-Peptidase Inhibitor Conjugates and Methods of Using Same
US20100168011A1 (en) * 2006-12-12 2010-07-01 Amylin Pharmaceuticals, Inc. Pharmaceutical Formulations and Methods for Making the Same
US20110200623A1 (en) * 2008-07-23 2011-08-18 Hanmi Holdings Co., Ltd. Polypeptide complex comprising non-peptidyl polymer having three functional ends

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE UNIPROTKB/SWISS-PROT 1 May 2005 (2005-05-01), accession no. 706DO *

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* Cited by examiner, † Cited by third party
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US10758592B2 (en) 2012-10-09 2020-09-01 Sanofi Exendin-4 derivatives as dual GLP1/glucagon agonists
US9670261B2 (en) 2012-12-21 2017-06-06 Sanofi Functionalized exendin-4 derivatives
US9745360B2 (en) 2012-12-21 2017-08-29 Sanofi Dual GLP1/GIP or trigonal GLP1/GIP/glucagon agonists
US10253079B2 (en) 2012-12-21 2019-04-09 Sanofi Functionalized Exendin-4 derivatives
US9751926B2 (en) 2013-12-13 2017-09-05 Sanofi Dual GLP-1/GIP receptor agonists
US9789165B2 (en) 2013-12-13 2017-10-17 Sanofi Exendin-4 peptide analogues as dual GLP-1/GIP receptor agonists
US9694053B2 (en) 2013-12-13 2017-07-04 Sanofi Dual GLP-1/glucagon receptor agonists
US9750788B2 (en) 2013-12-13 2017-09-05 Sanofi Non-acylated exendin-4 peptide analogues
US9758561B2 (en) 2014-04-07 2017-09-12 Sanofi Dual GLP-1/glucagon receptor agonists derived from exendin-4
US9771406B2 (en) 2014-04-07 2017-09-26 Sanofi Peptidic dual GLP-1/glucagon receptor agonists derived from exendin-4
US9775904B2 (en) 2014-04-07 2017-10-03 Sanofi Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists
EP3139955A4 (fr) * 2014-04-30 2018-06-27 President and Fellows of Harvard College Protéines de fusion pour le traitement du cancer et procédés associés
US10308697B2 (en) 2014-04-30 2019-06-04 President And Fellows Of Harvard College Fusion proteins for treating cancer and related methods
US10538566B2 (en) 2014-04-30 2020-01-21 President And Fellows Of Harvard College Fusion proteins for treating cancer and related methods
US11014973B2 (en) 2014-04-30 2021-05-25 President And Fellows Of Harvard College Fusion proteins for treating cancer and related methods
US9932381B2 (en) 2014-06-18 2018-04-03 Sanofi Exendin-4 derivatives as selective glucagon receptor agonists
US10806797B2 (en) 2015-06-05 2020-10-20 Sanofi Prodrugs comprising an GLP-1/glucagon dual agonist linker hyaluronic acid conjugate
US9982029B2 (en) 2015-07-10 2018-05-29 Sanofi Exendin-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists
US11123405B2 (en) 2015-12-23 2021-09-21 The Johns Hopkins University Long-acting GLP-1R agonist as a therapy of neurological and neurodegenerative conditions

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