WO2014014819A2 - Méthodes de traitement de troubles du métabolisme du glucose - Google Patents

Méthodes de traitement de troubles du métabolisme du glucose Download PDF

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WO2014014819A2
WO2014014819A2 PCT/US2013/050485 US2013050485W WO2014014819A2 WO 2014014819 A2 WO2014014819 A2 WO 2014014819A2 US 2013050485 W US2013050485 W US 2013050485W WO 2014014819 A2 WO2014014819 A2 WO 2014014819A2
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peptide
seq
agr2
antibody
scg3
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WO2014014819A3 (fr
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Daniel David Kaplan
Maziyar SABERI
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NGM Biopharmaceuticals Inc
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NGM Biopharmaceuticals Inc
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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
    • C07K14/475—Growth factors; Growth regulators
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to, among other things, peptides and antibodies having glucose modulating activity, and associated methods and uses in treatment of diabetes and other metabolic-related disorders.
  • Patients who have a glucose metabolism disorder can suffer from hyperglycemia, hyperinsulinemia, and/or glucose intolerance.
  • An example of a disorder that is often associated with the aberrant levels of glucose and/or insulin is insulin resistance, in which liver, fat, and muscle cells lose their ability to respond to normal blood insulin levels.
  • Obesity is most commonly caused by excessive food intake coupled with limited energy expenditure and/or lack of physical exercise. Obesity increases the likelihood of development of various diseases, such as diabetes mellitus, hypertension, atherosclerosis, coronary artery disease, sleep apnea, gout, rheumatism and arthritis. Moreover, mortality risk directly correlates with obesity, such that, for example, a body-mass index in excess of 40 results in an average decreased life expectancy of more than 10 years.
  • the present disclosure contemplates the use of the agents described herein, and compositions thereof, to treat and/or prevent various diseases, disorders and conditions, and/or the symptoms thereof.
  • the diseases, disorders and conditions, and/or the symptoms thereof pertain to metabolic-related disorders, while in other embodiments they pertain to glucose metabolism disorders.
  • the agents, and compositions thereof can be used for the treatment and/or prevention of diabetes (e.g., Type 2 diabetes), insulin resistance and diseases, disorders and conditions characterized by insulin resistance, decreased insulin production, hyperglycemia, hypoinsulinemia, and metabolic syndrome.
  • Some of the agents, and compositions thereof may also be useful in, for example, subjects who may be overweight or obese.
  • the agents of the present disclosure are referred to herein as "Modulators".
  • peptide subsequences derived from the Agr2, Cnpy4, Scg3 and Smoc2 gene products have been identified.
  • the nomenclature “ms-p” is generally used (e.g., Smoc2 ms-p and Agr2 ms-p4); when reference is made to human peptides, the nomenclature “hu-p” is generally used (e.g., Smoc2 ms-p and Agr2 ms-p4).
  • Three human Agr2 peptides (Agr2 hu-(p4, p5, p6)), one human Cnpy4 peptide (Cnpy4 hu-p), one human Scg3 peptide (Scg3 hu-p) and one human Smoc2 peptide (Smoc2 hu-p) have been identified (see Figures 3A, 9, 14A and 19A, respectively).
  • Three murine Agr2 peptides (Agr2 ms-(p4, p5, p6)), one murine Cnpy4 peptide (Cnpy4 ms-p), one murine Scg3 peptide
  • Agr2, Cnpy4, Scg3 and/or Smoc2 - related peptides is meant to include the disclosed Agr2, Cnpy4, Scg3 and/or Smoc2 human and murine subsequences (e.g., Agr2(hu-p4, hu-p5, hu-p6, ms-p4, ms-p5, ms-p6), Cnpy4(hu-1, ms-1), Scg3(hu-1, ms-1) and Smoc2(hu-pl, ms-pl)), as well as homologues, variants, fragments and other modified forms thereof.
  • Agr2(hu-p4, hu-p5, hu-p6, ms-p4, ms-p5, ms-p6) Cnpy4(hu-1, ms-1), Scg3(hu-1, ms-1) and Smoc2(hu-p
  • Agr2, Cnpy4, Scg3 and Smoc2 - related peptides is meant to refer to both the human and the murine peptides.
  • data regarding the activity (or lack thereof) of the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides were generated using mouse peptides. The corresponding human peptides are believed to have comparable activity based upon their sequence homology to the mouse peptides.
  • the present disclosure contemplates Agr2, Cnpy4, Scg3 and Smoc2 - related peptides, and the nucleic acid molecules which encode them, from other species (e.g., non-human primates (e.g., chimpanzees), dogs, rats, etc.).
  • Modulators refers to the one or more Agr2, Cnpy4, Scg3 and Smoc2 - related peptides and agents that stimulate, increase, activate, facilitate, enhance activation, sensitize or up-regulate the function or activity, either directly or indirectly, of one or more of Agr2, Cnpy4, Scg3 and Smoc2 - related peptides.
  • Modulators are agents that effect a desired biological response (e.g., lowering of glucose and/or body weight) by the same mechanism of action as the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides (e.g., agents that modulate the same signaling pathway as the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides, in a manner analogous to that of the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides, and are capable of eliciting a biological response comparable to (or greater than) that of the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides).
  • Modulators include other agonists such as antibodies and fragments thereof, small molecule agonist compounds, and other agonistic peptides structurally
  • compositions are provided that comprise one or more Modulators of the present disclosure, which compositions are useful in treating or preventing a metabolic disorder.
  • the compositions modulate aberrant glucose and/or insulin levels in a subject and may be used to treat or prevent diabetes mellitus (e.g., Type I and Type II diabetes and gestational diabetes).
  • diabetes mellitus e.g., Type I and Type II diabetes and gestational diabetes.
  • the compositions comprising the Modulators described herein may also be used to treat, for example, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome, and may also be used to treat or prevent various body weight - related disorders (e.g., obesity).
  • a use or method of treatment of a subject includes
  • a subject such as a subject having, or at risk of having, a disease or disorder treatable by a Agr2, Cnpy4, Scg3 and Smoc2 - related peptide, in an amount effective for treating the disease or disorder.
  • a subject such as a subject having, or at risk of having, a disease or disorder treatable by a Agr2, Cnpy4, Scg3 and Smoc2 - related peptide
  • a method includes administering a Agr2, Cnpy4, Scg3 and Smoc2 - related peptide to a subject, such as a subject having a hyperglycemic condition, insulin resistance,
  • hyperinsulinemia glucose intolerance or metabolic syndrome, in an amount effective for treating the disease or disorder.
  • one embodiment of the present disclosure relates to a peptide comprising a subsequence of human Agr2, Cnpy4, Scg3 or Smoc2 (as depicted in Figures 3 A, 9, 14A, and 19A, respectively), or a variant thereof; or a subsequence of murine Agr2, Cnpy4, Scg3 or Smoc2 (as depicted in Figures 3B, 10, 15 A, and 20A, respectively), or a variant thereof.
  • a peptide comprising: a subsequence of a human Agr2 peptide or a mouse Agr2 peptide as depicted in Figure 4, or a variant thereof, wherein the subsequence is fewer than 100 amino acids in length; a sequence comprising XiD X 2 TVKX 3 GX 4 (SEQ ID NO: 36); VFAEX 5 KEIQKLAEQFVLLNLX 6 YETTD (SEQ ID NO: 37); HLSPDGQYVP (SEQ ID NO: 3); IX 7 FX 8 VDPSLTVRADITG (SEQ ID NO: 38);
  • IX 7 FX 8 VDPSLTVRADIT (SEQ ID NO: 39); LYAYEPX 9 DXi 0 ALLXnDNM (SEQ ID NO: 40); or YSNRLYAYEPX 9 DX 10 ALLX 11 DNM (SEQ ID NO: 41); wherein each of Xi-Xn is a semi- conserved residue; at least 85% amino acid identity to a subsequence of a human Agr2 peptide or a mouse Agr2 peptide as depicted in Figure 4, wherein the peptide is fewer than 100 amino acids in length; or a sequence comprises XiD X 2 TVKX 3 GX 4 (SEQ ID NO: 36);
  • VFAEX 5 KEIQKLAEQFVLLNLX 6 YETTD SEQ ID NO: 37
  • HLSPDGQYVP SEQ ID NO: 3
  • IX 7 FX 8 VDPSLTVRADITG SEQ ID NO: 38
  • IX 7 FX 8 VDPSLTVRADIT SEQ ID NO: 39
  • LYAYEPX 9 DX 10 ALLX 11 DNM SEQ ID NO: 40
  • YSNRLYAYEPX 9 DXi 0 ALLXnDNM SEQ ID NO: 41
  • Xi is R or K
  • X 2 is T or I
  • X 3 P or S
  • X 4 is A or S
  • X 5 N or H
  • X 6 is I or V
  • X 7 is I, L, M or V
  • X 8 is V or is absent
  • X 9 is A or S
  • X 10 is T or I
  • Xn H, Y or L.
  • the peptide comprises Agr2 hu-p4, Agr2 hu-p5, Agr2 hu-p6, Agr2 ms-p4, Agr2 ms-p5 or Agr2 ms-p6.
  • Some embodiments of the present disclosure relate to a peptide comprising: a subsequence of a human Cnpy4 peptide or a mouse Cnpy4 peptide as depicted in Figure 11, or a variant thereof, wherein the subsequence is fewer than 100 amino acids in length; the sequence XiX 2 X 3 KEX 4 X 5 X 6 DTERLPSKCEVCKLLSX 7 ELQX 8 X 9 LSRT (SEQ ID NO: 42), wherein each of X1-X9 is a semi-conserved residue; at least 85% amino acid identity to a subsequence of a human Cnpy4 peptide or a mouse Cnpy4 peptide as depicted in Figure 11 , wherein the peptide is fewer than 100 amino acids in length; or the sequence
  • XiX 2 X 3 KEX 4 X 5 X 6 DTERLPSKCEVCKLLSX 7 ELQX 8 X 9 LSRT (SEQ ID NO: 42), wherein Xi is G or E; X 2 is M, A or T; X 3 is L, T or S; X 4 is E or is absent; X 5 is D or E; X 6 is D or A; X 7 is T, L or M; and X 8 and X 9 are E or A.
  • the peptide comprises Cnpy4 hu-p or Cnpy4 mu-p.
  • a peptide comprising: a subsequence of a human Scg3 peptide or a mouse Scg3 peptide as depicted in Figure 16, or a variant thereof, wherein the subsequence is fewer than 100 amino acids in length; the sequence
  • the peptide comprises Scg3 hu-p or Scg3 mu-p.
  • Still further embodiments of the present disclosure relate to a peptide comprising: a subsequence of a human Smoc2 peptide or a mouse Smoc2 peptide as depicted in Figure 21 , or a variant thereof, wherein the subsequence is fewer than 100 amino acids in length; the sequence CVKKFVEYCDXi NDKSrX 2 VQELMGCLGVX 3 X 4 EX 5 G (SEQ ID NO: 44), wherein Xi - X 5 are semi-conserved residues.
  • the peptide comprises Smoc2 hu-p or Smoc2 mu-p.
  • a variant of the peptides described herein comprises an amino acid sequence having at least 85% amino acid identity, at least 90% amino acid identity, at least 93% amino acid identity, at least 95% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to a subsequence of human or murine Agr2, Cnpy4, Scg3 or Smoc2.
  • the peptide comprises fewer than 100 amino acid residues, fewer than 75 amino acid residues, fewer than 50 amino acid residues, fewer than 25 amino acid residues, fewer than 20 amino acid residues, fewer than 15 amino acid residues, or fewer than 10 amino acid residues of human or murine Agr2, Cnpy4, Scg3 or Smoc2.
  • a peptide of the present disclosure comprises: a variant of a human AGR2 polypeptide of Figure 3 A or a variant of a murine AGR2 polypeptide of Figure 3B; a variant of a human CNPY4 polypeptide of Figure 9 or a variant of a murine CNPY4 polypeptide of Figure 10; a variant of a human SCG3 polypeptide of Figure 14A or a variant of a murine SCG3 polypeptide of Figure 15 A; or a variant of a human SMOC2 polypeptide of Figure 19A or a variant of a murine SMOC2 polypeptide of Figure 20A.
  • the aforementioned variants comprise an amino acid sequence having at least 85% amino acid identity, at least 90% amino acid identity, at least 93% amino acid identity, at least 95% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to the amino acid sequence of: a human or murine AGR2 polypeptide of Figure 3 A or Figure 3B, respectively; a human or murine CNPY4 polypeptide of Figure 9 or Figure 10, respectively; a human or murine SCG3 polypeptide of Figure 14A or Figure 15 A, respectively; or a human or murine SMOC2 polypeptide of Figure 19A or Figure 20A, respectively.
  • a peptide disclosed above is isolated.
  • the peptide comprises a CONH 2 group instead of a COOH group at the carboxyl terminus of the peptide.
  • nucleic acid molecules encoding the aforementioned peptides.
  • a nucleic acid molecule is operably linked to an expression control element that confers expression of the nucleic acid molecule encoding the peptide in vitro, in a cell or in vivo.
  • a vector e.g., a viral vector
  • Some embodiments include transformed or host cells that express one or more of the aforementioned peptides.
  • aforementioned peptides is formulated to yield a pharmaceutical composition, wherein the composition also includes one or more pharmaceutically acceptable diluents, carriers or excipients.
  • the pharmaceutical compositions may be used in combination with other therapeutically active agents or compounds (e.g., glucose lowering agents) as described herein in order to treat or prevent the diseases, disorders and conditions as contemplated by the present disclosure.
  • a pharmaceutical composition also includes at least one additional prophylactic or therapeutic agent.
  • Still further embodiments of the present disclosure comprise an antibody that binds specifically to one of the aforementioned peptides.
  • the antibody comprises a light chain variable region and a heavy chain variable region present in separate polypeptides or in a single polypeptide.
  • An antibody of the present disclosure binds the peptide with an affinity of from about 10 7 M - " 1 to about 1012 M - " 1 in certain embodiments.
  • the antibody comprises a heavy chain constant region of the isotype IgGl, IgG2, IgG3, or IgG4.
  • the antibody is detectably labeled, while it is a Fv, scFv, Fab, F(ab') 2 , or Fab' in other embodiments.
  • the present disclosure also contemplates antibodies that comprise a covalently linked non-peptide polymer (e.g., a poly(ethylene glycol) polymer).
  • the antibody comprises a covalently linked moiety selected from a lipid moiety, a fatty acid moiety, a polysaccharide moiety, and a carbohydrate moiety.
  • the antibody is a single chain Fv (scFv) antibody in some embodiments, and the scFv is multimerized in others.
  • scFv single chain Fv
  • the antibodies of the present disclosure may be, but are not limited to, monoclonal antibodies, polyclonal antibodies, or humanized antibodies.
  • compositions comprising an antibody as described above formulated with at least one pharmaceutically acceptable excipient, carrier or diluent.
  • Such pharmaceutical compositions may also contain at least one additional prophylactic or therapeutic agent.
  • a sterile container that contains one of the above-mentioned pharmaceutical compositions and optionally one or more additional components.
  • the sterile container may be a syringe.
  • the sterile container is one component of a kit; the kit may also contain, for example, a second sterile container that contains at least one prophylactic or therapeutic agent.
  • the present disclosure contemplates a method of treating or preventing a glucose metabolism disorder in a subject (e.g., a human) by administering to the subject a therapeutically effective amount of a Modulator (as defined herein).
  • the Modulator may be, for example, one of the aforementioned peptides, a small molecule compound (e.g., an agonist), an agonistic peptide distinguishable from the peptides described herein, or an antibody.
  • the treating or preventing results in a reduction in plasma glucose in the subject or an increase in glucose tolerance in the subject.
  • the treating or preventing may result in a lower insulin levels because the Modulator (e.g., a peptide of the present disclosure) improves insulin sensitivity, or in higher insulin levels because the Modulator (e.g., a peptide of the present disclosure) stimulates insulin secretion; a Modulator acting via either mechanism may result in improved glucose homeostasis.
  • the Modulator e.g., a peptide of the present disclosure
  • the Modulator e.g., a peptide of the present disclosure
  • a Modulator acting via either mechanism may result in improved glucose homeostasis.
  • the glucose metabolism disorder is diabetes mellitus.
  • the subject is obese.
  • the administering is by parenteral (e.g., subcutaneous) injection.
  • FIG. 1 shows anterior gradient homolog 2 (Agr2) gene expression in
  • EECs enteroendocrine cells
  • ECs enterocytes isolated from the indicated segments of the mouse gastrointestinal tract. Hashed bars represent expression in EECs, while solid bars represent expression in ECs.
  • FIG. 2 is a table depicting Agr2 murine peptides (ms pl-ms p7) (Top to Bottom; SEQ ID NOS: 1 - 7) and their activity in reducing blood glucose levels.
  • a "+” indicates that the peptide caused a significant (p ⁇ 0.05) reduction in blood glucose levels (i.e., p4 (peptide ms-p4), p5 (peptide ms-p5), and p6 (peptide ms-p6) during an oral glucose tolerance test, while a "-” indicates that the peptide did not cause a significant change in blood glucose levels (i.e., pi (peptide ms-pl), p2 (peptide ms-p2), p3 (peptide ms-p3), and p7 (peptide ms-p7)).
  • Figure 3 A shows the full-length human Agr2 amino acid sequence (SEQ ID NO: 8) (the regions encompassing hu-p4, hu-p5 and hu-p6 are highlighted in gray) and the nucleic acid sequence encoding full-length human Agr2 (SEQ ID NO: 9) (the coding region is highlighted in gray).
  • Figure 3B shows the full-length murine Agr2 amino acid sequence (SEQ ID NO: 10) (the regions encompassing ms-p4, ms-p5, and ms-p6 are highlighted in gray) and the nucleic acid sequence encoding full-length murine Agr2 (SEQ ID NO: 11) (the coding region is highlighted in gray).
  • Figure 4 shows a multiple sequence alignment of Agr2 amino acid sequences from the indicated species. Regions from macaque (SEQ ID NO: 12) , human (SEQ ID NO: 8) , dog (SEQ ID NO: 13) , mouse (SEQ ID NO: 10) , and rat (SEQ ID NO: 14) comprising the Agr2 p4, p5 and p6-related peptides are highlighted in gray. Residue positions that are fully conserved are indicated by (*), whereas residue positions that are semi-conserved are indicated by (.).
  • Figure 5A depicts the effect of a single bolus i.p. injection of murine Agr2(p4)
  • mice i.e., ms p4 (10 mg/kg; gray squares) and vehicle control (black squares) on basal (fasted) plasma glucose (FPG) concentration and on oral glucose tolerance in high-fat fed mice.
  • Figure 5C depicts the effect of a single bolus i.p. injection of murine Agr2(p4)
  • mice basal (fasted) plasma insulin concentration (FPI) and on glucose-stimulated insulin secretion (GSIS) in high-fat fed mice.
  • Figure 6A depicts the effect of a single bolus i.p. injection of murine Agr2(p5)
  • mice received a single bolus i.p. injection of peptide or vehicle control, and at minO glucose (lg/kg) in PBS was administered orally.
  • FIG. 6B depicts the data from Figure 6A expressed as the percent change in plasma glucose concentration normalized to baseline (min-30). Briefly, high-fat fed mice received a single bolus i.p.
  • Figure 6C depicts the effect of a single bolus i.p. injection of murine Agr2(p5)
  • mice (lOmg/kg; gray squares) and vehicle control (black squares) on basal (fasted) plasma insulin concentration (FPI) and on glucose-stimulated insulin secretion (GSIS) in high- fat fed mice. FPI concentrations were determined in untreated mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p. injection of peptide or vehicle control, and at minO glucose
  • Figure 7A depicts the effect of a single bolus i.p. injection of murine Agr2(p6) (i.e., ms p6) (10 mg/kg; gray squares) and vehicle control (black squares) on basal (fasted) plasma glucose (FPG) concentration and on oral glucose tolerance in high-fat fed mice.
  • FPG concentrations were determined in untreated mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p. injection of peptide or vehicle control, and at minO glucose (lg/kg) in PBS was administered orally.
  • mice basal (fasted) plasma insulin concentration (FPI) and on glucose-stimulated insulin secretion (GSIS) in high-fat fed mice.
  • Figure 8 shows canopy homolog 4 (Cnpy4) gene expression in enteroendocrine cells (EECs) and enterocytes (ECs) isolated from the indicated segments of the mouse gastrointestinal tract. Hashed bars represent expression in EECs, while solid bars represent expression in ECs.
  • Figure 9 shows the full-length human Cnpy4 amino acid sequence (SEQ ID NO: 15) (the region encompassing the Cnpy4 human peptide (Cnpy4 hu-p) is highlighted in gray) and the nucleic acid sequence encoding full-length human Cnpy4 (SEQ ID NO: 16) (the coding region is highlighted in gray).
  • Figure 10 shows the full-length murine Cnpy4 amino acid sequence (SEQ ID NO: 17) (the region encompassing the Cnpy4 murine peptide (CNPY ms-p) is highlighted in gray) and the nucleic acid sequence encoding full-length murine Cnpy4 (SEQ ID NO: 18) (the coding region is highlighted in gray).
  • Figure 11 shows a multiple sequence alignment of Cnpy4 amino acid sequences from the indicated species. Regions from chimpanzee (SEQ ID NO: 19), human (SEQ ID NO: 15), dog (SEQ ID NO: 20), mouse (SEQ ID NO: 17), and rat (SEQ ID NO: 21) comprising the Cnpy4 - related peptides are highlighted in gray. Residue positions that are fully conserved are indicated by (*), whereas residue positions that are semi-conserved are indicated by (.).
  • Figure 12A depicts the effect of a single bolus i.p. injection of murine Cnpy4 ms- p (10 mg/kg; gray squares) and vehicle control (black squares) on basal (fasted) plasma glucose (FPG) concentration and on oral glucose tolerance in high-fat fed mice.
  • FPG concentrations were determined in untreated mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p. injection of peptide or vehicle control, and at minO glucose (lg/kg) in PBS was administered orally.
  • Figure 12C depicts the effect of a single bolus i.p. injection of murine Cnpy4 ms- p (lOmg/kg; gray squares) and vehicle control (black squares) on basal (fasted) plasma insulin concentration (FPI) and on glucose-stimulated insulin secretion (GSIS) in high-fat fed mice.
  • FIG 13 shows secretogranin-3 ("Scg3") gene expression in enteroendocrine cells (EECs) and enterocytes (ECs) isolated from the indicated segments of the mouse gastrointestinal tract. Hashed bars represent expression in EECs, while solid bars, if present, represent expression in ECs.
  • EECs enteroendocrine cells
  • ECs enterocytes
  • Figure 14A shows the full-length human Scg3 amino acid sequence (SEQ ID NO: 22) and the region encompassing the Scg3 human peptide (Scg3 hu-p) (highlighted in gray).
  • Figure 14B shows the nucleic acid sequence encoding full-length human Scg3 (SEQ ID NO: 23) (the coding region is highlighted in gray).
  • Figure 15A shows the full-length murine Scg3 amino acid sequence (SEQ ID NO: 24) and the region encompassing the Scg3 murine peptide (Scg3 ms-p) (highlighted in gray).
  • Figure 15B shows the nucleic acid sequence encoding full-length murine Scg3 (SEQ ID NO: 25) (the coding region is highlighted in gray).
  • Figure 16 shows a multiple sequence alignment of Scg3 amino acid sequences from the indicated species. Regions from chimpanzee (SEQ ID NO: 26), human (SEQ ID NO: 22), dog (SEQ ID NO: 27), mouse (SEQ ID NO: 24), and rat (SEQ ID NO: 28) comprising the Scg3 - related peptides are highlighted in gray. Residue positions that are fully conserved are indicated by (*), whereas residue positions that are semi-conserved are indicated by (.).
  • Figure 17A depicts the effect of a single bolus i.p. injection of murine Scg3 ms-p
  • FPG basal plasma glucose
  • Figure 17C depicts the effect of a single bolus i.p. injection of murine Scg3 ms-p (lOmg/kg; gray squares) and vehicle control (black squares) on basal (fasted) plasma insulin concentration (FPI) and on glucose-stimulated insulin secretion (GSIS) in high-fat fed mice.
  • FIG 18 shows SPARC -related modular calcium-binding protein-2 ("Smoc2") gene expression in enteroendocrine cells (EECs) and enterocytes (ECs) isolated from the indicated segments of the mouse gastrointestinal tract. Hashed bars represent expression in EECs, while solid bars, if present, represent expression in ECs.
  • EECs enteroendocrine cells
  • ECs enterocytes
  • Figure 19A shows the full-length human Smoc2 amino acid sequence (SEQ ID NO: 29) and the region encompassing the Smoc2 human peptide (Smoc2 hu-p) (highlighted in gray).
  • Figure 19B shows the nucleic acid sequence encoding full-length human Smoc2 (SEQ ID NO: 30) (the coding region is highlighted in gray).
  • Figure 20A shows the full-length murine Smoc2 amino acid sequence (SEQ ID NO: 31) and the region encompassing the Smoc2 murine peptide (Smoc2 ms-p) (highlighted in gray).
  • Figure 20B shows the nucleic acid sequence encoding full-length murine Smoc2 (SEQ ID NO: 32) (the coding region is highlighted in gray).
  • Figure 21 shows a multiple sequence alignment of Smoc2 amino acid sequences from the indicated species. Regions from mouse (SEQ ID NO: 31), rat (SEQ ID NO: 33), chimpanzee (SEQ ID NO: 34), human (SEQ ID NO: 29), and dog (SEQ ID NO: 35) comprising the Smoc2 - related peptides are highlighted in gray. Residue positions that are fully conserved are indicated by (*), whereas residue positions that are semi-conserved are indicated by (.).
  • Figure 22A depicts the effect of a single bolus i.p. injection of murine Smoc2 ms-p (10 mg/kg; gray squares) and vehicle control (black squares) on basal (fasted) plasma glucose (FPG) concentration and on oral glucose tolerance in high-fat fed mice. FPG concentrations were determined in untreated mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p. injection of peptide or vehicle control, and at minO glucose (lg/kg) in PBS was administered orally.
  • murine Smoc2 ms-p (10 mg/kg; gray squares) and vehicle control (black squares)
  • FPG concentrations were determined in untreated mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p. injection of peptide or vehicle control, and at minO glucose (lg/kg) in PBS was administered orally.
  • Figure 22C depicts the effect of a single bolus i.p. injection of murine Smoc2 ms-p (lOmg/kg; gray squares) and vehicle control (black squares) on basal (fasted) plasma insulin concentration (FPI) and on glucose-stimulated insulin secretion (GSIS) in high-fat fed mice.
  • FPI concentrations were determined in untreated mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p. injection of peptide or vehicle control, and at minO glucose (lg/kg) in PBS was administered orally.
  • the present disclosure contemplates the use of the Modulators described herein, and compositions thereof, to treat and/or prevent various diseases, disorders and conditions, and/or the symptoms thereof.
  • the diseases, disorders and conditions, and/or the symptoms thereof pertain to metabolic-related disorders, while in other embodiments they pertain to glucose metabolism disorders.
  • the Modulators, and compositions thereof can be used for the treatment and/or prevention of diabetes (e.g., Type 2 diabetes), insulin resistance and diseases, disorders and conditions characterized by insulin resistance, decreased insulin production, hyperglycemia, hypoinsulinemia, and metabolic syndrome.
  • diabetes e.g., Type 2 diabetes
  • the Modulators, and compositions thereof may also be useful in, for example, subjects who may be overweight or obese.
  • the present disclosure contemplates the use of the Modulators to increase, enhance, or otherwise up-regulate, directly or indirectly, the activity of peptides subsequences derived from Agr2, Cnpy4, Scg3 and Smoc2.
  • the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides are secreted peptides, and additional characteristics of the peptides are described below.
  • Agr2, Cnpy4, Scg3 and Smoc2 encompass peptides and variants thereof that are encoded by the Agr2, Cnpy4, Scg3 and Smoc2 genes or homologues thereof, respectively.
  • ms-p is generally used (e.g., Smoc2 ms-p and Agr2 ms-p4); when reference is made to human peptides, the nomenclature “hu-p” is generally used (e.g., Smoc2 ms-p and Agr2 ms-p4).
  • examples of the peptides and variants include three human Agr2 peptides (Agr2 hu-(p4, p5, p6)), one human Cnpy4 peptide (Cnpy4 hu-p), one human Scg3 peptide (Scg3 hu-p) and one human Smoc2 peptide (Smoc2 hu-p) have been identified (see Figures 3 A, 9, 14A and 19A, respectively), while three murine Agr2 peptides (Agr2 ms-(p4, p5, p6)), one murine Cnpy4 peptide (Cnpy4 ms-p), one murine Scg3 peptide (Scg3 ms-p) and one murine Smoc2 peptide (Smoc2 ms-p) have also been identified (see Figures 3B, 10, 15A and 20A, respectively).
  • Agr2, Cnpy4, Scg3 and Smoc2 gene expression is prevalent in particular enteroendocrine cells (EECs) of the gastrointestinal tract and is also observed in some enterocytes (ECs) of the gastrointestinal tract.
  • EECs enteroendocrine cells
  • ECs enterocytes
  • patient or “subject” are used interchangeably to refer to a human or a nonhuman animal (e.g., a mammal).
  • a Modulator (such as administering a Modulator or a pharmaceutical composition comprising a Modulator) initiated after a disease, disorder or condition, or a symptom thereof, has been diagnosed, observed, and the like so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of a disease, disorder, condition afflicting a subject, or at least one of the symptoms associated with a disease, disorder, condition afflicting a subject.
  • treatment includes inhibiting (i.e., arresting the development or further development of the disease, disorder or condition or clinical symptoms association therewith) an active disease (e.g., so as to decrease the level of insulin and/or glucose in the bloodstream, to increase glucose tolerance so as to minimize fluctuation of glucose levels, and/or so as to protect against diseases caused by disruption of glucose homeostasis).
  • an active disease e.g., so as to decrease the level of insulin and/or glucose in the bloodstream, to increase glucose tolerance so as to minimize fluctuation of glucose levels, and/or so as to protect against diseases caused by disruption of glucose homeostasis.
  • in need of treatment refers to a judgment made by a physician or other caregiver that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of the physician's or caregiver's expertise.
  • prevent refers to a course of action (such as administering a Modulator or a pharmaceutical composition comprising a Modulator) initiated in a manner (e.g., prior to the onset of a disease, disorder, condition or symptom thereof) so as to prevent, suppress, inhibit or reduce, either temporarily or permanently, a subject's risk of developing a disease, disorder, condition or the like (as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof, generally in the context of a subject predisposed to having a particular disease, disorder or condition.
  • the terms also refer to slowing the progression of the disease, disorder or condition or inhibiting progression thereof to a harmful or otherwise undesired state.
  • in need of prevention refers to a judgment made by a physician or other caregiver that a subject requires or will benefit from preventative care. This judgment is made based on a variety of factors that are in the realm of a physician's or caregiver's expertise.
  • the phrase "therapeutically effective amount” refers to the administration of an agent (e.g., a Modulator) to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to a patient.
  • the therapeutically effective amount can be ascertained by measuring relevant physiological effects. For example, in the case of a hyperglycemic condition, a lowering or reduction of blood glucose or an improvement in glucose tolerance test can be used to determine whether the amount of an agent is effective to treat the hyperglycemic condition.
  • a therapeutically effective amount is an amount sufficient to reduce or decrease any level (e.g., a baseline level) of fasting plasma glucose (FPG), wherein, for example, the amount is sufficient to reduce a FPG level greater than 200 mg/dl to less than 200 mg/dl, wherein the amount is sufficient to reduce a FPG level between 175 mg/dl and 200 mg/dl to less than the starting level, wherein the amount is sufficient to reduce a FPG level between 150 mg/dl and 175 mg/dl to less than the starting level, wherein the amount is sufficient to reduce a FPG level between 125 mg/dl and 150 mg/dl to less than the starting level, and so on (e.g., reducing FPG levels to less than 125 mg/dl, to less than 120 mg/dl, to less than 115 mg/dl, to less than 110 mg/dl, etc.).
  • FPG fasting plasma glucose
  • the effective amount is an amount sufficient to reduce or decrease levels by more than about 10% to 9%, by more than about 9% to 8%, by more than about 8% to 7%, by more than about 7% to 6%, by more than about 6% to 5%, and so on. More particularly, a reduction or decrease of HbAIc levels by about 0.1%, 0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%), 33%), 35%), 40%), 45%, 50%>, or more is contemplated by the present disclosure.
  • the therapeutically effective amount can be adjusted in connection with the dosing regimen and diagnostic analysis of the subject's condition and the like.
  • the phrase "in a sufficient amount to effect a change” means that there is a detectable difference between a level of an indicator measured before (e.g., a baseline level) and after administration of a particular therapy.
  • Indicators include any objective parameter (e.g., level of glucose or insulin) or subjective parameter (e.g., subject's feeling of well-being).
  • glucose tolerance refers to the ability of a subject to control the level of plasma glucose and/or plasma insulin when glucose intake fluctuates.
  • glucose tolerance encompasses the subject's ability to reduce, within about 120 minutes, the level of plasma glucose back to a level determined before the intake of glucose.
  • the terms “diabetes” and “diabetic” refer to a progressive disease of carbohydrate metabolism involving inadequate production or utilization of insulin, frequently characterized by hyperglycemia and glycosuria.
  • the terms “pre-diabetes” and “pre-diabetic” refer to a state wherein a subject does not have the characteristics, symptoms and the like typically observed in diabetes, but does have characteristics, symptoms and the like that, if left untreated, may progress to diabetes. The presence of these conditions may be determined using, for example, either the fasting plasma glucose (FPG) test or the oral glucose tolerance test (OGTT). Both require that a subject fast for at least 8 hours prior to initiating the test.
  • FPG fasting plasma glucose
  • OGTT oral glucose tolerance test
  • a subject's blood glucose is measured after the conclusion of the fasting; generally, the subject fasts overnight and the blood glucose is measured in the morning before the subject eats.
  • a healthy subject would generally have a FPG concentration between 90 and about 100 mg/dl
  • a subject with "pre-diabetes” would generally have a FPG concentration between about 100 and about 125 mg/dl
  • a subject with "diabetes” would generally have a FPG level above about 126 mg/dl.
  • OGTT a subject's blood glucose is measured after fasting and again two hours after drinking a glucose-rich beverage.
  • a healthy subject generally has a blood glucose concentration below about 140 mg/dl
  • a pre-diabetic subject generally has a blood glucose concentration about 140 to about 199 mg/dl
  • a diabetic subject generally has a blood glucose concentration about 200 mg/dl or above.
  • glycemic values pertain to human subjects, normoglycemia, moderate hyperglycemia and overt hyperglycemia are scaled differently in murine subjects.
  • a healthy murine subject after a four-hour fast would generally have a FPG concentration between 100 to 150mg/dl
  • a murine subject with "pre-diabetes” would generally have a FPG concentration between 175 to 250 mg/dl
  • a murine subject with "diabetes” would generally have a FPG concentration between 250 to >600mg/dl.
  • insulin resistance refers to a condition where a normal amount of insulin is unable to produce a normal physiological or molecular response.
  • a hyper-physiological amount of insulin either endogenously produced or exogenously administered, is able to overcome the insulin resistance in whole or in part and produce a biologic response.
  • hyperinsulinemia refers to a condition in which there are elevated levels of circulating insulin when, concomitantly, blood glucose levels are either elevated or normal.
  • Hyperinsulinemia can be caused by insulin resistance which is associated with dyslipidemia such as high triglycerides, high cholesterol, high low-density lipoprotein (LDL) and low high-density lipoprotein (HDL); high uric acids levels; polycystic ovary syndrome; type II diabetes and obesity.
  • Hyperinsulinemia can be diagnosed as having a plasma insulin level higher than about 2 ⁇ /mL.
  • a Modulator e.g., a peptide of the present disclosure
  • administration of a Modulator to a subject may result in lower insulin levels because the Modulator improves insulin sensitivity, or in higher insulin levels because the Modulator stimulates insulin secretion; a Modulator acting via either mechanism may result in improved glucose homeostasis.
  • metabolic syndrome refers to an associated cluster of traits that includes, but is not limited to, hyperinsulinemia, abnormal glucose tolerance, obesity, redistribution of fat to the abdominal or upper body compartment, hypertension, dysfibrinolysis, and dyslipidemia characterized by high levels of triglycerides, low levels of high-density lipoprotein (HDL) cholesterol, and high levels of low-density lipoprotein (LDL) cholesterol.
  • Subjects having metabolic syndrome are at risk for development of Type 2 diabetes and, for example, atherosclerosis.
  • glucose metabolism disorder encompasses any disorder characterized by a clinical symptom or a combination of clinical symptoms that is associated with an elevated level of glucose and/or an elevated level of insulin in a subject relative to a healthy individual. Elevated levels of glucose and/or insulin may be manifested in the following diseases, disorders and conditions: hyperglycemia, type II diabetes (e.g., insulin-resistance diabetes), gestational diabetes, type I diabetes, insulin resistance, impaired glucose tolerance, hyperinsulinemia, impaired glucose metabolism, pre-diabetes, metabolic disorders (such as metabolic syndrome which is also referred to as syndrome X), hypoglycemia, and obesity, among others.
  • the Modulators of the present disclosure, and compositions thereof, can be used to, for example, achieve and/or maintain glucose homeostasis, e.g., to reduce glucose level in the bloodstream and/or to reduce insulin level to a range found in a healthy subject.
  • hyperglycemia refers to a condition in which an elevated amount of glucose circulates in the blood plasma of a subject relative to a healthy individual. Hyperglycemia can be diagnosed using methods known in the art, including measurement of fasting blood glucose levels as described herein.
  • Modemators refers to the one or more Agr2, Cnpy4, Scg3 and Smoc2 - related peptides and agents that stimulate, increase, activate, facilitate, enhance activation, sensitize or up-regulate the function or activity, either directly or indirectly, of one or more of the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides described herein.
  • Modulators are agents that effect a desired biological response (e.g., lowering of glucose and/or body weight) by the same mechanism of action as the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides described herein (e.g., agents that modulate the same signaling pathway as the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides described herein, in a manner analogous thereto, and are capable of eliciting a biological response comparable to (or greater than) that of the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides described herein).
  • Modulators include other agonists such as antibodies and fragments thereof, small molecule agonist compounds, and other agonistic peptides structurally distinguishable from the peptides disclosed herein.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the terms include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. While the aforementioned terms can be used interchangeably, in general the term “peptide” refers to a polymeric form of amino acids of less than about 50 amino acids in length.
  • homologues or “variants” are used interchangeably to refer to amino acid or DNA sequences that are similar to reference amino acid or nucleic acid sequences, respectively.
  • homologues may refer to nucleic acid or amino acid sequences in one species that are similar to nucleic acid or amino acid sequences in another species.
  • homologues may refer to nucleic acid or amino acid sequences in one species that are similar to nucleic acid or amino acid sequences in the same species.
  • Homologues or variants encompass naturally occurring DNA sequences and proteins encoded thereby and their isoforms.
  • Homologues also include known allelic or splice variants of a protein or gene. Homologues and variants also encompass nucleic acid sequences that vary in one or more bases from a naturally- occurring DNA sequence but still translate into an amino acid sequence that corresponds to the naturally-occurring protein due to degeneracy of the genetic code. Homologues and variants may also refer to those that differ from the naturally-occurring sequences by one or more conservative substitutions and/or tags and/or conjugates.
  • DNA DNA
  • nucleic acid nucleic acid molecule
  • polynucleotide polynucleotide
  • mRNA messenger RNA
  • cDNA complementary DNA
  • vectors vectors, probes, primers and the like.
  • Probe refers to a fragment of DNA or RNA corresponding to a gene or sequence of interest, wherein the fragment has been labeled radioactively (e.g., by incorporating
  • a probe can be used to, for example, label viral plaques, bacterial colonies or bands on a gel that contain the gene of interest.
  • a probe can be cloned DNA or it can be a synthetic DNA strand; the latter can be used to obtain a cDNA or genomic clone from an isolated protein by, for example, microsequencing a portion of the protein, deducing the nucleic acid sequence encoding the protein, synthesizing an oligonucleotide carrying that sequence, radiolabeling the sequence and using it as a probe to screen a cDNA library or a genomic library.
  • heterologous refers to two components that are defined by structures derived from different sources.
  • a “heterologous” peptide may include operably linked amino acid sequences that are derived from different peptides (e.g., a first component comprising a recombinant peptide and a second component derived from a native Agr2, Cnpy4, Scg3 or Smoc2 peptide described herein).
  • a heterologous polynucleotide may include in operably linked nucleic acid sequences that can be derived from different genes (e.g., a first component from a nucleic acid encoding a peptide according to an embodiment disclosed herein and a second component from a nucleic acid encoding a carrier peptide).
  • heterologous nucleic acids include expression constructs in which a nucleic acid comprising a coding sequence is operably linked to a regulatory element (e.g., a promoter) that is from a genetic origin different from that of the coding sequence (e.g., to provide for expression in a host cell of interest, which may be of different genetic origin than the promoter, the coding sequence or both).
  • a T7 promoter operably linked to a polynucleotide encoding a Agr2(p4, p5, p6), Cnpy4, Scg3 or Smoc2 peptide described herein is said to be a heterologous nucleic acid.
  • heterologous can refer to the presence of a nucleic acid (or gene product, such as a peptide) that is of a different genetic origin than the host cell in which it is present.
  • operably linked refers to linkage between molecules to provide a desired function.
  • “operably linked” in the context of nucleic acids refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second polynucleotide, wherein the expression control sequence affects transcription and/or translation of the second polynucleotide.
  • “operably linked” refers to a functional linkage between amino acid sequences (e.g., of different domains) to provide for a described activity of the peptide.
  • N-terminus (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the extreme amino and carboxyl ends of the peptide, respectively, while the terms “N-terminal” and “C-terminal” refer to relative positions in the amino acid sequence of the peptide toward the N-terminus and the C-terminus, respectively, and can include the residues at the N-terminus and C-terminus, respectively.
  • Immediately N-terminal or “immediately C-terminal” refers to a position of a first amino acid residue relative to a second amino acid residue where the first and second amino acid residues are covalently bound to provide a contiguous amino acid sequence.
  • “Derived from”, in the context of an amino acid sequence or polynucleotide sequence is meant to indicate that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid (e.g., a naturally occurring Agr2, Cnpy4, Scg3 or Smoc2 polypeptide or a Agr2, Cnpy4, Scg3 or Smoc2-encoding nucleic acid), and is not meant to be limiting as to the source or method in which the protein or nucleic acid is made.
  • the term “derived from” includes homologues or variants of reference amino acid or DNA sequences.
  • isolated refers to a peptide of interest that, if naturally occurring, is in an environment different from that in which it may naturally occur. "Isolated” is meant to include peptides that are within samples that are substantially enriched for the peptide of interest and/or in which the peptide of interest is partially or substantially purified. Where the peptide is not naturally occurring, “isolated” indicates that the peptide has been separated from an environment in which it was made by either synthetic or recombinant means.
  • Enriched means that a sample is non-naturally manipulated (e.g., by a scientist or a clinician) so that a peptide of interest is present in a) a greater concentration (e.g., at least 3-fold greater, at least 4-fold greater, at least 8-fold greater, at least 64-fold greater, or more) than the concentration of the peptide in the starting sample, such as a biological sample (e.g., a sample in which the peptide naturally occurs or in which it is present after administration), or b) a greater concentration than the environment in which the peptide was made (e.g., as in a bacterial cell).
  • a biological sample e.g., a sample in which the peptide naturally occurs or in which it is present after administration
  • a greater concentration than the environment in which the peptide was made e.g., as in a bacterial cell.
  • substantially pure indicates that a component (e.g., a peptide) makes up greater than about 50% of the total content of the composition and typically, greater than about 60% of the total peptide content. More typically, “substantially pure” refers to compositions in which at least 75%, at least 85%, at least 90% or more of the total composition is the component of interest. In some cases, the peptide will make up greater than about 90%, or greater than about 95% of the total content of the composition.
  • a component e.g., a peptide
  • antibodies refer to glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Antibodies are described in detail hereafter.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • An "isolated" antibody is one which has been separated and/or recovered from contaminant components of its natural environment; such contaminant components are materials which might interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • contaminant components are materials which might interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • Agr2, Cnpy4, Scg3 and Smoc2 encompass peptides and variants thereof that are encoded by the Agr2, Cnpy4, Scg3 and Smoc2 genes or homologues thereof, respectively.
  • Agr2 (anterior gradient protein), also referred to as Xenopus laevis secreted cement gland protein XAG homolog, prostate cancer hereditary 8 (HPC8), GOB-4, PDIA17, and prol272, is a member of the protein-disulfide isomerase (PDI) family. Members of this family are similar to secreted proteins encoded by the cement gland-specific genes XAG-1 and XAG-2, expressed in the anterior region of dorsal ectoderm of Xenopus laevis. Agr2 is over-expressed in several types of cancer (e.g., pancreatic cancer) and is believed to play a role in cancer development.
  • PDI protein-disulfide isomerase
  • Agr2 polypeptide is found in mammals (e.g. human, dog, and mouse).
  • Figure 4 sets forth a multiple sequence alignment of Agr2 amino acid sequences from several species.
  • Agr2 - related nucleic acid sequences encoding a variety of different Agr2 - related polypeptides are known and available in the art and include the following (listed with their corresponding GenBank accession numbers): 1) Homo sapiens: amino acid sequence: NP 006399; nucleotide sequence: NM 006408; 2) Mus musculus: amino acid sequence: NP 035913; nucleotide sequence: NM 011783; 3) Rattus norvegicus: amino acid sequence: NP 001100195; nucleotide sequence: NM 001106725; 4) Macaca mulatta: amino acid sequence: NP 001181233;
  • nucleotide sequence NM 001194304; and 5
  • Canis lupus familiaris amino acid sequence: XP 539450; nucleotide sequence: XM 539450.
  • the corresponding human peptides are believed to have comparable activity based upon their sequence homology to the mouse peptides; thus, hu- p4, hu-p5 and hu-p6 are believed to be active.
  • the seven human and seven murine Agr2 subsequences are as follows:
  • Agr2 (hu-p2) (SEQ ID NO: 46): VFAENKEIQKLAEQFVLLNLVYETTD-COOH;
  • Agr2 (hu-p4) (SEQ ID NO: 47): IMFVDPSLTVRADITG-COOH;
  • Agr2 (hu-p5) (SEQ ID NO: 48): IMF VDP SLT VRADIT-C ONH 2 ;
  • Agr2 (hu-p6) (SEQ ID NO: 49): LYAYEPADTALLLDNM-COOH;
  • Agr2 (hu-p7) (SEQ ID NO: 50): YSNRLYAYEPADTALLLDNM-COOH;
  • Agr2 (ms-pl) (SEQ ID NO: 1): KDTTVKSGA-COOH;
  • Agr2 (ms-p2) (SEQ ID NO: 2): VFAEHKEIQKLAEQFVLLNLVYETTD-COOH;
  • Agr2 (ms-p3) (SEQ ID NO: 3): HLSPDGQYVP-COOH;
  • Agr2 (ms-p4) (SEQ ID NO: 4): IVFVDPSLTVRADITG-COOH;
  • Agr2 (ms-p5) (SEQ ID NO: 5): IVFVDPSLTVRADIT-CONH 2 ;
  • Agr2 (ms-p7) (SEQ ID NO: 7): YSNRLYAYEPSDTALLYDNM-COOH.
  • One embodiment of the present disclosure relates to a peptide comprising any one of: a subsequence of human Agr2 or a murine isoform of Agr2 as depicted in Figure 4, or a variant thereof; or a sequence comprising XiD X 2 TVKX 3 GX 4 (SEQ ID NO: 36);
  • VFAEX 5 KEIQKLAEQFVLLNLX 6 YETTD SEQ ID NO: 37
  • HLSPDGQYVP SEQ ID NO: 3
  • IX 7 FX 8 VDPSLTVRADITG SEQ ID NO: 38
  • IX 7 FX 8 VDPSLTVRADIT SEQ ID NO: 39
  • LYAYEPX 9 DX 10 ALLX 11 DNM SEQ ID NO: 40
  • YSNRLYAYEPX 9 DXi 0 ALLXnDNM SEQ ID NO: 41
  • the peptide comprises a variant of a human Agr2 peptide of Figure 3 A, a variant of a murine Agr2 peptide of Figure 3B, or a variant of the highlighted AGR subsequences shown in Figure 3A or Figure 3B.
  • the variant of a human Agr2 peptide comprises an amino acid sequence having at least 85% amino acid identity, at least 90% amino acid identity, at least 93% amino acid identity, at least 95% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to the amino acid sequence of a human Agr2 peptide of Figure 3 A, or the highlighted subsequence shown in Figure 3A.
  • the Agr2 peptide comprises an amino acid sequence comprising XiD X 2 TVKX 3 GX 4 (SEQ ID NO: 36); VFAEX 5 KEIQKLAEQFVLLNLX 6 YETTD (SEQ ID NO: 37); HLSPDGQYVP (SEQ ID NO: 3); IX 7 FX 8 VDPSLTVRADITG (SEQ ID NO: 38); IX 7 FX 8 VDPSLTVRADIT (SEQ ID NO: 39); LYAYEPX 9 DXi 0 ALLXnDNM (SEQ ID NO: 40); or YSNRLYAYEPX 9 DXi 0 ALLXnDNM (SEQ ID NO: 41), wherein X 1 - X n are as defined above (e.g., semi-conserved residues, optionally residues as indicated), and has fewer than 100 amino acid residues, fewer than 75 amino acid residues, fewer than 50 amino acid residues, fewer than 25 amino acid
  • the Agr2 peptide comprises an amino acid sequence comprising XiD X 2 TVKX GX 4 (SEQ ID NO: 36); VFAEX 5 KEIQKLAEQFVLLNLX 6 YETTD (SEQ ID NO: 37); HLSPDGQYVP (SEQ ID NO: 3); IX 7 FX 8 VDPSLTVRADITG (SEQ ID NO: 38); LYAYEPX 9 DXi 0 ALLXnDNM (SEQ ID NO: 40); or YSNRLYAYEPX 9 DXi 0 ALLXnDNM (SEQ ID NO: 41), wherein Xi - Xn are as defined above and wherein the sequence comprises a CONH 2 group instead of a COOH group at the carboxyl terminus of the peptide.
  • murine Agr2(ms-p4) significantly improves oral glucose tolerance but not basal (fasted) glucose concentration (see Figure 5A), and these findings are also observed when the effects of murine Agr2(ms-p4) on oral glucose tolerance and basal glucose concentration are expressed as the percent change when normalized to baseline (see Figure 5B).
  • Murine Agr2(ms-p4) significantly increases both fasting plasma insulin (FPI) concentration and glucose-stimulated insulin secretion (GSIS) concentration (see Figure 5C).
  • FPI fasting plasma insulin
  • GSIS glucose-stimulated insulin secretion
  • Figure 6 indicates the effect of murine Agr2(ms-p5) on glucose and insulin concentrations.
  • Murine Agr2(ms-p5) does not significantly improve basal (fasted) plasma glucose concentration or oral glucose tolerance (see Figure 6A), but when the data from Figure 6A are expressed as the percent change in plasma glucose concentration normalized to baseline, Agr2(ms-p5) significantly improves oral glucose tolerance at the 60- and 120-minute time points (see Figure 6B).
  • Murine Agr2(ms-p5) does not significantly increase either FPI or GSIS concentrations (see Figure 6C), nor is there a significant increase in either parameter when the data from Figure 6C are expressed as the percent change in plasma insulin concentration normalized to baseline (see Figure 6D).
  • murine Agr2(ms-p6) significantly improves both basal (fasted) glucose concentration and oral glucose tolerance (see Figure 7A), and these effects are also observed when the data from Figure 7A are expressed as the percent change in plasma glucose concentration normalized to baseline (see Figure 7B).
  • Murine Agr2(ms-p6) significantly increases glucose-stimulated insulin secretion (GSIS) concentration at the 15-minute time point (see Figure 7C).
  • GSIS glucose-stimulated insulin secretion
  • murine AGPv(ms-p6) significantly increases both FPI and GSIS at the 15- and 60-minute time points (see Figure 7D).
  • Cnpy4 (canopy 4 homolog), also known as canopy 4 homolog (zebrafish), MGC40499, PRAT4B and Protein Associated with Tlr4, has been shown to play a role in the regulation of the cell surface expression of Toll-like Receptor 4 (TLR4).
  • TLR4 Toll-like Receptor 4
  • the toll-like receptors recognize microbial products and induce immune responses. (See Konno K, Biochem Biophys Res Commun. 339(4): 1076-82 (Jan 27 2006)).
  • FIG. 9 The full-length human Cnpy4 amino acid sequence and the nucleic acid sequence encoding full-length human Cnpy4 are depicted in Figure 9.
  • Figure 9 the amino acid sequence of human peptide Cnpy4 hu-pl is highlighted in gray, and the coding region of the nucleic acid molecule is highlighted in gray.
  • Figure 10 depicts the full-length murine Cnpy4 amino acid sequence (the amino acid sequence of murine Cnpy4 ms-1 is highlighted in gray), and the nucleic acid sequence encoding full-length murine Cnpy4 (the coding region is highlighted in gray).
  • Cnpy4 polypeptide is found in mammals (e.g. human, dog, and mouse).
  • Cnpy4 - related nucleic acid sequences encoding a variety of different Cnpy4 - related polypeptides are known and available in the art and include the following (listed with their corresponding GenBank accession numbers): 1) Homo sapiens: amino acid sequence:
  • NP_689968 nucleotide sequence: NM_152755; 2) Mus musculus: amino acid sequence:
  • the human Cnpy4 subsequence and the murine Cnpy4 subsequence are as follows:
  • One embodiment of the present disclosure relates to a peptide comprising any one of: a subsequence of human Cnpy4 or a murine isoform of Cnpy4 as depicted in Figure 11, or a variant thereof; or the sequence XiX 2 X 3 KEX 4 X 5 X 6 DTERLPSKCEVCKLLSX 7 ELQX 8 X 9 LSRT (SEQ ID NO: 42), wherein each of X 1 -X 9 are semi-conserved residues, which residues in some embodiments are as follows: Xi is G or E; X 2 is M, A or T; X 3 is L, T or S; X 4 is E or is absent; X 5 is D or E; X 6 is D or A; X 7 is T, L or M; and X 8 and X 9 are E or A.
  • the peptide comprises a carboxyl-terminal modification, e.g., instead of a carboxy
  • the peptide comprises a variant of a human Cnpy4 peptide of Figure 9, a variant of a murine Cnpy4 peptide of Figure 10, or a variant of the highlighted Cnpy4 subsequence shown in Figure 9 or 10.
  • the variant of a human Cnpy4 peptide comprises an amino acid sequence having at least 85% amino acid identity, at least 90% amino acid identity, at least 93% amino acid identity, at least 95% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to the amino acid sequence of a human Cnpy4 peptide of Figure 9, or the highlighted subsequence shown in Figure 9.
  • the Cnpy4 peptide comprises the amino acid sequence XiX 2 X 3 KEX 4 X 5 X 6 DTERLPSKCEVCKLLSX 7 ELQX 8 X 9 LSRT (SEQ ID NO: 42), where X X 9 are as defined above (e.g., semi-conserved residues, , optionally residues as indicated), and has fewer than 100 amino acid residues, fewer than 75 amino acid residues, fewer than 50 amino acid residues, fewer than 25 amino acid residues, or fewer than 20 amino acid residues.
  • the Cnpy4 peptide comprises the amino acid sequence
  • Scg3 secretogranin-3
  • FLJ90833, FACE2, SGIII and FLJ30921 is a member of the chromogranin/secretogranin family of neuroendocrine secretory proteins.
  • the granins may serve as precursors for biologically-active peptides.
  • Scg3 Genetic variations in the Scg3 gene may influence the risk of obesity through possible regulation of secretion of hypothalamic neuropeptides (e.g., orexin, melanin-concentrating hormone (MCH), neuropeptide Y (NPY), and POMC). More particularly, functional single- nucleotide polymorphisms in the Scg3 gene that form secretory granules with appetite-related neuropeptides have been shown to be associated with obesity. Thus, Scg3 may be a potential regulator of food intake based on its capacity to accumulate appetite-related hormones into secretory granules. (See, e.g., Tanabe A, et al, J Clin Endocrinol Metab.
  • FIG. 14A The full-length human Scg3 amino acid sequence is depicted in Figure 14A, and the nucleic acid sequence encoding full-length human Scg3 is depicted in Figure 14B (the coding region of the nucleic acid molecule is highlighted in gray).
  • Figure 14 A the amino acid sequence of human peptide Scg3 hu-pl is highlighted in gray.
  • Figure 15A depicts the full-length murine Scg3 amino acid sequence (the amino acid sequence of murine Scg3 ms-1 is highlighted in gray), and
  • Figure 15B depicts the nucleic acid sequence encoding full-length murine Scg3 (the coding region is highlighted in gray).
  • Scg3 polypeptide is found in mammals (e.g. human, dog, and mouse), and Figure 16 sets forth a multiple sequence alignment of Scg3 amino acid sequences from several species.
  • Scg3 - related nucleic acid sequences encoding a variety of different Scg3 - related polypeptides are known and available in the art and include the following (listed with their corresponding GenBank accession numbers): 1) Homo sapiens: amino acid sequence: NP 037375; nucleotide sequence: NM_013243; 2) Mus musculus: amino acid sequence: NP_033156; nucleotide sequence: NM 009130; 3) Rattus norvegicus: amino acid sequence: NP 446308; nucleotide sequence: NM 053856; 4) Pan troglodytes: amino acid sequence: XP 510407; nucleotide sequence: XM_510407; and 5) Canis lupus familiaris: amino acid sequence: XP
  • nucleotide sequence XM_535482.
  • the human Scg3 subsequence and the murine Scg3 subsequence are as follows:
  • One embodiment of the present disclosure relates to a peptide comprising any one of: a subsequence of human Scg3 or a murine isoform of Scg3 as depicted in Figure 16, or a variant thereof; or the sequence QSIRSXiPX 2 DNX 3 LNVX 4 DX 5 DSTKN (SEQ ID NO: 43), where Xi - X 5 are semi-conserved residues, which residues in some embodiments are as follows: Xi is S or P; X 2 is F, L or Y; X 3 is K, Q or R; X 4 is E or D; and X 5 is V or A.
  • the peptide comprises a carboxyl-terminal modification, e.g., instead of a carboxyl group at the carboxyl terminus, the peptide comprises a CONH 2 group.
  • the peptide comprises a variant of a human Scg3 peptide of Figure 14A, a variant of a murine Scg3 peptide of Figure 15 A, or a variant of the highlighted Scg3 subsequence shown in Figure 14A or Figure 15 A.
  • the variant of a human Scg3 peptide comprises an amino acid sequence having at least 85% amino acid identity, at least 90% amino acid identity, at least 93% amino acid identity, at least 95% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to the amino acid sequence of a human Scg3 peptide of Figure 14A, or the highlighted subsequence shown in Figure 14A.
  • the Scg3 peptide comprises the amino acid sequence QSIRSXiPX 2 DNX 3 LNVX 4 DX 5 DSTK (SEQ ID NO: 43), where Xi - X 5 are as defined above (e.g., semi-conserved residues, optionally residues as indicated), and has fewer than 100 amino acid residues, fewer than 75 amino acid residues, fewer than 50 amino acid residues, fewer than 25 amino acid residues, or fewer than 20 amino acid residues.
  • the Scg3 peptide comprises the amino acid sequence QSIRSXiPX 2 DNX 3 LNVX 4 DX 5 DSTKN (SEQ ID NO: 43) comprising a CONH 2 group instead of a COOH group at the carboxyl terminus of the peptide.
  • murine Scg3(ms-p) significantly improves oral glucose tolerance (but not basal (fasted) glucose concentration) at 60 and 120 minutes (see Figure 17A); when the effect of murine Scg3(ms-p) on oral glucose tolerance is expressed as the percent change when normalized to baseline, there is a trend toward improvement of oral glucose tolerance (see Figure 17B).
  • Murine Scg3(ms-p) significantly increases both fasting plasma insulin (FPI) concentration and glucose-stimulated insulin secretion (GSIS) concentration at the 60-minute time point (see Figure 17C).
  • Smoc2 secreted modular calcium-binding protein-2
  • DTDP1 modular calcium-binding protein-2
  • MSTP140 MSTP140
  • MEK 8 SPARC (Secreted Protein Acidic And Rich in Cysteine)-related protein.
  • SPARC Secreted Protein Acidic And Rich in Cysteine
  • Smoc2 has been shown to play a role in cell cycle control by promoting growth factor-induced cyclin Dl expression and DNA synthesis via integrin-linked kinase (ILK).
  • ILK integrin-linked kinase
  • FIG. 19A The full-length human Smoc2 amino acid sequence is depicted in Figure 19A, and the nucleic acid sequence encoding full-length human Smoc2 are depicted in Figure 19B (the coding region of the nucleic acid molecule is highlighted in gray).
  • Figure 19A the amino acid sequence of human peptide Smoc2 hu-pl is highlighted in gray.
  • Figure 20 A depicts the full- length murine Smoc2 amino acid sequence (the amino acid sequence of murine Smoc2 ms-1 is highlighted in gray)
  • Figure 20B depicts the nucleic acid sequence encoding full-length murine Smoc2 (the coding region is highlighted in gray).
  • Smoc2 polypeptide is found in mammals (e.g. human, dog, and mouse).
  • Figure 21 sets forth a multiple sequence alignment of Smoc2 amino acid sequences from several species.
  • Smoc2 - related nucleic acid sequences encoding a variety of different Smoc2 - related polypeptides are known and available in the art and include the following (listed with their corresponding GenBank accession numbers): 1) Homo sapiens: amino acid sequence:
  • NP 001159884 nucleotide sequence: NM 001166412; 2) Mus musculus: amino acid sequence: NP_071710; nucleotide sequence: NM_022315; 3) Rattus norvegicus: amino acid sequence: NP 001099685; nucleotide sequence: NM 001106215; 4) Pan troglodytes: amino acid sequence: XP 530989; nucleotide sequence: XM 530989; and 5) Canis lupus familiaris: amino acid sequence: NP_001171274; nucleotide sequence: NM_001177803.
  • the human Smoc2 subsequence and the murine Smoc2 subsequence are as follows:
  • one embodiment of the present disclosure relates to a peptide comprising any one of: a subsequence of human SMOC3 or a murine isoform of Smoc2 as depicted in Figure 21; or the sequence
  • Xi - X 5 are semi-conserved residues, which residues in some embodiments are as follows: Xi is V or M; X 2 is S or T; X is T or A; X 4 is R or K; and X 5 is E or D.
  • the peptide comprises a carboxyl-terminal modification, e.g., instead of a carboxyl group at the carboxyl terminus, the peptide comprises a CONH 2 group.
  • the peptide comprises a variant of a human Smoc2 peptide of Figure 19A, a variant of a murine Smoc2 peptide of Figure 20A, or a variant of the highlighted Smoc2 subsequence shown in Figure 19A or Figure 20A.
  • the variant of a human Smoc2 peptide comprises an amino acid sequence having at least 85% amino acid identity, at least 90% amino acid identity, at least 93% amino acid identity, at least 95% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to the amino acid sequence of a human Smoc2 peptide of Figure 19 A, or the highlighted subsequence shown in Figure 19A.
  • the Smoc2 peptide comprises the amino acid sequence CVKKFVEYCDXiNNDKSrX 2 VQELMGCLGVX 3 X 4 EX 5 G (SEQ ID NO: 44), where Xi - X 5 are as defined above (e.g., semi-conserved residues, optionally residues as indicated), and has fewer than 100 amino acid residues, fewer than 75 amino acid residues, fewer than 50 amino acid residues, fewer than 25 amino acid residues, or fewer than 20 amino acid residues.
  • the Smoc2 peptide comprises the amino acid sequence
  • Example 8 describes the glucoregulatory activity of murine Smoc2(ms-p).
  • Figure 22A shows the effect of a single bolus i.p. injection of Smoc2 ms-p (gray squares) and vehicle control (black squares) on basal (fasted) plasma glucose concentration and on oral glucose tolerance
  • Figure 22B shows the data from Figure 22A expressed as the percent change in plasma glucose concentration normalized to baseline (min-30).
  • Example 8 also shows the effect of a single bolus i.p.
  • Figure 22D shows the data from Figure 22C expressed as the percent change in plasma insulin concentration normalized to baseline (min-30).
  • Murine Smoc2(ms-p) significantly improves oral glucose tolerance at 120 minutes and shows a positive trend toward improving oral glucose tolerance at 60 minutes (see Figure 22A). Similar results are observed when the data from Figure 22A are expressed as the percent change in plasma glucose concentration normalized to baseline (see Figure 22B). Murine Smoc2(ms-p) did not significantly improve basal (fasted) plasma insulin (FPI) concentration or GSIS, but there is a trend toward improving GSIS at the 60 minute time point (see Figures 22C and 22D).
  • FPI plasma insulin
  • Agr2, Cnpy4, Scg3 and Smoc2 - related peptides is meant to include the disclosed Agr2, Cnpy4, Scg3 and Smoc2 human and murine subsequences (i.e., Agr2(hu-p4, hu-p5, hu-p6, ms-p4, ms-p5, ms-p6), Cnpy4(hu-1, ms-1), Scg3(hu-1, ms-1) and Smoc2(hu-pl, ms-pl)), as well as homologues, variants, fragments and other modified forms thereof.
  • Agr2(hu-p4, hu-p5, hu-p6, ms-p4, ms-p5, ms-p6 Cnpy4(hu-1, ms-1), Scg3(hu-1, ms-1) and Smoc2(hu-pl, m
  • Agr2, Cnpy4, Scg3 and Smoc2 - related nucleic acid molecules includes their naturally-occurring and non-naturally occurring isoforms, allelic variants and splice variants.
  • the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides also encompass peptides that have one or more alterations in the amino acid residues (e.g., at locations that are not conserved across variants or species) while retaining the conserved domains and having the same biological activity as the naturally-occurring Agr2, Cnpy4, Scg3 and Smoc2 - related peptides.
  • the Agr2, Cnpy4, Scg3 and Smoc2 - related nucleic acid sequences also encompass nucleic acid sequences that vary in one or more bases from a naturally-occurring DNA sequence but still translate into an amino acid sequence that corresponds to an Agr2, Cnpy4, Scg3 and Smoc2 - related peptides due to degeneracy of the genetic code.
  • Agr2, Cnpy4, Scg3 and Smoc2 - related peptides may also refer to amino acid sequences that differ from the naturally-occurring sequences by one or more conservative substitutions, tags, or conjugates.
  • the present disclosure contemplates having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, usually no more than 20, 10, or 5 amino acid substitutions, where the substitution is usually a conservative amino acid substitution.
  • conservative amino acid substitution generally refers to substitution of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E.
  • Conservative amino acid substitutions preserve the activity of the polypeptide by replacing an amino acid(s) in the polypeptide with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or size of the side chain.
  • Guidance for substitutions, insertions, or deletions may be based on alignments of amino acid sequences of different variant polypeptides or polypeptides from different species.
  • the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides are generally active fragments (subsequences) containing contiguous amino acid residues derived from the full-length Agr2, Cnpy4, Scg3 and Smoc2 polypeptides.
  • the regions encompassing Agr2(hu-p4, hu-p5, hu-p6), Cnpy4(hu- 1), Scg3(hu-1) and Smoc2(hu-pl) are highlighted in gray in Figures 3 A, 9, 14A and 19A, respectively, while the regions encompassing Agr2(ms-p4, ms-p5, ms-p6), Cnpy4(ms-1), Scg3(ms-1) and Smoc2(ms-pl)) are highlighted in gray in Figures 3B, 10, 15A and 20A, respectively, respectively.
  • peptides and polypeptides may be from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, from about 40 amino acids to about 50 amino acids, from about 50 amino acids to about 75 amino acids, from about 75 amino acids to about 100 amino acids, from about 100 amino acids to about 150 amino acids, from about 150 amino acids to about 200 amino acids, or from about 200 amino acids up to the full-length peptide or polypeptide.
  • the Agr2, Cnpy4 is the length of contiguous amino acid residues of a peptide or polypeptidequence.
  • Scg3 and Smoc2 - related peptides have a length that is less than the full length of the naturally- occurring polypeptides.
  • the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides can have a length of less than 5 amino acids, from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 45 amino acids, from about 45 amino acids to about 50 amino acids, or from more than about 50 amino acids.
  • the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides contemplated by the present disclosure are less than about 35 to about amino acids in length, whereas in other embodiments they are less than about 20 to about 25 amino acids in length.
  • the Agr2, Cnpy4, Scg3 or Smoc2 - related peptides can have a defined sequence identity compared to a reference sequence over a defined length of contiguous amino acids (e.g., a "comparison window").
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci.
  • a suitable Agr2, Cnpy4, Scg3 or Smoc2 - related peptide can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%o, at least about 90%>, at least about 95%, at least about 98%>, or at least about 99%, amino acid sequence identity to a contiguous stretch of less than 5 amino acids, from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 12 amino acids, from about 12 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 22 amino acids, from about 22 amino acids to about 25 amino acids, from about 25 amino acids to about 27 amino acids, from about 27 amino acids to about 29 amino acids, or from more than about 29 amino acids of one of the following reference amino acid sequences previously set forth:
  • Agr2 (hu-p4) (SEQ ID NO: 47): IMFVDPSLTVRADITG-COOH;
  • Agr2 (hu-p5) (SEQ ID NO: 48): IMFVDPSLTVRADIT-CONH 2 ;
  • Agr2 (hu-p6) (SEQ ID NO: 49): LYAYEPADTALLLDNM-COOH;
  • Agr2 (ms-p4) (SEQ ID NO: 4): IVFVDPSLTVRADITG-COOH;
  • Agr2 (ms-p5) (SEQ ID NO: 5): IVFVDPSLTVRADIT-CONH 2 ;
  • a suitable Agr2, Cnpy4, Scg3 or Smoc2 - related peptide can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%), amino acid sequence identity to a contiguous stretch of less than about 5 amino acids, from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 12 amino acids, from about 12 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 22 amino acids, or from more than about 22 amino acids of the Agr2, Cnpy4, Scg3 or Smoc2 subsequences set forth above, and where the Agr2, Cnpy4, Scg3 or Smoc2 - related peptide has a length of less than 5 amino acids, from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 12 amino
  • a peptide of the present disclosure comprises: a variant of a human AGR2 polypeptide of Figure 3 A or a variant of a murine AGR2 polypeptide of Figure 3B; a variant of a human CNPY4 polypeptide of Figure 9 or a variant of a murine CNPY4 polypeptide of Figure 10; a variant of a human SCG3 polypeptide of Figure 14A or a variant of a murine SCG3 polypeptide of Figure 15 A; or a variant of a human SMOC2 polypeptide of Figure 19A or a variant of a murine SMOC2 polypeptide of Figure 20A.
  • the aforementioned variants comprise an amino acid sequence having at least 85% amino acid identity, at least 90% amino acid identity, at least 93% amino acid identity, at least 95% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to the amino acid sequence of: a human or murine AGR2 polypeptide of Figure 3 A or Figure 3B, respectively; a human or murine CNPY4 polypeptide of Figure 9 or Figure 10, respectively; a human or murine SCG3 polypeptide of Figure 14A or Figure 15 A, respectively; or a human or murine SMOC2 polypeptide of Figure 19A or Figure 20A, respectively.
  • the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides may be isolated from a natural source (e.g., in an environment other than its naturally-occurring environment) and also may be recombinantly made (e.g., in a genetically modified host cell such as bacteria; yeast; Pichia; insect cells; and the like), where the genetically modified host cell is modified with a nucleic acid comprising a nucleotide sequence encoding the peptide.
  • the Agr2, Cnpy4, Scg3 or Smoc2 - related peptides may also be synthetically produced (e.g., by cell-free chemical synthesis). Methods of productions are described in more detail below.
  • An Agr2, Cnpy4, Scg3 or Smoc2 - related peptide may be generated using recombinant techniques to manipulate different Agr2, Cnpy4, Scg3 or Smoc2 - related nucleic acids known in the art to provide constructs capable of encoding the Agr2, Cnpy4, Scg3 or Smoc2 - related peptides. It will be appreciated that, when provided a particular amino acid sequence, the ordinary skilled artisan will recognize a variety of different nucleic acid molecules encoding such amino acid sequence in view of her background and experience in, for example, molecular biology.
  • the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides effect a decrease in systemic glycemia that may be attributable to, for example, increased insulin production and/or secretion.
  • the present disclosure contemplates other Modulators having comparable physiological activity.
  • Modulators refers to agents that, for example, stimulate, increase, activate, facilitate, enhance activation, sensitize or up-regulate the function or activity of one or more Agr2, Cnpy4, Scg3 or Smoc2 - related peptides.
  • Modulators encompass the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides and agents that operate through the same mechanism of action as the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides (e.g., agents that modulate the same signaling pathway as the Agr2, Cnpy4, Scg3 and/or Smoc2 - related peptides in a manner analogous to that of the peptides) and are capable of eliciting a biological response comparable to (or greater than) that of the Agr2, Cnpy4, Scg3 and/or Smoc2 - related peptides.
  • a Modulator may also be, for example, a small molecule agonist compound, or other bioorganic molecule.
  • the Modulator is a small molecule agonist compound.
  • the Modulator is an agonistic peptide structurally distinguishable from the Agr2, Cnpy4, Scg3 or Smoc2 - related peptides but having comparable activity.
  • the skilled artisan is able to identify such peptides having desired properties through the use of, for example, the methods described herein.
  • a Agr2, Cnpy4, Scg3 and Smoc2 - related peptide includes one or more linkages other than peptide bonds, e.g., at least two adjacent amino acids are joined via a linkage other than an amide bond.
  • one or more amide bonds within the backbone of a Agr2, Cnpy4, Scg3 or Smoc2 - related peptide can be substituted.
  • One or more amide linkages in a Agr2, Cnpy4, Scg3 or Smoc2 - related peptide can also be replaced by, for example, a reduced isostere pseudopeptide bond (see, e.g., Couder et al. (1993) Int. J. Peptide Protein Res. 41 : 181-184). Such replacements and how to effect them are known to those of ordinary skill in the art.
  • One or more amino acid substitutions can be made in a Agr2, Cnpy4, Scg3 or Smoc2 - related peptide.
  • alkyl-substituted hydrophobic amino acids including alanine, leucine, isoleucine, valine, norleucine, (S)-2-aminobutyric acid, (S)-cyclohexylalanine or other simple alpha-amino acids substituted by an aliphatic side chain from Ci-Cio carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions;
  • aromatic-substituted hydrophobic amino acids including phenylalanine, tryptophan, tyrosine, sulfotyrosine, biphenylalanine, 1-naphthylalanine, 2- naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, including amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy (from Ci- C 4 ) substituted forms of the above-listed aromatic amino acids, illustrative examples of which are: 2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4- methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5- methoxytrypto
  • amino acids containing basic side chains including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, including alkyl, alkenyl, or aryl- substituted (from Ci-Cio branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms (such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position for example.
  • heteroatoms such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon
  • N-epsilon-isopropyl-lysine 3-(4-tetrahydropyridyl)-glycine, 3-(4- tetrahydropyridyl)-alanine, ⁇ , ⁇ -gamma, gamma'-diethyl-homoarginine.
  • [00181] d) substitution of acidic amino acids, including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of 2,4- diaminopriopionic acid, ornithine or lysine and tetrazole-substituted alkyl amino acids;
  • an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide comprises one or more naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids or D- enantiomers of an amino acid.
  • an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide can comprise only D-amino acids.
  • an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide can comprise one or more of the following residues: hydroxyproline, ⁇ -alanine, o- aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2,3-diaminopropionic acid, a-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, naphthylalanine, pyridylalanine 3-benzothienyl alanine, 4-chlorophenylalanine, 2- fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenyl
  • homoarginine N-acetyl lysine, 2,4-diamino butyric acid, rho-aminophenylalanine, N- methylvaline, homocysteine, homoserine, ⁇ -amino hexanoic acid, ⁇ -aminohexanoic acid, ⁇ - aminoheptanoic acid, ⁇ -aminooctanoic acid, ⁇ -aminodecanoic acid, ⁇ -aminotetradecanoic acid, cyclohexylalanine, ⁇ , ⁇ -diaminobutyric acid, ⁇ , ⁇ -diaminopropionic acid, ⁇ -amino valeric acid, and 2,3-diaminobutyric acid.
  • a cysteine residue or a cysteine analog can be introduced into an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide to provide for linkage to another peptide via a disulfide linkage or to provide for cyclization of the Agr2, Cnpy4, Scg3 or Smoc2 - related peptide.
  • Methods of introducing a cysteine or cysteine analog are known in the art (see, e.g., U.S. Pat. No.
  • An Agr2, Cnpy4, Scg3 or Smoc2 - related peptide can be cyclized.
  • One or more cysteine or cysteine analogs can be introduced into an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide, where the introduced cysteine or cysteine analog can form a disulfide bond with a second introduced cysteine or cysteine analog.
  • Other means of cyclization include introduction of an oxime linker or a lanthionine linker (see, e.g., U.S. Patent No. 8,044,175). Any
  • a cyclizing bond can be generated with any combination of amino acids (or with an amino acid and -(CH 2 ) n -CO- or -(CH 2 ) n -C 6 H 4 -CO-) with functional groups which allow for the introduction of a bridge.
  • Some examples are disulfides, disulfide mimetics such as the -(CH 2 ) n -carba bridge, thioacetal, thioether bridges (cystathionine or lanthionine) and bridges containing esters and ethers.
  • n can be any integer, but is frequently less than ten.
  • hydroxymethyl derivatives O-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides.
  • one or more L-amino acids in an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide is replaced with a D-amino acid.
  • an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide is a retroinverso analog.
  • Retro-inverso peptide analogs are isomers of linear peptides in which the direction of the amino acid sequence is reversed (retro) and the chirality, D- or L-, of one or more amino acids therein is inverted (inverso), e.g., using D-amino acids rather than L-amino acids.
  • inverso e.g., Jameson et al. (1994) Nature 368:744; and Brady et al. (1994) Nature 368:692.
  • An Agr2, Cnpy4, Scg3 or Smoc2 - related peptide can include a "Protein
  • PTD Transduction Domain
  • a PTD attached to another molecule facilitates the molecule traversing a membrane, for example, going from extracellular space to intracellular space, or cytosol to within an organelle.
  • a PTD is covalently linked to the amino terminus of an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide, while in other
  • a PTD is covalently linked to the carboxyl terminus of the peptide.
  • exemplary PTDs include, but are not limited to, a minimal undecapeptide PTD (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO: 57); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes
  • Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 57); RKKRRQRRR (SEQ ID NO: 62); RKKRRQRR (SEQ ID NO: 63); YARAAARQARA (SEQ ID NO: 64); THRLPRRRR (SEQ ID NO: 65); and GGRRARRRRRR (SEQ ID NO: 66).
  • the carboxyl group can also be esterified with primary, secondary or tertiary alcohols such as, e.g., methanol, branched or unbranched Ci-C 6 -alkyl alcohols, e.g., ethyl alcohol or tert-butanol.
  • the carboxyl group can also be amidated with primary or secondary amines such as ammonia, branched or unbranched Ci-C6-alkylamines or Ci-C 6 di-alkylamines, e.g., methylamine or dimethylamine.
  • primary or secondary amines such as ammonia, branched or unbranched Ci-C6-alkylamines or Ci-C 6 di-alkylamines, e.g., methylamine or dimethylamine.
  • the amino group can be present in a form protected by amino-protecting groups conventionally used in peptide chemistry such as, e.g., Fmoc, Benzyloxy-carbonyl (Z), Boc, or Alloc.
  • Alkyl residues can be straight chained, branched or cyclic (e.g., ethyl, isopropyl and cyclohexyl, respectively).
  • an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide can include one or more modifications that enhance a property desirable in a protein formulated for therapy (e.g., serum half-life), that enable the raising of antibodies for use in detection assays (e.g., epitope tags), that provide for ease of protein purification, etc.
  • modifications include, but are not limited to, pegylation (covalent attachment of one or more molecules of polyethylene glycol (PEG), or derivatives thereof); N-glycosylation and polysialylation; albumin fusion; albumin binding through a conjugated fatty acid chain (acylation); Fc-fusion proteins; and fusion with a PEG mimetic.
  • Pegylation The clinical effectiveness of protein therapeutics is often limited by short plasma half-life and susceptibility to protease degradation.
  • Studies of various therapeutic proteins e.g., filgrastim
  • conjugating or linking the polypeptide sequence to any of a variety of non-proteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes (e.g., typically via a linking moiety covalently bound to both the protein and the non-proteinaceous polymer, e.g., a PEG).
  • PEG polyethylene glycol
  • polypropylene glycol polypropylene glycol
  • polyoxyalkylenes e.g., typically via a linking moiety covalently bound to both the protein and the non-proteinaceous polymer, e.g., a PEG.
  • PEG-conjugated biomolecules have been shown to possess clinically useful properties, including better physical and thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulating half-life and decreased clearance, reduced immunogenicity and antigenicity, and reduced toxicity.
  • PEGs suitable for conjugation to a polypeptide sequence are generally soluble in water at room temperature, and have the general formula R(0-CH 2 -CH 2 ) n O-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons.
  • the PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star-PEGs" and multi-armed PEGs are contemplated by the present disclosure.
  • the molecular weight of a PEG used in the present disclosure is not restricted to any particular range, but certain embodiments have a molecular weight between 500 and 20,000, while other
  • embodiments have a molecular weight between 4,000 and 10,000.
  • Such compositions can be produced by reaction conditions and purification methods know in the art.
  • conjugates may be separated from unmodified protein sequences and from conjugates having other numbers of PEGs attached.
  • fraction is then identified which contains the conjugate having, for example, the desired number of PEGs attached, purified free from unmodified protein sequences and from conjugates having other numbers of PEGs attached.
  • PEG may be bound to a polypeptide of the present disclosure via a terminal reactive group (a "spacer").
  • the spacer is, for example, a terminal reactive group which mediates a bond between the free amino or carboxyl groups of one or more of the polypeptide sequences and polyethylene glycol.
  • An example of a PEG molecule, modified to include a spacer, that may be bound to the free amino group of a polypeptide is N-hydroxysuccinylimide polyethylene glycol, which may be prepared by activating succinic acid ester of polyethylene glycol with N-hydroxysuccinylimide.
  • Another activated polyethylene glycol which may be bound to a free amino group is 2,4-bis(0-methoxypolyethyleneglycol)-6-chloro-s-triazine, which may be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride.
  • Examples of an activated polyethylene glycol bound to the free carboxyl group of a polypeptide include polyoxyethylenediamine.
  • the conjugation reaction can be carried out in solution at a pH of from 5 to 10, at a temperature from 4°C to room temperature, for 30 minutes to 20 hours, utilizing a molar ratio of reagent to protein of from 4: 1 to 30: 1.
  • Reaction conditions may be selected to direct the reaction towards producing predominantly a desired degree of substitution.
  • high temperature, neutral to high pH e.g., pH>7
  • longer reaction time tend to increase the number of PEGs attached.
  • Various means known in the art may be used to terminate the reaction.
  • the reaction is terminated by acidifying the reaction mixture and freezing at, e.g., -20°C.
  • PEG Mimetics Recombinant PEG mimetics have been developed that retain the attributes of PEG (e.g., enhanced serum half- life) while conferring several additional advantageous properties.
  • simple polypeptide chains comprising, for example, Ala, Glu, Gly, Pro, Ser and Thr
  • PEG protein of interest
  • This obviates the need for an additional conjugation step during the manufacturing process.
  • established molecular biology techniques enable control of the side chain composition of the polypeptide chains, allowing optimization of immunogenicity and manufacturing properties.
  • glycosylation is meant to broadly refer to the enzymatic process that attaches glycans to proteins, lipids or other organic molecules.
  • the use of the term “glycosylation” in conjunction with the present disclosure is generally intended to mean adding or deleting one or more carbohydrate moieties (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites to the native sequence.
  • the term includes qualitative changes in the
  • glycosylation pattern of the native sequence involving a change in the nature and proportions of the various carbohydrate moieties present.
  • Glycosylation can dramatically affect the physical properties of proteins and can also be important in protein stability, secretion, and subcellular localization. Proper
  • glycosylation can be essential for biological activity.
  • some genes from eucaryotic organisms when expressed in bacteria (e.g., E. coli) which lack cellular processes for glycosylating proteins, yield proteins that are recovered with little or no activity by virtue of their lack of glycosylation.
  • the addition of glycosylation sites can be accomplished by altering the amino acid sequence.
  • the alteration to the polypeptide may be made by, for example, the addition of, or substitution by, one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites).
  • the structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type may be different.
  • One type of sugar that is commonly found in both is N-acetylneuraminic acid (hereafter referred to as sialic acid).
  • Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycoprotein.
  • a particular embodiment of the present disclosure comprises the generation and use of N-glycosylation variants.
  • polypeptide sequences of the present disclosure may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Removal of carbohydrates may be accomplished chemically or enzymatically, or by substitution of codons encoding amino acid residues that are glycosylated. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases.
  • DHFR Dihydrofolate reductase
  • CHO Chinese Hamster Ovary
  • PSA biodegradable a-(2 ⁇ 8) linked polysialic acid
  • Albumin Fusion and Conjugation with Other Molecules include, for example, thyroglobulin; albumins such as human serum albumin (HAS); tetanus toxoid; Diphtheria toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses; influenza virus hemaglutinin, influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B virus core protein and surface antigen; or any combination of the foregoing.
  • albumins such as human serum albumin (HAS); tetanus toxoid; Diphtheria toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses; influenza virus hemaglutinin, influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (
  • Fusion of albumin to one or more polypeptides of the present disclosure can, for example, be achieved by genetic manipulation, such that the DNA coding for HSA, or a fragment thereof, is joined to the DNA coding for the one or more polypeptide sequences.
  • a suitable host can be transformed or transfected with the fused nucleotide sequences in the form of, for example, a suitable plasmid, so as to express a fusion polypeptide.
  • the expression may be effected in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo from, for example, a transgenic organism.
  • the expression of the fusion protein is performed in mammalian cell lines, for example, CHO cell lines. Transformation is used broadly herein to refer to the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells.
  • albumin itself may be modified to extend its circulating half-life.
  • Fusion of the modified albumin to one or more Agr2, Cnpy4, Scg3 or Smoc2 - related peptides can be attained by the genetic manipulation techniques described above or by chemical conjugation; the resulting fusion molecule has a half-life that exceeds that of fusions with unmodified albumin. (See, e.g., WO2011/051489).
  • albumin - binding strategies have been developed as alternatives to direct fusion, including albumin binding through a conjugated fatty acid chain (acylation). Because serum albumin is a transport protein for fatty acids, these natural ligands with albumin-binding activity have been used for half-life extension of small protein therapeutics.
  • insulin determir an approved product for diabetes, comprises a myristyl chain conjugated to a genetically-modified insulin, resulting in a long-acting insulin analog.
  • Another type of modification involves conjugation of one or more additional components or molecules at the N- and/or C-terminus of a polypeptide sequence, such as another protein (e.g., a protein having an amino acid sequence heterologous to the subject protein), or a carrier molecule.
  • a polypeptide sequence such as another protein (e.g., a protein having an amino acid sequence heterologous to the subject protein), or a carrier molecule.
  • another protein e.g., a protein having an amino acid sequence heterologous to the subject protein
  • a carrier molecule e.g., a protein having an amino acid sequence heterologous to the subject protein
  • a conjugate modification may result in a polypeptide sequence that retains its inherent activity but also has an additional or complementary function or activity of the second molecule.
  • a polypeptide sequence may be conjugated to a molecule to, e.g., facilitate solubility, storage, in vivo or shelf half-life or stability, reduction in immunogenicity, delayed or controlled release in vivo, etc.
  • Other functions or activities include a conjugate that reduces toxicity relative to an unconjugated polypeptide sequence, a conjugate that targets a type of cell or organ more efficiently than an unconjugated polypeptide sequence, or a drug to further counter the causes or effects associated with a disorder or disease as set forth herein (e.g., diabetes).
  • a polypeptide may also be conjugated to large, slowly metabolized
  • macromolecules such as proteins; polysaccharides, such as sepharose, agarose, cellulose and cellulose beads; polymeric amino acids such as polyglutamic acid and polylysine; amino acid copolymers; inactivated virus particles; inactivated bacterial toxins such as toxoid from diphtheria, tetanus, cholera and leukotoxin molecules; inactivated bacteria; and dendritic cells.
  • polysaccharides such as sepharose, agarose, cellulose and cellulose beads
  • polymeric amino acids such as polyglutamic acid and polylysine
  • amino acid copolymers amino acid copolymers
  • inactivated virus particles inactivated bacterial toxins such as toxoid from diphtheria, tetanus, cholera and leukotoxin molecules
  • inactivated bacteria inactivated bacteria
  • dendritic cells dendritic cells.
  • Additional candidate components and molecules for conjugation include those suitable for isolation or purification.
  • Particular non-limiting examples include binding molecules, such as biotin (biotin-avidin - specific binding pair), an antibody, a receptor, a ligand, a lectin, or molecules that comprise a solid support, including, for example, plastic or
  • polystyrene beads plates or beads, magnetic beads, test strips, and membranes.
  • cation exchange chromatography may be used to separate conjugates by charge difference, which effectively separates conjugates into their various molecular weights.
  • a cation exchange column can be loaded and then washed with ⁇ 20 mM sodium acetate, pH ⁇ 4, and then eluted with a linear (0 M to 0.5 M) NaCl gradient buffered at a pH from about 3 to 5.5, e.g., at pH ⁇ 4.5.
  • the content of the fractions obtained by cation exchange chromatography may be identified by molecular weight using conventional methods, for example, mass spectroscopy, SDS-PAGE, or other known methods for separating molecular entities by molecular weight.
  • Fc-fusion Molecules In certain embodiments, the amino- or carboxyl- terminus of a polypeptide sequence of the present disclosure can be fused with an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half- life of biopharmaceuticals, and thus the
  • biopharmaceutical product may require less frequent administration.
  • Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells that line the blood vessels, and, upon binding, the Fc fusion molecule is protected from degradation and re-released into the circulation, keeping the molecule in circulation longer.
  • This Fc binding is believed to be the mechanism by which endogenous IgG retains its long plasma half-life.
  • More recent Fc- fusion technology links a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical as compared to traditional Fc-fusion conjugates.
  • Suitable linkers include "flexible linkers" which are generally of sufficient length to permit some movement between the modified polypeptide sequences and the linked components and molecules.
  • the linker molecules are generally about 6-50 atoms long.
  • the linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.
  • Suitable linkers can readily be selected and can be of any suitable length, such as 1 (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 amino acids (e.g., Gly).
  • Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (for example, (GS) n , GSGGS n (SEQ ID NO: 67) and GGGS n (SEQ ID NO: 68), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • Glycine and glycine-serine polymers are relatively unstructured, and therefore may serve as a neutral tether between components.
  • Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO: 69), GGSGG (SEQ ID NO: 70), GSGSG (SEQ ID NO: 71), GSGGG (SEQ ID NO: 72), GGGSG (SEQ ID NO: 73), and GSSSG (SEQ ID NO: 74).
  • a peptide of the present disclosure can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis).
  • a polypeptide is chemically synthesized
  • the synthesis may proceed via liquid phase or solid-phase.
  • Solid-phase peptide synthesis allows the incorporation of unnatural amino acids and/or peptide/protein backbone modification.
  • Various forms of SPPS such as Fmoc and Boc, are available for synthesizing peptides of the present disclosure. Details of the chemical synthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10; and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8).
  • Solid phase peptide synthesis may be performed as described hereafter.
  • the a functions (Na) and any reactive side chains are protected with acid-labile or base-labile groups.
  • the protective groups are stable under the conditions for linking amide bonds but can be readily cleaved without impairing the peptide chain that has formed.
  • Suitable protective groups for the a-amino function include, but are not limited to, the following: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), o-chlorbenzyloxycarbonyl, bi-phenylisopropyloxycarbonyl, tertamyloxycarbonyl (Amoc), a,a-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl, 2-cyano-t-butoxy-carbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), l-(4,4-dimethyl-2,6- dioxocylohex-l-ylidene)ethyl (Dde) and the like.
  • Suitable side chain protective groups include, but are not limited to: acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl), benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl, 2- chlorobenzyl, 2-chlorobenzyloxycarbonyl (2-CIZ), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)ethyl (Dde), isopropyl, 4-methoxy-2,3-6- trimethylbenzylsulfonyl (Mtr), 2,3,5,7,8-pentamethylchromine,
  • the C-terminal amino acid is coupled to a suitable support material.
  • suitable support materials are those which are inert towards the reagents and reaction conditions for the step-wise condensation and cleavage reactions of the synthesis process and which do not dissolve in the reaction media being used.
  • Examples of commercially- available support materials include styrene/divinylbenzene copolymers which have been modified with reactive groups and/or polyethylene glycol; chloromethylated
  • TentaGel® derivatized with 4-benzyloxybenzyl-alcohol (Wang-anchor) or 2-chlorotrityl chloride can be used if it is intended to prepare the peptidic acid.
  • polystyrene (1%) divinylbenzene or TentaGel® derivatized with 5-(4'-aminomethyl)-3',5'- dimethoxyphenoxy)valeric acid (PAL-anchor) or p-(2,4-dimethoxyphenyl-amino methyl)- phenoxy group (Rink amide anchor) can be used.
  • the linkage to the polymeric support can be achieved by reacting the C-terminal Fmoc-protected amino acid with the support material with the addition of an activation reagent in ethanol, acetonitrile, ⁇ , ⁇ -dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N-methylpyrrolidone or similar solvents at room temperature or elevated temperatures (e.g., between 40°C and 60°C) and with reaction times of, e.g., 2 to 72 hours.
  • an activation reagent in ethanol, acetonitrile, ⁇ , ⁇ -dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N-methylpyrrolidone or similar solvents at room temperature or elevated temperatures (e.g., between 40°C and 60°C) and with reaction times of, e.g., 2 to 72 hours.
  • the coupling of the Na-protected amino acid (e.g., the Fmoc amino acid) to the PAL, Wang or Rink anchor can, for example, be carried out with the aid of coupling reagents such as ⁇ , ⁇ '-dicyclohexylcarbodiimide (DCC), ⁇ , ⁇ '-diisopropylcarbodiimide (DIC) or other carbodiimides, 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU) or other uronium salts, o-acyl-ureas, benzotriazol-l-yl-tris-pyrrolidino-phosphonium
  • DCC ⁇ , ⁇ '-dicyclohexylcarbodiimide
  • DIC ⁇ , ⁇ '-diisopropylcarbodiimide
  • TBTU 2-(lH-benzotriazol-l-
  • hexafluorophosphate PyBOP or other phosphonium salts, N-hydroxysuccinimides, other N- hydroxyimides or oximes in the presence or in the absence of 1-hydroxybenzotriazole or 1- hydroxy-7-azabenzotriazole, e.g., with the aid of TBTU with addition of HOBt, with or without the addition of a base such as, e.g., diisopropylethylamine (DIEA), triethylamine or N- methylmorpholine, e.g., diisopropylethylamine with reaction times of 2 to 72 hours (e.g., 3 hours in a 1.5- to 3-fold excess of the amino acid and the coupling reagents, e.g., in a 2-fold excess, and at temperatures between about 10°C and 50°C, e.g., 25°C, in a solvent such as
  • the Na-protected amino acid e.g., the Fmoc amino acid
  • the Na-protected amino acid can be coupled to the 2- chlorotrityl resin in dichloromethane with the addition of DIEA with reaction times of 10 to 120 minutes, e.g., 20 minutes, but is not limited to the use of this solvent and this base.
  • the successive coupling of the protected amino acids can be carried out according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer.
  • the next protected amino acid in a 3 to 10-fold excess is coupled to the previous amino acid in an inert, non-aqueous, polar solvent such as dichloromethane, DMF or mixtures of the two and at temperatures between about 10°C and 50°C, e.g., at 25°C.
  • Cleavage can be carried out with trifluoroacetic acid or other strongly acidic media with addition of 5%-20% V/V of scavengers such as dimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol, anisole ethanedithiol, phenol or water, e.g., 15% v/v dimethylsulfide/ethanedithiol/m-cresol 1 : 1 : 1, within 0.5 to 3 hours, e.g., 2 hours.
  • scavengers such as dimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol, anisole ethanedithiol, phenol or water, e.g., 15% v/v dimethylsulfide/ethanedithiol/m-cresol 1 : 1 : 1, within 0.5 to 3 hours, e
  • Peptides with fully protected side chains are obtained by cleaving the 2-chlorotrityl anchor with glacial acetic acid/trifluoroethanol/dichloromethane 2:2:6.
  • the protected peptide can be purified by chromatography on silica gel. If the peptide is linked to the solid phase via the Wang anchor and if it is intended to obtain a peptide with a C- terminal alkylamidation, the cleavage can be carried out by aminolysis with an alkylamine or fluoroalkylamine. The aminolysis is carried out at temperatures between about -10°C and 50°C, e.g., about 25°C, and reaction times between about 12 and 24 hours, e.g., about 18 hours.
  • the peptide can be cleaved from the support by re-esterification, e.g., with methanol.
  • the acidic solution that is obtained may be admixed with a 3- to 20-fold amount of cold ether or n-hexane, e.g., a 10-fold excess of diethyl ether, in order to precipitate the peptide and hence to separate the scavengers and cleaved protective groups that remain in the ether.
  • a further purification can be carried out by re -precipitating the peptide several times from glacial acetic acid.
  • the precipitate that is obtained can be taken up in water or tert- butanol or mixtures of the two solvents, e.g., a 1 : 1 mixture of tert-butanol/water, and freeze-dried.
  • the peptide obtained can be purified by various chromatographic methods, including ion exchange over a weakly basic resin in the acetate form; hydrophobic adsorption
  • non-derivatized polystyrene/divinylbenzene copolymers e.g., Amberlite® XAD
  • adsorption chromatography on silica gel e.g., on silica gel
  • ion exchange chromatography e.g., on carboxymethyl cellulose
  • distribution chromatography e.g., on Sephadex® G-25
  • countercurrent distribution chromatography e.g., on Sephadex® G-25
  • HPLC high pressure liquid chromatography
  • HPLC reversed- phase HPLC on octyl or octadecylsilylsilica (ODS) phases.
  • the peptide may be produced as an intracellular protein or as a secreted protein, using any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E. coli) or a yeast host cell, respectively.
  • a prokaryotic or eukaryotic cell such as a bacterial (e.g., E. coli) or a yeast host cell, respectively.
  • Other examples of eukaryotic cells that may be used as host cells include insect cells, mammalian cells, and/or plant cells.
  • mammalian host cells may include human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells (e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos 7 and CV1) and hamster cells (e.g., CHO cells).
  • human cells e.g., HeLa, 293, H9 and Jurkat cells
  • mouse cells e.g., NIH3T3, L cells, and C127 cells
  • primate cells e.g., Cos 1, Cos 7 and CV1
  • hamster cells e.g., CHO cells.
  • a variety of host- vector systems suitable for the expression of a peptide may be employed according to standard procedures known in the art. See, e.g., Sambrook et al, 1989 Current Protocols in Molecular Biology Cold Spring Harbor Press, New York and Ausubel et al. 1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation,
  • the method for transfer can be selected so as to provide for stable expression of the introduced polypeptide-encoding nucleic acid.
  • the polypeptide-encoding nucleic acid can be provided as an inheritable episomal element (e.g., a plasmid) or can be genomically-integrated.
  • a variety of appropriate vectors for use in production of a peptide of interest are available commercially.
  • Vectors can provide for extra-chromosomal maintenance in a host cell or can provide for integration into the host cell genome.
  • the expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression where the coding region is operably-linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7 and the like).
  • Expression constructs generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest.
  • a selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector.
  • the expression construct may include additional elements.
  • the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selectable genes are well known in the art and will vary with the host cell used.
  • Isolation and purification of a protein can be accomplished according to methods known in the art.
  • a protein can be isolated from a lysate of cells genetically modified to express the protein constitutively and/or upon induction, or from a synthetic reaction mixture by immune-affinity purification, which generally involves contacting the sample with an anti-protein antibody, washing to remove non-specifically bound material, and eluting the specifically bound protein.
  • the isolated protein can be further purified by dialysis and other methods normally employed in protein purification methods.
  • the protein may be isolated using metal chelate chromatography methods. Proteins may contain
  • the peptides may be prepared in substantially pure or isolated form (e.g., free from other polypeptides).
  • the peptides can be present in a composition that is enriched for the peptide relative to other components that may be present (e.g., other polypeptides or other host cell components).
  • Purified peptide may be provided such that the peptide is present in a composition that is substantially free of other expressed proteins, e.g., less than 90%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1%,
  • composition is made up of other expressed proteins.
  • Antibodies are antibodies to antibodies
  • the present disclosure provides antibodies, including isolated antibodies, that specifically bind a polypeptide of the present disclosure.
  • antibody encompasses intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody binding fragments including Fab and F(ab)'2, provided that they exhibit the desired biological activity.
  • the basic whole antibody structural unit comprises a tetramer, and each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” chain (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Human light chains are classified as kappa and lambda, whereas human heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively.
  • Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab') 2 , Fv, and single-chain antibodies.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of
  • each light chain has a variable domain at one end (VL) and a constant domain at its other end.
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids.
  • the antibody chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper- variable regions, also called complementarity-determining regions or CDRs.
  • the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FRl, CDRl, FR2, CDR2, FR3, and CDR3 and FR4.
  • An intact antibody has two binding sites and, except in bifunctional or bispecific antibodies, the two binding sites are the same.
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments.
  • binding fragments may be produced by enzymatic or chemical cleavage of intact antibodies. Digestion of antibodies with the enzyme papain results in two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc” fragment which has no antigen-binding activity. Digestion of antibodies with the enzyme pepsin results in a F(ab') 2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab') 2 fragment has the ability to crosslink antigen.
  • Fab refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.
  • Fv refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites.
  • this region includes a dimer of one heavy-chain and one light-chain variable domain in non-covalent association.
  • one heavy-chain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. While the six CDRs, collectively, confer antigen- binding specificity to the antibody, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen.
  • CDRs complementarity determining regions
  • hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a CDR and/or those residues from a "hypervariable loop".
  • epitopic determinants refers to binding sites for antibodies on protein antigens.
  • Epitopic determinants usually comprise chemically active surface groupings of molecules such as amino acids or sugar side chains, as well as specific three dimensional structural and charge characteristics.
  • An antibody is said to bind an antigen when the dissociation constant is ⁇ 1 ⁇ , ⁇ 100 nM, or ⁇ 10 nM.
  • An increased equilibrium constant (“K D ”) means that there is less affinity between the epitope and the antibody, whereas a decreased equilibrium constant means that there is a higher affinity between the epitope and the antibody.
  • An antibody with a K D of "no more than" a certain amount means that the antibody will bind to the epitope with the given K D or more strongly. Whereas K D describes the binding
  • potency describes the effectiveness of the antibody itself for a function of the antibody. There is not necessarily a correlation between an equilibrium constant and potency; thus, for example, a relatively low K D does not automatically mean a high potency.
  • the term "selectively binds" in reference to an antibody does not mean that the antibody only binds to a single substance, but rather that the K D of the antibody to a first substance is less than the K D of the antibody to a second substance.
  • An antibody that exclusively binds to an epitope only binds to that single epitope.
  • antibodies that contain rodent e.g., mouse or rat
  • variable and/or constant regions are sometimes associated with, for example, rapid clearance from the body or the generation of an immune response by the body against the antibody.
  • fully human antibodies can be generated through the introduction of human antibody function into a rodent so that the rodent produces fully human antibodies.
  • human and “fully human” antibodies can be used interchangeably herein.
  • the term “fully human” can be useful when distinguishing antibodies that are only partially human from those that are completely, or fully, human. The skilled artisan is aware of various methods of generating fully human antibodies.
  • Chimeric or otherwise humanized antibodies can be utilized. Chimeric antibodies have a human constant region and a murine variable region, and, as such, human anti-chimeric antibody responses may be observed in some patients. Therefore, it is advantageous to provide fully human antibodies against multimeric enzymes in order to avoid possible human anti-mouse antibody or human anti-chimeric antibody responses.
  • Fully human monoclonal antibodies can be prepared, for example, by the generation of hybridoma cell lines by techniques known to the skilled artisan. Other preparation methods involve the use of sequences encoding particular antibodies for transformation of a suitable mammalian host cell, such as a CHO cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example, packaging the
  • polynucleotide in a virus or into a viral vector
  • transducing a host cell with the virus (or vector) or by transfection procedures known in the art Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene -mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to ,CHO cells, HeLa cells, and human hepatocellular carcinoma cells.
  • the antibodies of the present disclosure can be used diagnostically and/or
  • the antibodies can be used as a diagnostic by detecting the level of one or more peptides of the present disclosure in a subject, and either comparing the detected level to a standard control level or to a baseline level in a subject determined previously (e.g., prior to any illness).
  • the antibodies can be used as a therapeutic to modulate the activity of one or more peptides of the present disclosure, thereby having an effect on a condition or disorder associated with the one or more peptides.
  • dAbs are the smallest functional binding units of human antibodies (IgGs) and have favorable stability and solubility characteristics.
  • the technology entails a dAb(s) conjugated to HSA (thereby forming a "AlbudAb”; see, e.g., EP1517921B,
  • a molecule of interest e.g., a peptide of the present disclosure
  • AlbudAbs are often smaller and easier to manufacture in microbial expression systems, such as bacteria or yeast, than current technologies used for extending the serum half- life of peptides. As HSA has a half-life of about three weeks, the resulting conjugated molecule improves the half-life of the molecule of interest. Use of the dAb technology may also enhance the efficacy of the molecule of interest.
  • the present disclosure provides methods for treating or preventing hyperglycemia, hyperinsulinemia, glucose intolerance, glucose metabolism disorders, obesity, as well as other metabolic and metabolic-associated diseases, disorders and conditions by the administration of the Modulators, or compositions thereof, as described herein. Such methods may also have an advantageous effect on one or more of symptoms associated with a disease, disorder or condition by, for example, decreasing the severity or the frequency of a symptom.
  • a subject may be a candidate for the treatment or prevention of hyperglycemia, hyperinsulinemia, glucose intolerance, and/or glucose disorders by the methods provided herein.
  • various diagnostic methods known in the art may be utilized. Such methods include those described elsewhere herein (e.g., fasting plasma glucose (FPG) evaluation and the oral glucose tolerance test (oGTT)).
  • FPG fasting plasma glucose
  • oGTT oral glucose tolerance test
  • a subject may be considered obese or overweight by assessment of the subject's Body Mass Index (BMI).
  • BMI Body Mass Index
  • An adult having a BMI in the range of 18.5 to 24.9 kg/m is considered to have a normal weight; an adult having a BMI between 25 and 29.9 kg/m may be considered overweight (pre -obese); an adult who has a BMI of 30 kg/m or higher may be considered obese.
  • the Modulators of the present disclosure may be in the form of compositions suitable for administration to a subject.
  • compositions are "pharmaceutical compositions" comprising one or more Modulators and one or more pharmaceutically acceptable or
  • physiologically acceptable diluents, carriers or excipients In certain embodiments, the
  • Modulators are present in a therapeutically acceptable amount.
  • compositions may be used in the methods of the present disclosure; thus, for example, the pharmaceutical compositions can be administered ex vivo or in vivo to a subject in order to practice the therapeutic and prophylactic methods and uses described herein.
  • compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration; exemplary routes of
  • compositions may be used in combination with other therapeutically active agents or compounds (e.g., glucose lowering agents) as described herein in order to treat or prevent the diseases, disorders and conditions as contemplated by the present disclosure.
  • other therapeutically active agents or compounds e.g., glucose lowering agents
  • compositions typically comprise a therapeutically effective amount of at least one of the Modulators contemplated by the present disclosure and one or more pharmaceutically and physiologically acceptable formulation agents, such as pharmaceutically and physiologically acceptable diluents, carriers or excipients.
  • diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethyl- or n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, fillers, bulking agents, detergents, buffers, vehicles, and/or adjuvants.
  • a suitable vehicle may be physiological saline solution or citrate -buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Those skilled in the art will readily recognize a variety of buffers that could be used in the
  • Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof.
  • the buffer components are water soluble materials such as phosphoric acid, tartaric acids, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof.
  • Acceptable buffering agents include, for example, a Tris buffer, N-(2-Hydroxyethyl)piperazine- N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), and N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).
  • HEPES N-(2-Hydroxyethyl)piperazine- N'-(2-ethanesulfonic acid)
  • MES 2-(N-Morpholino)ethanesulfonic acid
  • MES 2-(N- Morpholino)ethanesulfonic acid sodium salt
  • MOPS 3-(N-Morpholino)propanesulf
  • a pharmaceutical composition After a pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in a ready to use form, a lyophilized form requiring
  • the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, ampoule, syringe, or autoinjector (similar to, e.g., an
  • a multi-use container e.g., a multi-use vial
  • Any drug delivery apparatus may be used to deliver the Agr2, Cnpy4, Scg3 and Smoc2 - related peptides, including implants (e.g., implantable pumps) and catheter systems, both of which are well known to the skilled artisan.
  • Depot injections which are generally administered subcutaneously or intramuscularly, may also be utilized to release the peptides disclosed herein over a defined period of time. Depot injections are usually either solid- or oil- based and generally comprise at least one of the formulation components set forth herein.
  • One of ordinary skill in the art is familiar with possible formulations and uses of depot injections.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents mentioned herein.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
  • Acceptable diluents, solvents and dispersion media include water, Ringer's solution, isotonic sodium chloride solution, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS), ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables. Prolonged absorption of particular injectable formulations can be achieved by including an agent that delays absorption (e.g., aluminum monostearate or gelatin).
  • compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microbeads or elixirs.
  • Pharmaceutical compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain, for example, one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.
  • the tablets, capsules and the like suitable for oral administration may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time-delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by techniques known in the art to form osmotic therapeutic tablets for controlled release.
  • Additional agents include biodegradable or biocompatible particles or a polymeric substance such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides, polyglycolic acid, ethylene -vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers,
  • the oral agent can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, by the use of hydroxymethylcellulose or gelatin-microcapsules or poly (methylmethacrolate)
  • colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Methods of preparing liposomes are described in, for example, U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. Methods for the preparation of the above-mentioned formulations will be apparent to those skilled in the art and are commercially available.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose
  • water or an oil medium for example, peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture thereof.
  • excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia.
  • Dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxy-ethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate.
  • the aqueous suspensions may also contain one or more preservatives.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of, for example, water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one
  • the pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth; naturally- occurring phosphatides, for example, soy bean, lecithin, and esters or partial esters derived from fatty acids; hexitol anhydrides, for example, sorbitan monooleate; and condensation products of partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
  • Formulations can also include carriers to protect the composition against rapid degradation or elimination from the body, such as a controlled-release formulation, including implants, liposomes, hydrogels, prodrugs and microencapsulated delivery systems.
  • a time delay material such as glyceryl monostearate or glyceryl stearate, alone or in combination with a wax, may be employed.
  • the present disclosure contemplates the administration of an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide in the form of suppositories for rectal administration.
  • the suppositories can be prepared by mixing the pharmaceutical agent with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the agent.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter and polyethylene glycols.
  • Modulators contemplated by the present disclosure may be in the form of any other suitable pharmaceutical composition (e.g., sprays for nasal or inhalation use) currently known or developed in the future.
  • suitable pharmaceutical composition e.g., sprays for nasal or inhalation use
  • the concentration of an Agr2, Cnpy4, Scg3 or Smoc2 - related peptide in a formulation can vary widely (e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20%> to 50%> or more by weight) and will usually be selected primarily based on fluid volumes, viscosities, and subject-based factors in accordance with, for example, the particular mode of administration selected.
  • the present disclosure contemplates the administration of the disclosed Modulators (e.g., Agr2, Cnpy4, Scg3 or Smoc2 - related peptides), and compositions thereof, in any appropriate manner.
  • Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (injection or implant), intraperitoneal, intracisternal, intraarticular, intracerebral (intraparenchymal and intracerebro ventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), sublingual and inhalation.
  • Depot injections are usually either solid- or oil-based and generally comprise at least one of the formulation components set forth herein.
  • One of ordinary skill in the art is familiar with possible formulations and uses of depot injections.
  • an antibody or antibody fragment of the present disclosure is stored at 10 mg/ml in sterile isotonic aqueous saline solution for injection at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to
  • the antibody is administered by intravenous infusion over the course of 1 hour at a dose of between 0.2 and 10 mg/kg. In other embodiments, the antibody is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via subcutaneous bolus injection.
  • the present disclosure contemplates the use of the Modulators (e.g., Agr2, Cnpy4, Scg3 or Smoc2 - related peptides) in combination with one or more active therapeutic agents or other prophylactic or therapeutic modalities.
  • the various active agents frequently have different mechanisms of action.
  • Such combination therapy may be especially advantageous by allowing a dose reduction of one or more of the agents, thereby reducing or eliminating the adverse effects associated with one or more of the agents; furthermore, such combination therapy may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder, or condition.
  • kits therapies that can be administered separately, e.g., formulated separately for separate administration (e.g., as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a "co- formulation").
  • the Modulators are administered or applied sequentially, e.g., where one agent is administered before one or more other agents.
  • the Modulators are administered simultaneously, e.g., where two or more agents are administered at or about the same time; the two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether the two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for purposes of the present disclosure.
  • Modulators of the present disclosure can be used in combination with other agents useful in the treatment, prevention, suppression or amelioration of the diseases, disorders or conditions set forth herein, including those that are normally administered to subjects suffering from hyperglycemia, hyperinsulinemia, glucose intolerance, and other glucose metabolism disorders.
  • the present disclosure contemplates combination therapy with numerous agents (and classes thereof), including 1) insulin, insulin mimetics and agents that entail stimulation of insulin secretion, including sulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) and meglitinides (e.g., repaglinide (PRANDIN) and nateglinide (STARLIX)); 2) biguanides (e.g., metformin (GLUCOPHAGE)) and other agents that act by promoting glucose utilization, reducing hepatic glucose production and/or diminishing intestinal glucose output; 3) alpha-glucosidase inhibitors (e.g., acarbose and miglitol) and other agents that slow down carbohydrate digestion and consequently absorption from the gut and reduce postprandial hyperglycemia; 4) thiazolidinedione
  • the present disclosure contemplates combination therapy with agents and methods for promoting weight loss, such as agents that stimulate metabolism or decrease appetite, and modified diets and/or exercise regimens to promote weight loss.
  • the Modulators of the present disclosure may be used in combination with one or more other agent in any manner appropriate under the circumstances.
  • treatment with the at least one active agent and at least one Modulator of the present disclosure is maintained over a period of time.
  • treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable), while treatment with the Modulator of the present disclosure is maintained at a constant dosing regimen.
  • treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable), while treatment with the Modulator of the present disclosure is reduced (e.g., lower dose, less frequent dosing or shorter treatment regimen).
  • treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable), and treatment with the Modulator of the present disclosure is increased (e.g., higher dose, more frequent dosing or longer treatment regimen).
  • treatment with the at least one active agent is maintained and treatment with the Modulator of the present disclosure is reduced or discontinued (e.g., lower dose, less frequent dosing or shorter treatment regimen).
  • treatment with the at least one active agent and treatment with the Modulator of the present disclosure are reduced or discontinued (e.g., lower dose, less frequent dosing or shorter treatment regimen).
  • the Modulators e.g., Agr2, Cnpy4, Scg3 or Smoc2 - related peptides
  • the Modulators may be administered to a subject in an amount that is dependent upon, for example, the goal of the administration (e.g., the degree of resolution desired); the age, weight, sex, and health and physical condition of the subject to be treated; the nature of the Modulator, and/or formulation being administered; the route of administration; and the nature of the disease, disorder, condition or symptom thereof (e.g., the severity of the dysregulation of glucose/insulin and the stage of the disorder).
  • the dosing regimen may also take into consideration the existence, nature, and extent of any adverse effects associated with the agent(s) being
  • Effective dosage amounts and dosage regimens can readily be determined from, for example, safety and dose-escalation trials, in vivo studies (e.g., animal models), and other methods known to the skilled artisan.
  • dosing parameters dictate that the dosage amount be less than an amount that could be irreversibly toxic to the subject (e.g., the maximum tolerated dose, "MTD”) and not less than an amount required to produce a measurable effect in the subject.
  • MTD maximum tolerated dose
  • Such amounts are determined by, for example, the pharmacokinetic and pharmacodynamic parameters associated with absorption, distribution, metabolism, and excretion (“ADME”), taking into consideration the route of administration and other factors.
  • An effective dose is the dose or amount of an agent that produces a therapeutic response or desired effect in some fraction of the subjects taking it.
  • the "median effective dose” or ED50 of an agent is the dose or amount of an agent that produces a therapeutic response or desired effect in 50% of the population that takes it.
  • the ED50 is commonly used as a measure of reasonable expectance of an agent's effect, it is not necessarily the dose that a clinician might deem appropriate taking into consideration all relevant factors.
  • the effective amount is more than the calculated ED 50, in other situations the effective amount is less than the calculated ED50, and in still other situations the effective amount is the same as the calculated ED 5 o.
  • an effective dose of the Modulators of the present disclosure may be an amount that, when administered in one or more doses to a subject, produces a desired result relative to a healthy subject.
  • an effective dose may be one that, when administered to a subject having elevated plasma glucose and/or plasma insulin, achieves a desired reduction relative to that of a healthy subject by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than 80%.
  • An appropriate dosage level will generally be about 0.001 to 100 mg/kg patient body weight per day, which can be administered in single or multiple doses.
  • the dosage level will be about 0.01 to about 25 mg/kg per day, and in other embodiments about 0.05 to about 10 mg/kg per day.
  • a suitable dosage level may be about 0.01 to 25 mg/kg per day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day. Within this range, the dosage may be 0.005 to 0.05, 0.05 to 0.5 or 0.5 to 5.0 mg/kg per day.
  • compositions can be provided in the form of tablets, capsules and the like containing from 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • the compounds may be administered on a regimen of 1 to 4 times per day, and often once or twice per day.
  • the dosage of the Modulators of the present disclosure may be repeated at an appropriate frequency which may be in the range of, for example, once per day to once every three months, depending on the pharmacokinetics of the Modulators (e.g. half-life of the antibody in the circulation) and the pharmacodynamic response (e.g. the duration of the therapeutic effect of the Modulator).
  • the Modulator is an antibody or a fragment thereof, or a peptide or variant thereof
  • dosing is repeated between, for example, once per week and once every 3 months.
  • the antibody is administered approximately once per month.
  • the dosage of the disclosed Modulators is contained in a "unit dosage form".
  • unit dosage form refers to physically discrete units, each unit containing a predetermined amount of a Modulator of the present disclosure, either alone or in combination with one or more additional agents, sufficient to produce the desired effect. It will be appreciated that the parameters of a unit dosage form will depend on the particular agent and the effect to be achieved.
  • kits comprising the disclosed Modulators (e.g., Agr2, Cnpy4, Scg3 or Smoc2 - related peptides), and pharmaceutical compositions thereof.
  • the kits are generally in the form of a physical structure housing various components, as described below, and may be utilized, for example, in practicing the methods described herein (e.g., administration of a Modulator to a subject in need of restoring glucose homeostasis).
  • a kit can include one or more of the Modulators disclosed herein (provided in, e.g., a sterile container), which may in the form of a pharmaceutical composition suitable for administration to a subject.
  • the Modulators can be provided in a form that is ready for use or in a form requiring, for example, reconstitution or dilution prior to administration.
  • the kit may also include buffers, pharmaceutically acceptable excipients, and the like, packaged with or separately from the Modulators.
  • the kit may contain the several agents separately or they may already be combined in the kit.
  • Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package.
  • a kit of the present disclosure can be designed for conditions necessary to properly maintain the components housed therein (e.g., refrigeration or freezing).
  • a kit may contain a label or packaging insert including identifying information for the components therein and instructions for their use (e.g., dosing parameters, clinical pharmacology of the active ingredient(s), including mechanism of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.). Labels or inserts can include manufacturer information such as lot numbers and expiration dates.
  • the label or packaging insert may be, e.g., integrated into the physical structure housing the components, contained separately within the physical structure, or affixed to a component of the kit (e.g., an ampoule, tube or vial).
  • Exemplary instructions include those for reducing or lowering blood glucose, treatment of hyperglycemia, treatment of diabetes, etc., with the disclosed Modulators and pharmaceutical compositions thereof.
  • Labels or inserts can additionally include, or be incorporated into, a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVDROM/ RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory-type cards.
  • a computer readable medium such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVDROM/ RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory-type cards.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided.
  • Body Weight was determined prior to all metabolic tests using an automated digital scale (Sartorius LE5201, Sartorius Mechatronics Corp., Bohemia, NY) equipped with Software Wedge (Sartorius YSW05, Sartorius Mechatronics Corp., Bohemia, NY).
  • mice received a single bolus i.p. injection of peptide or vehicle control at min-30.
  • glucose lg/kg
  • PBS PBS
  • Plasma glucose concentration was determined at -30, 0, 15, 60 and 120 minutes by the Mutarotase-GOD enzymatic assay (Wako Diagnostics Inc, Richmond, VA). All measurements were made by taking blood samples from the tail vein.
  • oGTT was used to assess glucose tolerance.
  • mice received a single bolus i.p. injection of peptide or vehicle control at min-30.
  • mice received glucose (lg/kg) in PBS orally.
  • Plasma insulin concentration was determined at -30, 0, 15 and 60 minutes by a mouse ultra-sensitive enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH). All measurements were made by taking blood samples from the tail vein. GSIS was used to assess insulin secretion in response to a glucose challenge.
  • EECs and ECs were isolated from the mouse duodenum, jejunum, ileum and colon. Agr2 gene expression was then measured in FACS-sorted cells using SOLiD deep sequencing technology as directed by the manufacturer (Applied Biosystems, Foster City, CA, USA).
  • Agr2 gene expression data (measured in units of FPKM) are set forth in Figure 1. As indicated in Figure 1 , Agr2 gene expression is most prevalent in EECs of the ileum and colon and is observed to a lesser extent in EECs of the jejunum and duodenum, whereas Agr2 gene expression is most prevalent in ECs of the ileum and jejunum, with low or undetectable expression observed in the duodenum and colon.
  • Example 2 Effect of Murine Agr2(p4 ), Agr2(p5) and Agr2(p6) on Glucose Homeostasis in High-fat Fed Obese, Insulin-resistant Mice
  • Figure 5A shows the effect of a single bolus i.p. injection of Agr2-p4 (gray squares) and vehicle control (black squares) on basal (fasted) plasma glucose concentration and on oral glucose tolerance
  • Figure 5B shows the data from Figure 5A expressed as the percent change in plasma glucose concentration normalized to baseline (min-30).
  • Agr2-p4 significantly improved oral glucose tolerance but not basal glucose concentration.
  • Basal (fasted) plasma insulin (FPI) concentrations were evaluated in a manner similar to that utilized for plasma glucose. Briefly, FPI concentrations were determined in untreated high-fat fed mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p.
  • FIG. 5C shows the effect of a single bolus i.p. injection of Agr2-p4 (gray squares), and vehicle control (black squares) on FPI concentration and on glucose-stimulated insulin secretion (GSIS), whereas Figure 5D shows the data from Figure 5C expressed as the percent change in plasma insulin concentration normalized to baseline (min-30).
  • FIG. 7A-d set forth the data generated for Agr2-p6.
  • Figure 7A shows the effect of a single bolus i.p.
  • FIG. 7A shows the data from Figure 7A expressed as the percent change in plasma glucose concentration normalized to baseline (min-30).
  • Agr2-p6 significantly improved both basal glucose concentration and oral glucose tolerance.
  • FPI and GSIS concentrations were also evaluated as previously described.
  • Agr2-p6 significantly increased GSIS at 15 minutes.
  • EECs and ECs were isolated from the mouse duodenum, jejunum, ileum and colon. Cnpy4 gene expression was then measured in FACS-sorted cells using SOLiD deep sequencing technology as directed by the manufacturer (Applied Biosystems, Foster City, CA, USA).
  • Cnpy4 gene expression data (measured in units of FPKM) are set forth in Figure 8.
  • Cnpy4 gene expression is most prevalent in EECs of the colon, is observed to a lesser extent in EECs of the duodenum and ileum, and is either below the level of detection or is not expressed in the jejunum. Cnpy4 gene expression in ECs is either below the level of detection or is not expressed in the duodenum, jejunum, ileum or colon.
  • mice weighing approximately 50 g were injected i.p. with a single dose of Cnpy4 ms-p or vehicle control, and plasma glucose and insulin concentrations were determined.
  • Basal (fasted) plasma glucose (FPG) concentrations were determined in untreated mice following a 4-hour fast (min-30).
  • Plasma glucose concentrations were determined at -30, 0, 15, 60 and 120 minutes.
  • Figure 12A shows the effect of a single bolus i.p. injection of Cnpy4 ms-p (gray squares) and vehicle control (black squares) on basal (fasted) plasma glucose concentration and on oral glucose tolerance
  • Figure 12B shows the data from Figure 12A expressed as the percent change in plasma glucose concentration normalized to baseline (min-30).
  • Cnpy4 ms-p did not significantly improve FPG concentration or oral glucose tolerance, but there is a trend towards improving both parameters.
  • Basal (fasted) plasma insulin (FPI) and GSIS concentrations were evaluated in a manner similar to that utilized for plasma glucose. Briefly, FPI concentrations were determined in untreated high-fat fed mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p. injection of Cnpy4 ms-p or vehicle control, and at minO glucose (1 g/kg) in PBS was administered orally. Plasma insulin concentrations were determined at -30, 0, 15 and 60 minutes.
  • Figure 12C shows the effect of a single bolus i.p.
  • FIG. 12D shows the data from Figure 12C expressed as the percent change in plasma insulin concentration normalized to baseline (min-30). As indicated in Figures 12C and 12D, Cnpy4 ms-p did not significantly improve either FPI or GSIS
  • EECs and ECs were isolated from the mouse duodenum, jejunum, ileum and colon. Scg3 gene expression was then measured in FACS-sorted cells using SOLiD deep sequencing technology as directed by the manufacturer (Applied Biosystems, Foster City, CA, USA).
  • Scg3 gene expression data (measured in units of FPKM) are set forth in Figure 13. As indicated in Figure 13, Scg3 gene expression is prevalent in EECs of the jejunum and is either below the level of detection or is not expressed in the duodenum, ileum and colon. Scg3 gene expression in ECs is either below the level of detection or is not expressed in the duodenum, jejunum, ileum or colon.
  • Example 6 Effect of Murine Scg3 ms-p on Glucose Homeostasis in High-fat Fed Obese, Insulin-resistant Mice
  • mice received a single bolus i.p. injection of Scg3 ms-p (lOmg/kg) or vehicle control, and at minO glucose (1 g/kg) in PBS was administered orally. Plasma glucose concentrations were determined at -30, 0, 15, 60 and 120 minutes.
  • Figure 17A shows the effect of a single bolus i.p. injection of Scg3 ms-p (gray squares) and vehicle control (black squares) on basal (fasted) plasma glucose concentration and on oral glucose tolerance
  • Figure 17B shows the data from Figure 17A expressed as the percent change in plasma glucose concentration normalized to baseline (min-30).
  • Scg3 ms-p significantly improved oral glucose tolerance at 60 and 120 minutes.
  • the effect of murine Scg3 ms-p on oral glucose tolerance is expressed as the percent change when normalized to baseline, there is a trend toward improvement of oral glucose tolerance (see Figure 17B).
  • Basal (fasted) plasma insulin (FPI) and GSIS concentrations were evaluated in a manner similar to that utilized for plasma glucose. Briefly, FPI concentrations were determined in untreated high-fat fed mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p. injection of Scg3 ms-p or vehicle control, and at minO glucose (1 g/kg) in PBS was administered orally. Plasma insulin concentrations were determined at -30, 0, 15 and 60 minutes.
  • Figure 17C shows the effect of a single bolus i.p.
  • FIG. 17D shows the data from Figure 17C expressed as the percent change in plasma insulin concentration normalized to baseline (min-30).
  • Scg3 statistically improved FPI and GSIS at 60 minutes.
  • Scg3 ms-p significantly improved both FPI and GSIS at 15 and 60 minutes (see Figure 17D).
  • n 6 mice per group; p-values determined by Student's t-test (2- tailed distribution) comparing peptide- with vehicle-treated mice are indicated on the graph.
  • EECs and ECs were isolated from the mouse duodenum, jejunum, ileum and colon. Smoc2 gene expression was then measured in FACS-sorted cells using SOLiD deep sequencing technology as directed by the manufacturer (Applied Biosystems, Foster City, CA, USA).
  • Smoc2 gene expression data (measured in units of FPKM) are set forth in Figure 18. As indicated in Figure 18, Smoc2 gene expression is prevalent in EECs of the ileum and duodenum, is present in a lesser in the jejunum, and is either below the level of detection or is not expressed in the colon. Smoc2 gene expression in ECs is either below the level of detection or is not expressed in the duodenum, jejunum, ileum or colon.
  • Example 8 Effect of Murine Smoc2 ms-p on Glucose Homeostasis in High-fat Fed Obese, Insulin-resistant Mice
  • mice weighing approximately 50 g were injected i.p. with a single dose of Smoc2 ms-p or vehicle control, and plasma glucose and insulin concentrations were determined.
  • Basal (fasted) plasma glucose (FPG) concentrations were determined in untreated mice following a 4-hour fast (min-30).
  • Plasma glucose concentrations were determined at -30, 0, 15, 60 and 120 minutes.
  • Figure 22A shows the effect of a single bolus i.p. injection of Smoc2 ms-p (gray squares) and vehicle control (black squares) on basal (fasted) plasma glucose concentration and on oral glucose tolerance
  • Figure 22B shows the data from Figure 22A expressed as the percent change in plasma glucose concentration normalized to baseline (min-30).
  • Smoc2 ms-p significantly improved oral glucose tolerance at 120 minutes and also shows a positive trend toward improving oral glucose tolerance at the 60 minute time point.
  • Basal (fasted) plasma insulin (FPI) and GSIS concentrations were evaluated in a manner similar to that utilized for plasma glucose. Briefly, FPI concentrations were determined in untreated high-fat fed mice following a 4-hour fast (min-30). Thereafter, mice received a single bolus i.p. injection of Smoc2 ms-p or vehicle control, and at minO glucose (1 g/kg) in PBS was administered orally. Plasma insulin concentrations were determined at -30, 0, 15 and 60 minutes.
  • Figure 22C shows the effect of a single bolus i.p.
  • FIG. 22D shows the data from Figure 22C expressed as the percent change in plasma insulin concentration normalized to baseline (min-30).
  • Murine Smoc2 ms-p did not significantly improve basal (fasted) plasma insulin (FPI) concentration or GSIS, but there is a trend toward improving GSIS at the 60 minute time point (see Figures 22C and 22D).
  • n 6 mice per group; p-values determined by Student's t-test (2 -tailed distribution) comparing peptide- with vehicle-treated mice are indicated on the graph.

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Cited By (2)

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WO2017070744A1 (fr) * 2015-10-30 2017-05-04 Monash University Méthodes et compositions permettant d'améliorer le métabolisme du glucose
CN110520439A (zh) * 2016-11-08 2019-11-29 迈阿密大学 抗分泌粒蛋白iii(scg3)抗体及其用途

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EP1898897A2 (fr) * 2005-07-07 2008-03-19 Sirtris Pharmaceuticals, Inc. Methodes et compositions associees pour le traitement ou la prevention de l'obesite, de troubles d'insulino-resistance et de troubles associes aux mitochondries
US20100239589A1 (en) * 2009-02-23 2010-09-23 Salk Institute For Biological Studies Methods and Compositions for Ameliorating Diabetes and Symptoms Thereof

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Publication number Priority date Publication date Assignee Title
WO2017070744A1 (fr) * 2015-10-30 2017-05-04 Monash University Méthodes et compositions permettant d'améliorer le métabolisme du glucose
JP2018533578A (ja) * 2015-10-30 2018-11-15 モナシュ ユニバーシティー グルコース代謝改善のための方法及び組成物
US20180362602A1 (en) * 2015-10-30 2018-12-20 Monash University Methods and compositions for improving glucose metabolism
US10703786B2 (en) 2015-10-30 2020-07-07 The University Of Melbourne Methods and compositions for improving glucose metabolism
CN110520439A (zh) * 2016-11-08 2019-11-29 迈阿密大学 抗分泌粒蛋白iii(scg3)抗体及其用途
JP2019535256A (ja) * 2016-11-08 2019-12-12 ユニバーシティ オブ マイアミ 抗セクレトグラニンｉｉｉ（ｓｃｇ３）抗体およびその使用
EP3538553A4 (fr) * 2016-11-08 2020-11-25 University of Miami Anticorps anti-sécrétogranine iii (scg3) et leurs utilisations
US11414482B2 (en) 2016-11-08 2022-08-16 University Of Miami Anti-secretogranin III (SCG3) antibodies and uses thereof
JP7136468B2 (ja) 2016-11-08 2022-09-13 ユニバーシティ オブ マイアミ 抗セクレトグラニンｉｉｉ（ｓｃｇ３）抗体およびその使用
CN110520439B (zh) * 2016-11-08 2024-03-08 迈阿密大学 抗分泌粒蛋白iii(scg3)抗体及其用途

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