WO2013103981A1 - Compositions et procédés d'utilisation des polypeptides fndc5 - Google Patents

Compositions et procédés d'utilisation des polypeptides fndc5 Download PDF

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Publication number
WO2013103981A1
WO2013103981A1 PCT/US2013/020566 US2013020566W WO2013103981A1 WO 2013103981 A1 WO2013103981 A1 WO 2013103981A1 US 2013020566 W US2013020566 W US 2013020566W WO 2013103981 A1 WO2013103981 A1 WO 2013103981A1
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Prior art keywords
polypeptide
sequence
fndc5
isolated polypeptide
conjugate
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PCT/US2013/020566
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English (en)
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Jasbir S. Seehra
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Ember Therapeutics, Inc.
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Publication of WO2013103981A1 publication Critical patent/WO2013103981A1/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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Fibronectin type III domain-containing protein (Fndc5) is a 196-amino acid fibronectin type III domain containing protein transmembrane protein. Fndc5 is proteolytically cleaved in vivo, with the cleaved portion secreted into the circulation.
  • the invention features, an isolated polypeptide, e.g., consisting, or consisting essentially, of:
  • a functional fragment e.g., a fragment having biological activity, e.g., an a activity described herein, of any of (a), (b), (c), or (d),
  • SEQ ID NO: 1 is:
  • sequence of (a) is selected from Column 1 of Table 1.
  • sequence of (a) is selected from Column 2 of Table 1.
  • sequence of (a) is selected from Column 3 of Table 1.
  • sequence of (a) is selected from Column 4 of Table 1.
  • sequence of (a) is selected from Column 5 of Table 1.
  • the isolated polypeptide has other than a C at residue 87.
  • cysteine at position 87 has been deleted or replaced by another amino acid
  • position 87 is other than cysteine, e.g., an alanine, a glycine or a serine.
  • an isolated polypeptide comprises one or more additional amino acid modifications, e.g., at an amino acid 1, 2, 3, 4, or 5 amino acids upstream or downstream from the amino acid at the position corresponding to position 87, e.g., one or more modifications which function as a glycosylation site, e.g., the position corresponding to amino acid 87 is a serine and the position corresponding to position 85 is an asparagine.
  • the isolated polypeptide has one or more additional amino acid modifications, e.g., at an amino acid 1, 2, 3, 4, or 5 amino acids upstream or downstream from the amino acid at the position corresponding to position 87, e.g., the polypeptide or functional fragment has one or more modifications which function as a glycosylation site in the polypeptide or functional fragment, e.g., the position corresponding to amino acid 87 is a serine and the position corresponding to position 85 is an asparagine.
  • the amino acid residue at position 87 is not present (i.e., has been deleted).
  • the isolated polypeptide is conjugated, e.g., fused, to a second moiety (e.g., other than an fndc5 sequence or other than a sequence of SEQ ID NO: 1), e.g., a second moiety attached directly or via a linker, e.g., a peptide linker, e.g., a linker described herein, to the polypeptide.
  • the invention features, a preparation, e.g., a purified, isolated or pharmaceutically acceptable preparation, of two or more of the polypeptide species disclosed herein (or fragments, analogs or conjugates thereof described herein).
  • the preparation is heterogeneous at the C terminus of the polypeptide, e.g., it comprises:
  • the preparation is heterogeneous at the at the N terminus of the polypeptide, e.g., it comprises:
  • heterogeneous preparation comprises:
  • X an X th polypeptide having an X th residue selected from the residues D29-A33 of SEQ ID NO: 1 as the N terminal residue, wherein X can be the integer 4 or 5.
  • the preparation is heterogeneous at the C terminus and the N terminus of the polypeptide.
  • the preparation comprises 2, or more (e.g., 3, 4, 5, 6) of the polypeptides (or fragments, analogs or conjugates thereof described herein) of column 1, 2, 3, 4, or 5, of Table 1.
  • the preparation comprises 2, or more (e.g., 3, 4, 5, 6) of the polypeptides (or fragments, analogs or conjugates thereof described herein) of Column 1-5, e.g., from: columns 1 and 2; columns 1 and 3; columns 1 and 4; columns 1 and 5; columns 2 and 3; columns 2 and 4; columns 2 and 5; columns 3 and 4, columns 3 and 5; columns 4 and 5;
  • the invention features, an isolated fndc5 polypeptide or functional fragment thereof, e.g., a polypeptide or fragment described herein, having one or more modifications, e.g., substitutions or deletions, of an amino acid residue as compared to the amino acid residue in the corresponding position of a naturally occurring fndc5 polypeptide described herein (e.g., the fndc5 polypeptide of SEQ ID NO:2), e.g., a variant polypeptide having one or more cysteine residues modified, e.g., substituted or deleted cysteine residues.
  • Polypeptides with amino acid sequence modifications or variants, e.g., deletions or substitutions are referred to herein as analogs.
  • the polypeptide is a full length fndc5 polypeptide, see e.g., SEQ ID NO:2, or a fragment thereof, e.g., a naturally occurring fragment thereof, any fragment described herein, e.g. as disclosed in Table 1, as well as fragments having a C terminus of E140, any of which can have, e.g., a deletion or substitution of a naturally occurring C, e.g., a deletion, or residue other than C, at position 87.
  • polypeptide comprises a polypeptide described herein, e.g., a polypeptide of Table 1, wherein an amino acid residue at a position which corresponds to a cysteine in the fndc5 polypeptide of SEQ ID NO:2 is an amino acid other than cysteine in the polypeptide, wherein SEQ ID NO:2 is
  • residue number 29 the initial residue (D) of SEQ ID No: 1 is referred to as residue number 29.
  • cysteine at position 87 has been deleted from, or replaced by another amino acid.
  • polypeptide e.g., a fndc5 variant
  • polypeptide has an amino acid at position 87 other than cysteine, e.g., the fndc5 variant has an alanine, a glycine or a serine at that position.
  • polypeptide e.g., aFndc5 variant
  • polypeptide has one or more additional amino acid modifications, e.g., at an amino acid 1, 2, 3, 4, or 5 amino acids upstream or downstream from the amino acid at the position corresponding to position 87.
  • polypeptide e.g., Fndc5 variant
  • glycosylation site in the polypeptide e.g., Fndc5 variant, e.g., the position corresponding to amino acid 87 is a serine and the position corresponding to position 85 is an asparagine.
  • the isolated polypeptide or fragment thereof described herein has an amino acid other than a cysteine at a position that corresponds to amino acid residue 87 of the fndc5 polypeptide of SEQ ID NO:2.
  • the isolated polypeptide or functional fragment has one or more additional amino acid modifications, e.g., at an amino acid 1, 2, 3, 4, or 5 amino acids upstream or downstream from the amino acid at the position corresponding to position 87, e.g., the polypeptide or functional fragment has one or more modifications which function as a glycosylation site in the polypeptide or functional fragment, e.g., the position corresponding to amino acid 87 is a serine and the position corresponding to position 85 is an asparagine.
  • amino acid residue at position 87 of the polypeptide, e.g., fndc5 polypeptide, of SEQ ID NO:2 is not present (i.e., has been deleted) at the corresponding position in the isolated polypeptide or fragment.
  • the invention features, a conjugate, e.g., a fusion, comprising a polypeptide, e.g., an isolated polypeptide, described herein, e.g., an isolated peptide of Table 1, and a second moiety, e.g., a second moiety attached directly or via a linker, e.g., a peptide linker, e.g., a linker described herein, to the polypeptide.
  • a linker e.g., a peptide linker, e.g., a linker described herein
  • addition of the second moiety does not result in the isolated peptide comprising additional contiguous residues of SEQ ID NO: 1 or SEQ ID NO:2 at its C terminal end.
  • the second moiety comprises a polypeptide, e.g., a polypeptide other than an Fndc5 sequence.
  • the second moiety comprises one or more of a linker and an immunoglobulin fragment, e.g., an Fc region.
  • the conjugate comprises a linker which comprises one or more amino acids, e.g., the linker comprises six or less amino acids, e.g., the linker comprises one or more glycine residue, e.g., the linker has the amino acid sequence glycine-glycine-glycine.
  • the second moiety comprises an agent which increases plasma solubility and/or half life, e.g., as compared to the polypeptide in the absence of the second agent.
  • the second moiety comprises a non-peptide water soluble polymer, e.g., a polyethylene glycol moiety.
  • more than one non-peptide water soluble polymer e.g., polyethylene glycol moiety
  • polypeptide e.g., more or more polyethylene glycol moieties are attached to a primary amine of the polypeptide (e.g., one or more polyethylene glycol moieties are attached to an N-terminal amine and/or a lysine residue of the polypeptide).
  • the second moiety is a heterologous polypeptide.
  • heterologous polypeptide is selected from the group consisting of albumin, transferrin, an antibody, or a fragment thereof (e.g., an antibody fragment, e.g., an Fc polypeptide, e.g., an IgGl Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide).
  • an antibody fragment e.g., an Fc polypeptide, e.g., an IgGl Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide.
  • the heterologous polypeptide is an Fc polypeptide, or fragment or variant thereof, e.g., an Fc variant that has at least one amino acid modification in the Fc region as compared to a naturally occurring Fc polypeptide, (e.g., an Fc variant that modulates binding to an Fc receptor e.g., as compared to the binding of the parent Fc polypeptide to the Fc receptor).
  • the Fc variant has a modification at one or more amino acid positions:238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 288, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, (an Fc variant having one or more amino acid substitution described in USPN: 6737056, 7790858, 7785791, 7416727, 7371826, 7335742, 7332581, 7122637, PCT Publication No.: WO 2010/045193.
  • a conjugate comprises a polypeptide or functional fragment thereof described herein and a second moiety.
  • the conjugate has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more, in vivo, e.g., in humans.
  • the polypeptide or functional fragment is physically associated with a second moiety that improves serum residence time, e.g., a moiety described herein.
  • the second moiety is an IgG, e.g., an IgGl, IgG2, IgG3 or IgG4, that has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in humans.
  • the polypeptide or functional fragment thereof is modified to include, e.g., PEGylation, fusion to serum albumin (e.g., human serum albumin), conjugation to human serum albumin, fusion to transferring, conjugation to transferrin, HESylation (HESylation
  • the conjugate is a conjugate described herein.
  • the disclosure features a method providing a subject, e.g., a human, with a polypeptide disclosed herein, e.g., a method of treating or preventing a disorder or disease described herein a subject, e.g., a human subject.
  • the method comprises:
  • such disorders include, e.g., obesity or diabetes or of a comorbidity of obesity or an obesity related disorder, or an age related disorder, or a disorder in which one or more symptoms can be alleviated by exercise.
  • the subject to whom the compound is administered may be overweight, for example, obese.
  • the subject may be diabetic, for example having insulin resistance or glucose intolerance, or both.
  • the subject may have diabetes mellitus, for example, the subject may have Type II diabetes.
  • the subject may be overweight, for example, obese and have diabetes mellitus, for example, Type II diabetes.
  • Obesity related disorders that can be treated or prevented with the polypeptides and functional fragment include, but are not limited to, type 2 diabetes, cardiovascular disease;
  • cancers e.g., breast, ovarian, esophageal, colorectal, liver, pancreatic, gallbladder, stomach, endometrial, cervical, prostate, kidney, non-Hodgkin's lymphoma, multiple myeloma; arthritis, sleep apnea; obstructive sleep apnea; obesity hypoventilation syndrome; asthma; increased complications during general anaesthesia; stroke, bone problems, joint problems, e.g., pain, stiffness; toosteoarthritis; congestive heart failure; erectile dysfunction; urinary incontinence; chronic renal failure; hypogonadism; gastroesophageal reflux disease; fatty liver disease; cholelithiasis; stretch marks; acanthosis nigricans; lymphedema; cellulitis;
  • hirsutism intertrigo; low back pain; poor mobility; gout; depression; social stigmatization; multiple sclerosis; idiopathic intracranial hypertension; dementia; carpal tunnel syndrome;
  • migranes meralgia paresthetica
  • intrauterine fetal death birth defects; infertility; menstral disorders; polycyctic ovary disease; deep vein thrombosis; abnormal cholesterol levels; high blood pressure; ischemic heart disease; angina; myocardial infarction.
  • polypeptide, functional fragment or conjugate, or preparation is administered in a therapeutically effective amount.
  • the polypeptide, functional fragment, conjugate or composition or preparation is administered in combination with another treatment for the disorder, e.g., another treatment described herein.
  • the invention features, a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide or functional fragment thereof or a conjugate described herein, or a heterogeneous mixture described herein, and optionally, a pharmaceutically acceptable carrier.
  • the composition can be at least 10, 20, 30, 50, 75, 85, 90, 95, 98, 99, or 99.9% free of other protein species, e.g., protein species other than an Fncd5 polypeptide (or heterogeneous mixture described herein) and/or free of aggregates.
  • the invention features a kit comprising a polypeptide, functional fragment, conjugate or composition or preparation described herein.
  • the disclosure features a nucleic acid encoding a polypeptide, functional fragment or conjugate described herein. In some aspects, the disclosure features vectors and host cells comprising such nucleic acids.
  • obesity is a condition in which excess body fat may put a person at health risk (see Barlow and Dietz, Pediatrics 102:E29, 1998; National Institutes of Health,
  • BMI Body Mass Index
  • waist circumference is used to assess obesity. Excess abdominal fat is an important, independent assessment of the risks associated with obesity or being overweight. Waist circumference measurement is particularly useful in patients who are categorized as normal or overweight. It is not usually necessary to measure waist circumference in individuals with BMIs > 35 kg/m since it adds little to the predictive power of the disease risk classification of BMI. Men who have waist circumferences greater than 40 inches (102 cm), and women who have waist circumferences greater than 35 inches (90 cm), are at higher risk of diabetes, dyslipidemia, hypertension, and cardiovascular disease because of excess abdominal fat. Individuals with waist circumferences greater than these values should be considered one risk category above that defined by their BMI.
  • obesity affects both the morbidity and mortality of individuals.
  • an overweight or obese individual is at increased risk for heart disease, non-insulin dependent (type 2) diabetes, hypertension, stroke, cancer (e.g. endometrial, breast, prostate, and colon cancer), dyslipidemia, gall bladder disease, sleep apnea, reduced fertility, and
  • an individual who is "overweight” is an individual who weighs more than their ideal body weight.
  • An overweight individual can be obese, but is not necessarily obese.
  • an overweight individual is any individual who desires to decrease their weight.
  • an overweight individual is an individual with a BMI of 25.0 kg/m to 29.9 kg/m 2 .
  • polypeptide As used herein, the terms “polypeptide,” “peptide” and “protein” are used
  • a polypeptide is a polymer in which the monomers are amino acid residues which are joined together through amide bonds.
  • the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being most commonly used.
  • an alpha-amino acid will be assumed to be the
  • protein as used herein encompass any amino acid sequence and include modified sequences such as glycoproteins. The terms cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.
  • polypeptide fragment refers to a portion of a polypeptide, for example such a fragment which exhibits at least one useful sequence.
  • functional fragments of a polypeptide refers to all fragments of a polypeptide that retain an activity of the polypeptide, e.g., an activity described herein.
  • compositions produced artificially or naturally can be any composition or preparation that is removed from at least 90% of at least one component of a natural sample from which the isolated composition can be obtained.
  • compositions produced artificially or naturally can be any composition or preparation that is removed from at least 90% of at least one component of a natural sample from which the isolated composition can be obtained.
  • polypeptide or conjugate refers to a polypeptide or conjugate that is removed from at least 90% of at least one component of a natural sample from which the isolated polypeptide or conjugate can be obtained.
  • Polypeptides (or conjugates) can be "of at least" a certain degree of purity if the species or population of species of interest (e.g., for heterogeneous mixtures) is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight-weight basis.
  • preventing or to "prevent” a disease or disorder in a subject refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease or disorder is prevented, that is, administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) so that it protects the host against developing the unwanted condition.
  • a pharmaceutical treatment e.g., the administration of a drug, such that at least one symptom of the disease or disorder is prevented, that is, administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) so that it protects the host against developing the unwanted condition.
  • Preventing" a disease or disorder may also be referred to as “prophylaxis” or “prophylactic treatment.”
  • a "therapeutically effective dosage” preferably modulates a measurable parameter, e.g., an activity described herein, by a statistically significant degree or at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
  • a measurable parameter e.g., an activity described herein
  • the ability of a compound to modulate a measurable parameter e.g., a disease-associated parameter
  • this property of a composition can be evaluated by examining the ability of the compound to modulate a parameter in vitro.
  • Treating" a disease (or disorder) in a subject or “treating" a subject having a disease or disorder refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is cured, alleviated or decreased.
  • combination therapy or treating in combination refers to administration of a plurality of agents, e.g., wherein at least one agent, e.g., an isolated polypeptide disclosed herein, is administered to a subject, e.g., a human subject.
  • introduction of the agents into the subject can be at different times.
  • the agents are administered in overlapping regimens, or such that the subject is simultaneously exposed to both agents, or such that the response of the subject is better than would be seen with either agent administered alone.
  • Fndc5 is a 196-amino acid fibronectin type III domain containing protein transmembrane protein. N terminal sequence of mature Fndc5 is proteolytically cleaved in vivo, to provide a circulating polypeptide hormone, sometimes referred to as Irisin. Irisin results in an increase in the ratio of brown fat, which is associated with energy conversion, to white fat, which is associated with energy storage. When injected into obese, diabetic laboratory mice the hormone switches on genes that would help burn fat, just as would exercise. Irisin has also been shown to improve glucose tolerance, and provide weight loss, in mice fed a high-fat diet.
  • fibronectin domain of Fndc5 is a tight structure from D29- P122. Proteolytic cleavage, whether endogenous or occurring in preparation of Fndc5 polypeptides, would tend to be favored outside that tightly structured region, e.g., between residues 127 and 140, which is supported by observations of in vitro cleavage events.
  • Polypeptides disclosed herein allow for selection of specific fragments, or mixtures thereof, e.g., polypeptides with selected C terminal endpoints as well of preparations having mixtures of selected C terminal endpoints.
  • Polypeptides disclosed herein can be modified, e.g., by fusion to other polypeptides, e.g., polypeptides, e.g., immunoglobulin fragments, that can optimize drug characteristics, e.g., half- life.
  • Other modification include modifications that minimize the formation of covalent aggregates, e.g., alteration of the polypeptide, e.g., by removal (e.g., by mutation/insertion of glycosylation site) of the ability of a cysteine residue, e.g., cys87, to form covalent aggregates, providing for more stable preparations.
  • Alphabetic, numerical, or other headings do not imply hierarchy or sequence, order, or importance. They are merely for ease of reading and should not be used to limit the disclosure herein.
  • the present disclosure features, at least in part, an Fndc5 polypeptide, or fragment thereof, and conjugates comprising such polypeptides and fragments.
  • the polypeptides comprise a fndc5 sequence that ends at or before K139, with the balance of the C terminal portion being absent.
  • the disclosure also features pharmaceutical compositions, nucleic acid molecules encoding the Fndc5 polypeptides or conjugate, vectors, host cells, and methods of making the same are disclosed.
  • references to an Fndc5 polypeptide shall be understood to refer to fibronectin domain containing 5 protein and are intended to include fragments, variants, and derivatives thereof. Exemplary Fndc5 polypeptides are described herein.
  • the polypeptides, functional fragments, conjugates and compositions described herein can be used to treat or prevent a disorder.
  • disorders include, e.g., obesity or diabetes or of a co-morbidity of obesity or an obesity related disorder, or an age related disorder, or a disorder in which one or more symptoms can be alleviated by exercise.
  • the subject to whom the compound is administered may be overweight, for example, obese, e.g., a BMI greater than 25.
  • the subject may be diabetic, for example having insulin resistance or glucose intolerance, or both.
  • the subject may have diabetes mellitus, for example, the subject may have Type II diabetes.
  • the subject may be overweight, for example, obese and have diabetes mellitus, for example, Type II diabetes.
  • Obesity related disorders that can be treated or prevented with the polypeptides and functional fragment, include, but are not limited to, type 2 diabetes, cardiovascular disease; hypertension; and certain cancers, e.g., breast, ovarian, esophageal, colorectal, liver, pancreatic, gallbladder, stomach, endometrial, cervical, prostate, kidney, non-Hodgkin' s lymphoma, multiple myeloma; arthritis, sleep apnea; obstructive sleep apnea; obesity hypoventilation syndrome; asthma; increased complications during general anaesthesia; stroke, bone problems, joint problems, e.g., pain, stiffness; toosteoarthritis;
  • cancers e.g., breast, ovarian, esophageal, colorectal, liver, pancreatic, gallbladder, stomach, endometrial, cervical, prostate, kidney, non-Hodgkin' s lymphoma, multiple myeloma
  • hypogonadism gastroesophageal reflux disease; fatty liver disease; cholelithiasis; stretch marks; acanthosis nigricans; lymphedema; cellulitis; hirsutism; intertrigo; low back pain; poor mobility; gout; depression; social stigmatization; multiple sclerosis; idiopathic intracranial hypertension; dementia; carpal tunnel syndrome; migranes; meralgia paresthetica; intrauterine fetal death; birth defects; infertility; menstral disorders; polycyctic ovary disease; deep vein thrombosis; abnormal cholesterol levels; high blood pressure; ischemic heart disease; angina; myocardial infarction.
  • polypeptides are provided in Table 1. In embodiments these can be fused, at the C or N terminus to a non-Fndc-5 moiety, e.g., a polypeptide that enhances in vivo half life.
  • D29-T121 S30-T121.
  • the Fndc5 polypeptide can be a variant of a naturally occurring Fndc5 polypeptide, e.g., a naturally occurring murine Fndc5 polypeptide or a naturally occurring Fndc5 polypeptide (e.g., a human Fndc5 polypeptide of SEQ ID NO:2 or naturally occurring isoforms thereof) or a variant of a polypeptide disclosed herein, e.g., in Table 1.
  • the Fndc5 polypeptide can differ, e.g., from a naturally occurring Fndc5 polypeptide by, for example, a modification (e.g., a substitution, deletion or insertion) of one or more amino acid residues of the naturally occurring Fndc5 polypeptide.
  • the Fndc5 polypeptide can, e.g., have one or more conservative or non- conservative amino acid substitution.
  • a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • the Fdnc5 polypeptide includes one or more mutations (e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions) relative to a naturally occurring Fndc5 polypeptide or fragment of a naturally occurring Fndc5 polypeptide, e.g., a naturally occurring Fndc5 polypeptide or a fragment thereof described herein.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the Fndc5 polypeptide without abolishing or more preferably, without substantially altering a biological activity, whereas changing an "essential" amino acid residue results in a substantial loss of activity.
  • An Fndc5 polypeptide may have mutations (e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions) (e.g., at least one, two, three, or four, and/or less than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations) relative to a naturally occurring Fndc5 polypeptide described herein. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect biological properties, such as binding activity, can be predicted, e.g., by evaluating whether the mutation is conservative or by an activity assay, e.g., an activity assay described herein.
  • mutations e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions) (e.g., at least one, two, three, or four, and/or less than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations) relative to a
  • sequence identity is calculated as follows.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 100% of the length of the reference sequence.
  • sequences similar or homologous e.g., at least about 85% sequence identity
  • sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
  • a polypeptide can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a Fndc5 polypeptide or fragment described herein.
  • nucleic acid segments hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02. Particular binding proteins may show a difference, e.g., in specificity or binding that are statistically significant (e.g., P value ⁇ 0.05 or 0.02).
  • the terms “induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease” or the like, e.g., which denote distinguishable qualitative or quantitative differences between two states, may refer to a difference, e.g., a statistically significant difference, between the two states.
  • An Fndc5 polypeptide can be a full length polypeptide described herein or a functional fragment thereof.
  • An Fncd5 polypeptide, functional fragment thereof or conjugate can be recombinant protein.
  • the disclosure features a polypeptide (e.g., an isolated polypeptide), fragment or conjugate that has one or more activity described herein.
  • the polypeptide can include one or more of the following characteristics: (a) an amino acid sequence described herein, e.g., in Table 1 (b) an amino acid sequence at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to an amino acid sequence described herein; (c) an amino acid sequence having at least one, two, three, four modifications but less than twenty, fifteen, ten modifications to an amino acid sequence described herein; (d) a polypeptide or fragment described herein.
  • the Fndc5 polypeptide or conjugate may be an isolated polypeptide or conjugate (e.g., at least 70, 80, 90, 95, or 99% free of other proteins).
  • the composition comprises a heterogeneous mixture of Fndc5 polypeptides and/or conjugate.
  • the composition is isolated from cleavage fragments and/or aggregates that are inactive or partially active (e.g., in an activity assay described herein).
  • the composition is at least 70% free of such cleavage fragments and/or aggregates; in other embodiments the composition is at least 80%, at least 90%, at least 95%, at least 99% or even 100% free from cleavage fragments and/or aggregates that are inactive or partially active.
  • polypeptides of the invention have biological activity. Activity can be measured or evaluated in a number of ways.
  • Suitable cells for the analysis of the in vitro effect of an Fndc5 polypeptide or conjugate can be determined by one skilled in the art.
  • Suitable cells may include but are not limited to, primary subcutaneous white adipocytes derived from an animal; primary subcutaneous beige/brite adipocytes derived from an animal; primary subcutaneous brown adipocytes derived from an animal; primary white adipocytes derived from an animal; primary brown adipocytes derived from an animal; primary beige/brite adipocytes derived from an animal; preadipocyte cell line, e.g., SGBS, hMADs; adipocyte cell line; white adipose cell line, e.g., 3T3L1/3T3 F442A, Obl7, PFC6, TA, 1246, ST13; brown adipocyte cell line, e.g., BFC-1, HB2, RBM-Ad, C3H10T1/2, HIB, T37i
  • in vitro cell cultures can be grown in the presence of an Fndc5 polypeptide or conjugate, or a suitable control, for a predetermined length of time, e.g., 5 days, 6 days, or 7 days, etc; and gene expression analyzed.
  • the level of gene expression can be assayed by determining the level of mRNA expression of the gene of interest using quantitative real time reverse transcription polymerase chain reaction (q-RT-PCR), using standard q-RT-PCR techniques known to those skilled in the art.
  • q-RT-PCR quantitative real time reverse transcription polymerase chain reaction
  • specific genes sets may be analyzed.
  • the level of expression of genes associated with a brown/beige adipocyte phenotype e.g., UPC1, PRDM16, PGCl , Cidea, Dio2, and Elovl3and/or a white adipocyte phenotype, e.g., resistin, angiotensinogen, WDNMl-like, Serpina3k, Ednra, Psat, Anxal; can be assayed.
  • Cellular transcriptional changes caused by an Fndc5 polypeptide or conjugate expressed in vitro can also be assayed using gene expression arrays using techniques known to those skilled in the art; and immunohistochemistry techniques, also known to those skilled in the art.
  • the effect of an Fndc5 polypeptide or conjugate on in vitro cellular mitochondrial biogenesis can be measured using several techniques known to those skilled in the art.
  • In vitro cellular mitochondrial biogenesis can be measured by examining the level of mitochondrial DNA (mtDNA) using southern blot analysis as described in the literature and known to those skilled in the art.
  • Mitochondrial biogenesis can also be analyzed by examining the level of expression of genes involved in mitochondrial biogenesis, e.g., mTFA or NRF-1, using q-RT-PCR.
  • In vitro cellular mitochondrial biogenesis can also be analyzed by transmission electron microscopy (TEM) using standard protocols known to those skilled in the art. TEM can also be used to analyze mitochondria size and density of cristae, and TEM coupled with quantitative
  • mitochondrial density mitochondrial density
  • Oxygen consumption The effect of an Fndc5 polypeptide or conjugate on in vitro total cellular oxygen consumption can be measured using an oxygen electrode, e.g., Clark-type electrode, using standard procedures known to those skilled in the art. Oxygen consumption can also be used as measure of cellular respiration. In addition to total oxygen consumption or respiration;
  • uncoupled respiration can be determined by adding an ATP synthase inhibitor, e.g., oligomycin (2 ⁇ g/mL) to the cultured cells, using standard protocols known to those skilled in the art. It may be beneficial to evaluate levels of uncoupled respiration in response to cAMP, as a characteristic of brown adipocytes is a high rate of uncoupled respiration in response to cAMP. Levels of uncoupled respiration in response to cyclic AMP can be evaluated via treatment of the cultured cells with 0.5mM dibutyryl-cAMP for 12 hours prior to measuring oxygen
  • the cultured cells can be analyzed for expression of genes related to thermogenesis, e.g., UPC1 and PCGla.
  • an Fndc5 polypeptide or conjugate on glucose uptake in vitro can be analyzed by positron emission tomography (PET) with fluorodeoxyglucose ( 18 FDG), using standard PET scan techniques previously published and known to those skilled in the art.
  • PET positron emission tomography
  • 18 FDG fluorodeoxyglucose
  • Active brown adipose tissue functions as a repository for glucose disposal and thus features increased levels of glucose uptake.
  • PET measures glucose uptake, and can thus be used to detect functioning brown adipose tissue in vitro.
  • a suitable in vivo mouse model can be used to evaluate the effect of Fndc5 polypeptides and conjugates on the treatment of diet induced obesity and insulin resistant diabetes.
  • a suitable mouse model of diet induced obesity and insulin resistance is known in the art and can be obtained or rendered by feeding C57BALB/c mice a high fat diet containing a high percentage of calories from fat.
  • Mice can be intravenously injected with an isolated protein or an adenoviral vector expressing an Fndc5 polypeptide or conjugate, and Fndc5 polypeptide or conjugate expression and activity analyzed at predetermined time points post administration, e.g., 5 days, 7 days, 10 days, 14 days, 21 days, etc; post administration.
  • the level of expression of the Fndc5 polypeptide or conjugate can be analyzed in the mouse plasma derived from tail blood sampling using polyclonal antibodies, e.g., using ELISA.
  • the level of Fndc5 polypeptide or conjugate expression in the mouse fat pads can be assayed post mouse sacrifice, collection, and processing of the fat pad tissue and subsequent mRNA analysis via q-RT-PCR.
  • Experimental results can be compared to a control group of mice administered a control adenoviral vector expressing a suitable control protein or peptide.
  • Genes of interest can include, genes associated with a brown adipocyte phenotype, e.g., UPC1, PRDM16, PGCla, Cidea, Dio2, ElovB; a white adipocyte phenotype, e.g., resistin, angiotensinogen, WDNMl-like, Serpina3k, Ednra, Psat, Anxal; a beige adipocyte phenotype; genes associated with mitochondrial biogenesis, e.g., mTFA or NRF-1; genes associated with thermogenesis, e.g., UCP1, PGCla; or any other gene or genes of interest. Transcriptional changes caused by an Fndc5 polypeptide or conjugate can also be assayed using gene expression arrays using techniques known to those skilled in
  • mouse body weight can be measured using a suitable scale; and analysis of body fat content can be measured using a dual energy X-ray absorptiometry (DEXA) scanner, both utilizing standard protocols known to those skilled in the art.
  • DEXA dual energy X-ray absorptiometry
  • Metabolic Parameters Diet induced insulin resistant diabetes is associated with impaired insulin and glucose tolerance as well as elevated levels of fasting plasma insulin and glucose concentrations.
  • standard metabolic tests known to those skilled in the art can be performed using standard techniques.
  • In vivo insulin tolerance can be tested using a standard insulin tolerance test known to those skilled in the art. Briefly, mice fasted for several hours are injected intraperitoneally with insulin at approximately 0.8U/kg.
  • Fasting plasma insulin concentrations can be measured using a standard insulin enzyme linked immunosorbant assay (ELISA) using plasma derived from tail blood sampling at specific time points pre and post insulin administration.
  • ELISA insulin enzyme linked immunosorbant assay
  • In vivo glucose tolerance can be tested using a standard glucose tolerance test known to those skilled in the art. Briefly, mice fasted for several hours are injected intraperitoneally with glucose at approximately 2 g/kg. Fasting plasma glucose concentrations can be measured using a standard glucometer using plasma derived from tail blood sampling at specific time points pre and post glucose administration. Insulin resistance is indicated by elevated fasting plasma insulin and fasting plasma glucose concentrations, as well as impaired glucose and insulin tolerance.
  • an Fndc5 polypeptide or conjugate on glucose uptake in vivo can be analyzed by positron emission tomography (PET) with fluorodeoxyglucose ( 18 FDG), using standard PET scan techniques previously published and known to those skilled in the art.
  • PET positron emission tomography
  • 18 FDG fluorodeoxyglucose
  • Active brown adipose tissue functions as a repository for glucose disposal and thus features increased levels of glucose uptake.
  • PET measures glucose uptake, and can thus be used to detect functioning brown adipose tissue in vivo.
  • PET scans can be conducted on the mice or tissue derived from the mice using standard PET scan techniques previously published and known to those skilled in the art.
  • CLAM Comprehensive Lab Animal Monitoring System
  • the effect of an Fndc5 polypeptide or conjugate on energy intake in vivo can be measured by the level of food consumption.
  • the effect of an Fndc5 polypeptide or conjugate on energy expenditure in vivo can be measured using several techniques known to those skilled in the art.
  • Energy expenditure can be a measure of oxygen consumption, measured as described above; physical activity, which can be simultaneously, measured using the CLAM cages using standard protocols known to those skilled in the art; and/or thermogenesis, measured as described below.
  • thermogenesis can be evaluated by conducting a standard cold challenge assay, using standard protocols known to those skilled in the art. Briefly, mice can be exposed to a below thermoneutral temperature, e.g., 4°C, and their subsequent shivering response and core body temperature monitored at several predetermined time points pre and post the start of cold exposure. The mice can be analyzed for the display of signs of insensitivity to the cold, e.g., the ability to keep their body temperature around baseline; or sensitivity to the cold, e.g., a sustained drop in body temperature or hypothermia or lethal hypothermia.
  • the effect of an Fndc5 polypeptide or conjugate on mouse thermogenesis can also be measured by collecting brown fat tissue after several predetermined time points pre and post cold exposure and analyzing the gene expression of genes associated with mouse thermogenesis and brown fat cell thermogenesis, e.g., UCP1, PGCla.
  • mice acclimated to a below thermoneutral temperature e.g., 4°C
  • mice acclimated to a below thermoneutral temperature e.g., 4°C
  • thermogenesis e.g., UCP1, PGCla.
  • the invention provides a nucleic acid that encodes an Fndc5 polypeptide or conjugate described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing a peptide, e.g.,
  • mammalian cells e.g. CHO cells.
  • the disclosure features vectors, such as expression vectors, containing a nucleic acid encoding an Fndc5 polypeptide or conjugate described herein.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector.
  • the vector can be capable of autonomous replication or it can integrate into a host DNA.
  • Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.
  • a vector can include a nucleic acid in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vector typically includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed.
  • the term "regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors can be introduced into host cells to produce the Fndc5 polypeptide or conjugate, encoded by nucleic acids as described herein.
  • Recombinant expression vectors can be designed for expression of the Fndc5 polypeptide or conjugate in prokaryotic or eukaryotic cells.
  • an Fndc5 polypeptide or conjugate can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Purified recombinant Fndc5 polypeptides and conjugates can be tested in activity assays such as, e.g., the assays described herein.
  • the expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector, or a vector suitable for expression in mammalian cells.
  • the expression vector's control functions can be provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma,
  • Adenovirus 2 Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone ⁇ e.g., by means of a signal transduction pathway), or by a heterologous polypeptide ⁇ e.g., the tetracycline-inducible systems, "Tet-On” and "Tet-Off '; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).
  • a promoter regulated by a steroid hormone by a polypeptide hormone ⁇ e.g., by means of a signal transduction pathway
  • a heterologous polypeptide e.g., the tetracycline-inducible systems, "Tet-On” and "Tet-Off '; see, e.g., Clon
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type ⁇ e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific promoters include the albumin promoter (liver- specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid- specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), e.g., promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.
  • promoters are also encompassed, for example, the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the disclosure features a host cell which includes a nucleic acid molecule described herein, e.g., a nucleic acid molecule within a recombinant expression vector or a nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • a host cell and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell 23: 175-182).
  • bacterial cells such as E. coli
  • insect cells such as E. coli
  • yeast or mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell 23: 175-182).
  • COS-7 cells such as Chinese hamster ovary cells (CHO) or COS cells e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell 23: 175-182).
  • Other suitable host cells are known to those skilled in the art.
  • a host cell can be used to produce (i.e., express) an Fndc5 polypeptide or conjugate described herein. Accordingly, the disclosure also features methods for producing an Fndc5 polypeptide or conjugate using the host cells.
  • the method includes culturing the host cell (into which a recombinant expression vector encoding a protein has been introduced) in a suitable medium, such that an Fndc5 polypeptide or conjugate is produced.
  • the method further includes isolating an Fndc5 polypeptide or conjugate from the medium or the host cell.
  • the disclosure also features a pharmaceutical composition comprising an Fndc5
  • polypeptide or conjugate comprising an Fndc5 polypeptide (e.g., an Fndc5 polypeptide or conjugate described herein), or a preparation or composition described herein, e.g.,
  • the pharmaceutical compositions may take the form of a pharmaceutical
  • compositions according to the invention include those suitable for the route of administration such as, e.g., parenteral (including subcutaneous, intradermal,
  • intramuscular, intravenous, and intraarticular administration although the most suitable route may depend upon, for example, the condition and disorder of the recipient.
  • formulations may be conveniently presented in unit dosage form and may be
  • compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for- injection, e.g., immediately prior to use.
  • compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • An aqueous carrier may be, for example, an isotonic buffer solution at a pH of from about 3.0 to about 8.0, e.g., at a pH of from about 3.5 to about 7.4, for example from 3.5 to 6.0, for example from 3.5 to about 5.0.
  • Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate/acetic acid buffers.
  • Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.
  • the pharmaceutical composition may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Typical unit dosage compositions are those containing an effective dose, or an appropriate fraction thereof, of the active ingredient.
  • composition may include other agents conventional in the art having regard to the type of formulation in question.
  • sustained-release systems include suitable polymeric materials, for example semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules; suitable hydrophobic materials, for example as an emulsion in an acceptable oil; or ion exchange resins; and soluble derivatives thereof, for example, a sparingly soluble salt.
  • Preparations for administration can be suitably formulated to give controlled release of an Fndc5 polypeptide or conjugate, e.g., an Fndc5 polypeptide or conjugate described herein.
  • the pharmaceutical compositions may be in the form of particles comprising one or more of biodegradable polymers, polysaccharide jellifying and/or bioadhesive polymers, amphiphilic polymers, agents capable of modifying the interface properties of the particles of the compound featured in the invention. These compositions exhibit certain biocompatibility features which allow a controlled release of the active substance. See U.S. Patent No. 5,700,486.
  • a therapeutically effective amount of an Fndc5 polypeptide or conjugate described herein can be administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time.
  • a bolus administration of a compound featured in the invention is provided, followed by a time period wherein the Fndc5 polypeptide or conjugate is administered to the subject, followed by a second bolus administration.
  • pulse doses of an Fndc5 polypeptide or conjugate are administered during the course of a day, during the course of a week, or during the course of a month.
  • the Fndc5 polypeptide or conjugate is administered twice weekly, weekly, once every two weeks, once every three weeks or preferable once every month, month and a half or two months.
  • an Fndc5 polypeptide or conjugate will be dependent on the molecule utilized, the subject being treated, the severity and type of the affliction, and the manner and route of administration.
  • the disclosure features the use of an Fndc5 polypeptide or conjugate described herein for the treatment or prevention of a disorder.
  • disorders include obesity, diabetes, a co-morbidity of obesity, an obesity related disorder, an age related disorder, and a disorder in which one or more symptoms can be alleviated by exercise.
  • the subject to whom the compound is administered may be overweight, for example, obese.
  • the subject may be diabetic, for example having insulin resistance or glucose intolerance, or both.
  • the subject may have diabetes mellitus, for example, the subject may have Type II diabetes.
  • the subject may be overweight, for example, obese and have diabetes mellitus, for example, Type II diabetes.
  • the subject may have, or may be at risk of having, a disorder in which obesity or being overweight is a risk factor.
  • disorders include, but are not limited to, cardiovascular disease, for example hypertension, atherosclerosis, congestive heart failure, and dyslipidemia; stroke; gallbladder disease; osteoarthritis; sleep apnea; reproductive disorders for example, polycystic ovarian syndrome; cancers, for example breast, prostate, colon, endometrial, kidney, and esophagus cancer; varicose veins; acanthosis nigricans; eczema;
  • osteoarthritis osteoarthritis; orthopedic injury; insulin resistance, for example, type 2 diabetes and syndrome X; metabolic syndrome; and thromboembolic disease (see Kopelman (2000), Nature 404:635-43; Rissanen et al., British Med. J. 301, 835, 1990).
  • obesity is a recognized risk factor for increased incidence of complications of general anesthesia. (See e.g., Kopelman, Nature 404:635-43, 2000). In general, obesity reduces life span and carries a serious risk of co-morbidities such as those listed above.
  • Other diseases or disorders associated with obesity are birth defects, maternal obesity being associated with increased incidence of neural tube defects, carpal tunnel syndrome (CTS); chronic venous insufficiency (CVI); daytime sleepiness; deep vein thrombosis (DVT); end stage renal disease (ESRD); gout; heat disorders; impaired immune response; impaired respiratory function; infertility; liver disease; lower back pain; obstetric and gynecologic complications; pancreatititis; as well as abdominal hernias; acanthosis nigricans; endocrine abnormalities;
  • CTS carpal tunnel syndrome
  • CVI chronic venous insufficiency
  • DVT deep vein thrombosis
  • ESRD end stage renal disease
  • gout heat disorders; impaired immune response; impaired respiratory function; infertility; liver disease; lower back pain; obstetric and gynecologic complications; pancreatititis; as well as abdominal hernias; acanthosis nigricans
  • Chronic hypoxia and hypercapnia dermatological effects; elephantitis; gastroesophageal reflux; heel spurs; lower extremity edema; mammegaly which causes considerable problems such as bra strap pain, skin damage, cervical pain, chronic odors and infections in the skin folds under the breasts, etc.; large anterior abdominal wall masses, for example abdominal panniculitis with frequent panniculitis, impeding walking, causing frequent infections, odors, clothing difficulties, lower back pain; musculoskeletal disease; pseudo tumor cerebri (or benign intracranial hypertension), and sliding hiatil hernia.
  • Conditions or disorders associated with increased caloric intake include, but are not limited to, insulin resistance, glucose intolerance, obesity, diabetes, including type 2 diabetes, eating disorders, insulin-resistance syndromes, metabolic syndrome X, and Alzheimer's disease.
  • disorders in which one or more symptoms can be alleviated by exercise e.g., cardiovascular disease, neurodegenerative diseases, e.g., multiple sclerosis, Parkinson's disease and Alzheimer's disease; certain cancers, e.g. prostate, breast, colon; certain intestinal disorders, e.g., ulcers, irritable bowel syndrome, indigestion, diverticulosis, gastrointestinal bleeding; certain emotional disorders, e.g., depression, menopause related emotional symptoms, e.g., anxiety, stress, depression.
  • cardiovascular disease e.g., neurodegenerative diseases, e.g., multiple sclerosis, Parkinson's disease and Alzheimer's disease
  • certain cancers e.g. prostate, breast, colon
  • certain intestinal disorders e.g., ulcers, irritable bowel syndrome, indigestion, diverticulosis, gastrointestinal bleeding
  • certain emotional disorders e.g., depression, menopause related emotional symptoms, e.g., anxiety, stress, depression.
  • the disclosure also features Fndc5 polypeptides and conjugates for use as a medicament for the prevention or treatment of diseases and disorders described herein, e.g., obesity, or obesity related disorders, or disorders in which exercise can alleviate one or more symptoms, or age-related disorders.
  • diseases and disorders described herein e.g., obesity, or obesity related disorders, or disorders in which exercise can alleviate one or more symptoms, or age-related disorders.
  • an Fndc5 polypeptide or conjugate described herein is administered in combination with another agent, for example, a treatment for lipolysis, an antihypertension treatment, a treatment for dyslipidemia, and/or a treatment for type 2 diabetes.
  • Administered "in combination”, as used herein means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder or diagnosed as being at risk for the disorder and before the disorder has been cured or eliminated, or before the symptom or symptoms associated with risk for the disorder have been alleviated or eliminated or treatment has ceased for other reasons.
  • the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous" or "concurrent delivery".
  • the delivery of one treatment ends before the delivery of the other treatment begins.
  • the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
  • the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
  • Combination treatments can include: a treatment for lipolysis: e.g., a beta 3 agonist (e.g., Amibegron (SR-58611A), Solabegron (GW-427,353), Nebivolol, L-796,568, CL-316,243, LY- 368,842, Ro40-2148 and Octopamine); an anti-hypertension treatment e.g., diuretic, e.g., hydrochlorothiazide, Acetazolamide, Chlorthalidone, Hydrochlorothiazide, Indapamide, Metolazone, Amiloride hydrochloride, Bumetanide, Ethacrynic acid Furosemide,
  • a beta 3 agonist e.g., Amibegron (SR-58611A), Solabegron (GW-427,353), Nebivolol, L-796,568, CL-316,243, LY- 368,842, Ro40-2148 and
  • hydrochlorothiazide Losartan and hydrochlorothiazide, Methyldopa and hydrochlorothiazide, Metoprolol and hydrochlorothiazide, Nadolol and bendroflumethiazide, Propranolol and hydrochlorothiazide, Spironolactone and hydrochlorothiazide, Triamterene and
  • statins e.g., Atorvastatin (Lipitor), Fluvastatin (Lescol), Lovastatin (Mevacor), Pravastatin (Pravachol), Simvastatin (Zocor), Rosuvastatin (Crestor); Bile acid sequestrants, e.g., Questran and Questran Light, Colestid, WelChol; Niacin, e.g., Nicolar, Niaspan; Fibrates, e.g., Atromid, Tricor, Lopid, Lofibra (fenofibrate); Ezetimibe; Omega-3 fatty acids, e.g., Lovaza; Combination statin and niacin, e.g., Advicor (niacin-lovastatin); Combination cholesterol absorption inhibitor and statin, e.g.
  • Glimepiride (Amaryl), Glyburide (DiaBeta, Glynase); Dipeptidy peptidase-4 (DPP-4) inhibitors e.g., Saxagliptin (Onglyza), Sitagliptin (Januvia), Linagliptin (Tradjenta); Biguanides, e.g., Metformin (Fortamet, Glucophage, etc); Thiazolidinediones, e.g., Rosiglitazone (Avandia), Pioglitazone (Actos); Alpha-glucosidase inhibitors, e.g., Acarbose (Precose), Miglitol (Glyset); Amylin mimetics, e.g., Pramlintide (Symlin); Incretin mimetics, e.g., Exenatide (Byetta), Liraglutide (Victoza).
  • kits e.g., as a component of a kit.
  • the kit includes (a) an Fndc5 polypeptide, e.g., a composition (e.g., a pharmaceutical composition) that includes an Fndc5 polypeptide or conjugate, and, optionally (b) informational material.
  • the informational material can be descriptive, instructional, marketing or other material that relates to a method described herein and/or the use of an Fndc5 polypeptide or conjugate, e.g., for a method described herein.
  • the informational material of the kit is not limited in its form.
  • the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth.
  • the informational material relates to using the polypeptide, functional fragment or conjugate to treat, prevent, or diagnosis of disorders and diseases, e.g., disorders and diseases described herein.
  • the informational material can include instructions to administer a polypeptide, functional fragment or conjugate in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein).
  • the informational material can include instructions to administer a polypeptide, functional fragment or conjugate to a suitable subject, e.g., a human, e.g., a human having, or at risk for, a disorder or disease described herein.
  • the material can include instructions to administer a polypeptide, functional fragment or conjugate to a patient with a disorder or condition described herein, e.g., diabetes or obesity.
  • the informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in print but may also be in other formats, such as computer readable material.
  • a polypeptide, functional fragment or conjugate described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that a polypeptide, functional fragment or conjugate be substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • a polypeptide, functional fragment or conjugate is provided as a dried form, reconstitution generally is by the addition of a suitable solvent.
  • the solvent e.g., sterile water or buffer, can optionally be provided in the kit.
  • the kit can include one or more containers for the composition containing a polypeptide, functional fragment or conjugate described herein.
  • the kit contains separate containers, dividers or compartments for the composition and informational material.
  • the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in association with the container.
  • the separate elements of the kit are contained within a single, undivided container.
  • the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a polypeptide, functional fragment or conjugate.
  • the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a polypeptide, functional fragment or conjugate.
  • the containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
  • the kit optionally includes a device suitable for administration of the composition, e.g., a syringe or any such delivery device.
  • a device suitable for administration of the composition e.g., a syringe or any such delivery device.
  • the disclosure also features a method of providing a kit, e.g., by combining components described herein.

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  • Health & Medical Sciences (AREA)
  • Diabetes (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Obesity (AREA)
  • Endocrinology (AREA)
  • Emergency Medicine (AREA)
  • Child & Adolescent Psychology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un polypeptide Fndc5 Isolé ou des fragments fonctionnels correspondants, subissant une ou plusieurs modifications, par exemple, substitutions ou suppressions d'un résidu d'acide aminé par rapport au résidu d'acide aminé dans la position correspondante d'un polypeptide fndc5 se produisant naturellement. L'invention porte également sur un produit de clivage protéolytique in vivo dudit polypeptide Fndc5, dans lequel ledit produit de clivage est sécrété dans la circulation.
PCT/US2013/020566 2012-01-06 2013-01-07 Compositions et procédés d'utilisation des polypeptides fndc5 WO2013103981A1 (fr)

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US201261583886P 2012-01-06 2012-01-06
US61/583,886 2012-01-06

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111808186A (zh) * 2019-12-19 2020-10-23 山东大学第二医院 一种人源性分泌型fndc5蛋白及其制备方法和用途

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US20090191154A1 (en) * 2004-06-28 2009-07-30 Merck Patent Gmbh Assembly and folding of fc-interferon-beta fusion proteins

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DATABASE UNIPROT 19 October 2011 (2011-10-19), GIBBS, R.A. ET AL.: "FNDC5_RAT.", retrieved from http://www.uniprot.org/uniproUQ8K3V5.bct?version=46 accession no. 8K3V5 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111808186A (zh) * 2019-12-19 2020-10-23 山东大学第二医院 一种人源性分泌型fndc5蛋白及其制备方法和用途
CN111808186B (zh) * 2019-12-19 2021-08-03 山东大学第二医院 一种人源性分泌型fndc5蛋白及其制备方法和用途

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